Evaluation et application de méthodes de criblage in silico

Transcript

1 Evaluation et application de méthodes de criblage in silico Hélène Guillemain To cite this version: Hélène Guillemain. Evaluation et application de méthodes de criblage in silico. Sciences agricoles. Conservatoire national des arts et metiers - CNAM, Français.. HAL Id: tel Submitted on 16 Apr 2013 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 A A B C D B ABC D E F B BF F BB A B BC C D A B E B BC BE F D A EB B E F E B F F B B E B B E F F DF F DF A FBF B F C AB A CC D AEBAF EB AEBA D BA B E B FEB B B F EF B B F B B B FE B B EE B C F C B C B F C B C FB BD E B C F F D B B B E B C

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4 F BC B F B BC BC BC F ABC D E F F

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6 AB CD BEF E EA B E EA BEA FAE D A F AE E E E E D D BE D F E E D ED EBDE D E D AEA FA BE BED B E AEF E D E E E DAA FE E D E D E AEB FA F E D AE AA EA B EBF BEA BE D BBD A EDFE E E D E AEDFEC E D FBE DBA E D ED A E D E AE E F E AA EA B E E E D AE E E F E DA F E E D A E E E F E EDFBB E E DA D E E DE E AE E DE E F ED E D E AA EA B C E BE ABE D AE EA FA E F E F E F E E AE F E E F F E E DA D E AE B E F EB AE D BE DE E D E F E E D BE DBEB E A BE E E BE F B E E D DBE D E FE E D E E E E B DA E E B F E DAB E AE AEB E FD AE F E E E F D E AE A B E E B E E F DFE EA BE D A ECD E E A AE F DF E D F E EAF F E B F E AE D F BE D A BA E B E E BE A B E D E DEB F E EA B E E F E DE DA E D E E D A F E F E D AB E AE F E DFA E A A E F E D AEF EA BE D E E E E F ED E BE EA BE E E E BF E E AE E BA D E DE D D E E F E E AE EB E E F E DBE BE DAB E E D E B E E F DFE AEA FED A E E AE ED B E F EA FBE BEBAD D BE F E AE D F E AA E F E F E D E FE F EDFA EF E B E E E DE D E AE BED BE F E AEBF A E D AE AA EA B E AD AE E DBE AE D ED D A E AE AEA F F BEB FA F

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8 BBD C A E E B A EC BA FA E AD B E A E A E E D E AE BB E F D E D E E DE EA E F C E E A D D EC B DA E E C CE EC A E E B F EC B E E D A F E BB A E E E F FAE E E A D A B DB E F D E DBB DA E E D E D D E A E A DA E E D E DB E AFD E D E C E E A ECDADE D E E A A D E E D E C E E DA EC B D AE E E FBAE A D E D A CE E AE D E FD EC DA E E E DA E D D A BA E E AE BB E F D E D E E A F AF E DB E AFD E D E C E E A F AF ECDADE E E E F D FAE A E BA E F E B BE D A ED D

9 BF BE E DE A E E D AB E E D E E BAE E FBE E FBEFA B E AE BE A BE B BE BB A AE A E D F B E D FDA E E E A BEDE BE E E D A E BE A BE E D E EB E AE BE BE E BA F A E E DE D F E D FDA E E E C C E DE DA E B E F E BEB A BE E D B E AD AE DBEA F F BED D A E EA FBE BED A B E DE F BBD E DA F ED AF E E AAD A E FB F BEBA F AF BE AD BE AE A E B BE F EA A E E E DE A E BE B A BE E D B E EDFA E EDE A E BE E E E BE A F BE D FDA E BE A BEB F AE E D B EC E F BE A F BE AE E A E B B EA BE F E C E AE E EDFA ED A DA E BAE B E E BF D AE DE D D A E A E F E A E ED A B E EF E A A E F ED A EBF E E E A E AE EBF EB F BEDE A E A E D EF E A E E D E E B E D E E B E D DA E BE A B E E D E E FAE A E F E AD AEB FA E E A DA E E F BE F BEDE B EA D FA F ABE BE E D E EB E EB D A E C E A F BE D FDA E F E E A A E B E E

10 BF E ED D B EA E A F A E E AFD EB E EA E F E B E BB EA E F E E AFD EB E A BE DBE E DB ED ED D D E A BE D EA E E D FDA E EA BE E AE AFD EB E A BE E D FDA E EA EC CE DAD DB EB E D FDA E E BED B E D EB EB A BE EA EC CE DAD DB EDBEA E EB A E DA EFB E EA EC CE DBE AE D AE ED EA ED A B E BE FADA D E E E ABEA ED BBEA BE BBF E DBB D E E F BE D E E E EB D E D EBA F AF B EFB EA E B AEA E EB A E A E BED B E D EA DAE D FDA E A BEB EB E DB B E E D FDA E A BE D EA FBE E B E E C ED E E ED A DA E A E DBED B E B EFB E A BBE F B E DB E E F ED A A E D A D EDE AFD EB E F E DBE ED EA E α E EB D E F E A E B E A ED E D A A E E E DBE E A E BE BA DA EA E D F E E AFD EB E EA E F E B E BB ED A F E AFD EB E A BE E A E E B E AFD EB E E CEB D A E D FDA E A B E A BBE F E B E E

11 D E BE DA B E ABEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E EE E BBD EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E EE E BF EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E EE E BF E ED D BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E EE E D E BE DA BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E BA E BEAD DF EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E BA E BE F BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E D A EE E E A F A EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E A F A E D EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E D E E A A BE F BE DB EBF E DEBA F AF E F E D E E E EEEEEE E E E E E E D E E A A BE F BEE DB EBF E DEBA F AF E E DE A E E E EEEEEEEEEEEEEE E E E E E E D FDA E E A BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E A BE EA B EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E EC F E D A EE E E BF ADABEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E D FDA E E A BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E E DA EE E A A BE F BE A BE FE E D AED A BE E E EEEEEEEEEEEEE E E E E E FB EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E BF EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E E BF E ED D BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE E E E E

12 BA E BEAD DF D E E E A BE B F BE BEB BA BE AE BEB A BE E D B E AEF E F E E F E E E A AB E AEF E F AD E E A E F E E E D B E D E E E A BE B F BE BEB BA BE AE BEB A BE E D B E AEF E F E E F E E E A AB E AEF E F AD E E A EBF F E E E B E D E E E A BE B F BE BEB BA BE AE BEB A BE E D B E AEF E F E EBF F E E E D B E AEF E F AD E E A E F E E E D B E D E E E A BE B F BE BEB BA BE AE BEB A BE E D B E AEF E F E EBF F E E E D B E AEF E F AD E E A EBF F E E E B E D E E E D A F BE BB AE E E AE E AE E E A FBE D E F E AE E F E D F E BA F AF E B E B BA B E A AB E AE D B E D E E E D A F BE BB AE E E AE E AE E E A FBE D E F E AE E F E D F EBA F AF E BEB BA BE A ABE AE B D E E E D A F BE BB AE E E AE E AE E E A FBE D E F E AE E F E D F EBA F AF E BEB BA BE D BE AE D B D E E E D A F BE BB AE E E AE E AE E E A FBE D E F E AE E F E D F EBA F AF E BEB BA BE D BE AE B

13 BA E BE F B F E E E BD E E A BE EB E D BE E BBFBE E AE F E D A F E E E BE E DABE E BE F E E B E F E E E B ADA E FE D D E F E A A E F E E D E E E BE D A F BE E D B E EB AE B A BE E A E BE BED DA F BE E D E BE F ABE BE EAF F B E AE DE D E DA E E F F E E E B ADA E BE A D A BE A E EB A E E D B E FE D A F E DE AEB E D E BAD B E E C E E E B ADA E D A ED E E DF E AE B E A F E E E B ADA E E DEB A E E E F BE D BEF E A F E E E B B F E E E DA BE A E BE D BE B A BE E BE DF E B A BE E BE D BE DA BE E AE BE DF E DA BE E BE D F BE B A AE DE D E A E BE BF ADABEDAA FBE AE BE BF ADABE B BE F EF EB F E E BE AA AE E D F E DEB B A E E DE B A E E DE D F E A E B A E E AE DE D F E A E DA E F E E E BE E BA FA E BED A BE D BE BE E BE F BE F E D F E DBB E E D A E F E A E EB F E E E B ADA E E E F BE E E F E D E F E F E BA FA E E AE F E A D E DE F E E DE BA FA E E BAE E D E FDA E F E E E BE E E E E DA B E D E DE F E E BAE F E F E DE F E E F EF EB A E E D BE BE EB AE A F B E E DE F E E BAE F E F E DE F E E F EF EB A E E D BE E E DE F E E BAE F F E E E F BE E E D B E BE A BE EB AE B A BE D EF EA D AE E AE BE A BE E D E BE A B E BE A BE F F B E C E E AE B EB AE B A BE E F E F E DF E AE A E B A A F E E E F BE E B A E BE BA FA E EB BEDAA F BEDF ED A BE FB E AEDF E D A BE F B E D E E F E BE EB BA BE E DEC C E F E D F E BA FA E DE D A E

14 D F E B A E B E B B E B E A F D A E DF E E D A A E E DE E F E E E F BE E B A E BE BA FA E EB BEDAA F BEDF ED A BE FB E AEDF E D A BE F B E D E F E F E BE EB BA BE E DEC C E F E D F E BA FA E DE D A E D F E B A E BEB BEB EA F D AEDF E E D A A E E DE F E E E F BE BB AE EB E F E F E A D AE E E AE E A BE F B EBF E BE EB BA BE E DEC C E DE BA FA E E BED A BE BAE B A E E B E F E E E F BE E E F E F E A D AE E E AE E A BE F B E BF E BE EB BA BE E DEC C E DE BA FA E E BED A BE BAE B A E E B F E E E F BE E A A E F E F E A D AE E E AE E A BE F B E EB EBF E BE EB BA BE E DEC C E BE BE D BED EF E E D EB AE B A BE E D E BE A BE BEDFE DFE E DE D E B A AEF E BA FA E E BED A BE D BE DE D F

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16 E D A E A F A

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18 E A F A E D DE A E E D ABE BAEF E BBFBE A F EAD AEDFE DFE FEA BE F E E D AE BA E DE B EDFE AE F E D AE A ED B E A E E BE AE E D BE F BE D AD B B E E A E BE A BE AE BEFA B B ED B E F E E DE E E AF EC E EB E A BE AF B E A E DE DB E D E E E F E A DA E EA A BE EB E AE DE B EBF E E D E F E F E E E E ED BE F AEB F E BE E DE DB E D E E E BE A BE E D E E DFAE AE FE E F E E F FAE E F AE A EFA B B E BE A BE AA AE A DA E EA F BE D E BE B BE BE D F BE E A A BE F BE F BE A F B E B B E BE F BEA F BEB AE BF A E A B BED E A E BE F BED D AEF E F ED A A E BE DE E A AE A A BE EB B E DE F E DE FBE AA FB E BAEB A E F E A E E DB BE F E F BE F B DE DB E F E B BA E EA BA E D A A E AE DEA A E FE D DA D AEBF E F BE AEA BBFBE F BEBDE B A EB ED A A E AEBDEA A EBF ED DF E A BE C A E E B A EC BA FA E AD B E A E AE A E BEA BABE AA AE E A E BE ABE E DE F EBF E BE A F BE F D BE AE BE D B E DE D EA D FA F E B EA F E E B E D ED B E F EBDEA A ED F E AE F E E B E B AD AEF ED A A EB DA EBD BE ABEB D BE D F BE DBB E E AF E F E E E DE DB E E F E BAE EBF E E D BE A BE FBE BD B EB A BE F E E DE F E D DA EC BE B BE BBD A BE FE B E B AED BA BE F E A E EB F E A D E BE BBD BE F BE E DB E EB E AEBF E E FBE D BE A B E AA E BE E DA AB E BE A BE A D BE E B A E B E A E F EB AE A BE AE BE ABEA D FA F BE AE B D BEB AE BE B A E DE DB E E F E DE AA E DE D DA E BE BF ADABE BE F E AD BE A BE EF E D E E E B E BAE D E EF E D E A FEDFEA D A AE E EB E BA E E BB E BA AE FAED BE A E A E F E A E DFA BDA E E B EBF E E D E FE AE EBDE D BDA E E D AE A E D BE DE DB E E F E DB E E D D D E E EA E

19 A D AE BE ABEB D BE D BE AE AAD AE E AA E E D E E F BE DA BEA D FA F BE AE E F BE F DA B DE E AE BE A BE D D F BEA D A BE AE BE EA BA E A BE B BEBF E BE BE A A E D A E E DFB E BE ADA BEA F B E E E E E B BE F D AE A EA BA BE E D E D D E AF A E DE A E E D AE BAE D AEDBB BA E D E DA F E D BE FBA E D D FA F E F E E D E E BBFB E AAD AE EA BA E F F BE BE E B B EB E BE A BE AE DE F BBD E DA F E B B E BEDB ABEB AE AD BE FBEAD E D BE A F A D A E BE F E D DAEBF E E A D AE E DB E F E F DE E DFA BDA E E B EBF E E D E E E DFAEADF E E BAE E D A E EF E DF D B E B E BE D B BE DA F BE AE E D F E D A DA E FE E C A E D AE BE BE AD BE E DE A E E D A E D E EF E D EB F A E FEF E D E D A E FE B E F AEF E D E D A E BE BE FBE E DE D D A F E F E B A AE E E BED ABE E BBD E F E E D AE E D E E DFAE A EAD AEA D A E F E AF E E E E DE A F E AEB AE BBF BE BE F BE F EB AE BEBF E DE E A A EDEDFBB EF E D E AD E AEDB AE BAE E AD E D BE E D A EBF D A

20 F E E BD E E A BE EB E D BE E BBFBE E AE F E D A E BE A F B E E E F BE A A AEB A A BD BE BAE B E E E E E B B E D A E E BA D AE E E F E E BE E F BE BAD AE B F A E E DE DBB DA E E D E E BA E ABEA BE E A F BE E BE DB BE E BE EBA F AF BE F B E BE A F BE D BA BE E F ABEFA BD BE EB A B E BE A D AE FB AE BE F ABE F AED A BE AE E BE A F BE E D E DE D A E BE A F BE F BB AE BEBA F AF BE CE AE CE BE B B EC BE D BE D AE BEBA F AF BEA B BE BE F BE F AE E A E FA B BE E E E E D BE DEBA F AF E CED B E E E B A E DBE AEF E DA ED A E E DE F

21 E A F BE D B E DB BE EBA F AF BE F B BE DB BE EBA F AF BE F BEB AE BE DB BE E AE E DE F D AE BE B BE FE ED D F E E FE E FBA E B F E DA E BAE B E D BEF E A E E D E BA D AE E A AE A BE A F B E DE E BA E BAE BA AF E BEBF BAD BE A BE D BE BE F DF E AB E DAD F BE F BE AE B F BE A AE D B E E A ED B E FBE E E BE EBF BAD B E DE FBE A AE BEBF BAD BE D AE E F DF E AB E D ABEA F BE AE E A FBE E B E FBE E E BE EBF BAD BEB AED B E A B EC DFA BE DB BE E BE BA A EA BE F E E F E A AED AF AE E BE E B B E FE BA E D A D AE BA F EB FBE E E E D B E BE EB AE DBE FA BD BE A AE E DA F E D E BE E A AE DBE E BE F D AEA FBE BE F AB E A F BE D BA B BE A F BE D BA BE A AE BE F BE A AED AD BEDF BE E ABE F BB F B E DE FD A A E DE FD A E DE B A E AE E E E D F E F AE B AE B B E AAD AEF E D E F E BE ABE F ABEB F D A B E E DE B D FA AE D AE BE BE DA F BE F D AE BEBA F AF BE BE ABE B BE AE D AD AE F EFA BDA E D E BE DA B E AD BE E BE F BE AE F ED A A E F E F EBF E BE BE A A E D BE BE A F BE D D AE F E FAE F E E F E BE B B E AA ED A A E BAE DBEA F F BE F E DE A F E CE D B E BAEF E BE D BE D F BE E A A BE F B E A D AE FBE E E BE E B BEB A A F B EC DFA BE A F BEB AE D AE B B E A D AE E EA E DA B E E F E FBE A A B EA BE F E D E FBE E E F B E FE E B E E B B E D E AEDFBB E E D E BE BE E B BE D EB D A

22 E A F BE E F ABE F AED A B BE A F BE A AE BE B BE F E B F BE BE DA BE D A A E F EB AE F B E BE F BE B A BE D BE BE D F BE EB AE DBE AE B BE B F A E D BE DE B E E BE DA F BE F D AE BE F BE E BE A F BEFA BE AEFA BD BE E DA F E AD BE D F BE A AE BE D ABE E AE FE D B B E E DE C E C F ECDADE AE CC E F E A AE F E BAD AE FBE E E B BE F AED A BED E DEBA F AF E F E BE DA BE D AE E AE FD E E BA E BE A BE D F BE E D A E E DEBA F AF E F E D A A E F E E FD ADA A EF E B A E EB ED A EA D FA F E FE EB E DFE E AE E C EC BE DA BEB D BEB AE B BE E D A E FEC AE EC F E E C EF E A F E F E F E E D ABE FE F BE D D F AED A BE BBFBE E F DF E FE B EC BE BE A D A B E FBD E D E DA E FE E E D B E D A EB AE D AE B BE F E D F E B EC BE A F BE FBEB D B BEBF EF E D D E FEF E A E E D D E BEB AE D AE B B E E E F E D E E DA D E D E BA AFA E F E E FDA E A F BE F F AE B BE AE F E A BEBF E E C E AE E D E DE A F E E F E FDB AE E B BE D A B E F BBD AE F EBA F AF E C E BE A BE D F BE D E E E E E BE F D E AE BE DA BEBF EBDE D A A E BE BAD A BE E D B E BE BE C A E A F BE E D BE A F BE E D EB AE A AE BA BEDFE D E E DFAE AE AE F AE A EFA B BE E DA F E E E BA E FB F BE B E D AE E BE B BE B F BE E E D F BE E BB BE F D BEFA BD BE EB A B E F E D F BE E F ABE DAF B E E A E A E E AE E BE E DE A E BE D ABED F BE D E DE ED EC F E BADA E AD AE DB BEBF E BE F ABE DAF B E

23 E D F BE E B A E F E D AE BE B BEB A BE F E F E B D E F E E BE D B E E D E FE E B A E E D F BE D B BE BD AE BE B BE A A AED A BEBF EF E E D A F E FEF E D E E B E D E E E B E FB F BE A F BE B D B BEBF E BE DB BE D AE AE D E BE A F BE F BEDF E A BE E E D E BE D DF E F BE E D E AE AE E D E FE E BE A F BE E BE F D BE E D FA AE F E E B E BE B BE A B E AD BE A F BE A AE BE D ABE F D B E E AEB FB D AE FE D E E D ABEDE A E AE D E B E AE B E F E AE A E F E BE F BE E F E E E F AE A E B BE E AD AE DE D B E E E FE FB F BE D AB E BE D ABE F D BE AEF E A E F D E FBE D E F E BE B BE A B ED B E DE D A E F E F BE D D A BA F BE F B D A F BE E D B BE E E A E B AE E BE FEB A E E D B E BAE FBE AD A E BE D ABE AEF ED A E E D B E FBE D BE F E DE F D AE BEA F BEA F BE D E D E E B BE A B E E FBE D E A E E F BEDA BEB AE F BE D BE BE A D A BE A AED E DE E E F E A D EF E FBE D E E E D B E D EF A E E DBB E F D E E AE C E B A E D E E BE A F BE E D AB E E BA ED B EF E D E E E A F BE AE E AE EB A E DE A F E EA D D EB E E A E E A EF E A F EB D B EBF EF E E D A F E E DE DBE DA AEB E E F EA D D D AEBF E DE E E F BA E E F DF E BB BE F D BEB AE B E DABE E A F B E BA E FB F BE DABE A F BE F E B A E BE A A BE F B E BE FBE FA B BE F E BE A F BEB AE E C E E FE C

24 E E DAE E E E DAE E F E E F D FAE A E BA E E AE DE B ADA E F E F E D EF E D E E D D A B EB B E D EF E D AE DA BE AE E D B B E BEDA BE E D BD AE DBE D A E E DE E D F E DBB F EB AE B A BE A E AB E BE D B BE EB AE DBE B A BE FD E BEB AEB BE FE D DA F B EAD BE F E BE D B BE F BE E AEA BE EB AED BE E D E A E D EB DA E BEDA BE EB AE A B F E E BE E DABE E BE F E E B E D A E E F E ED AE A AE DBE D F A EBF E DE F E B A E D EF E E E D A F E FB F BE BE E F AE A E ABE F EF E E F E E F E E E E EF E F E E D E E F E E E BE F BE D A B E D BEF E D E A F E AA E ADA E AD AE E

25 D D A EC F ED DA BE F E AE A ED A BE E BE ADA BE E D F BE AE E B F B DE ADA E E D F EDE A E A F A E BE E E A BBD AE DEBA F AF E F E F E E D E E F ED A BEB D BE E E AE E E AE D F E E D F EDA E E DE F EF E E D F E BA E F EB DE E E F F EB AE DA E E D A E D A E E D AE BF A E E DA ED D AE E E D F E E FBE D E F E BA F E E E E AA E E F E F F E DA EA D E BAE B EB E BA EC E DE E D E E D F E D E DA E B E D D AE E FBE A AE E BAE B E F E DA E F E F E D DA E AA E D E EF E E E A E D EF E D EA E AD A E D BEF E A F E E D E AA E ADA E E AE DBE E D E DE B DA E A E F E F BEBA B B C F E F BEB AE BEBA B BE B F E E F E F BE F BEB BE B AE A F B E BE AE F E E DFA E D E D AEB DA D E E F BEDA B E BE B BED D AE BE A BE B F BE A B E E AD AE BBD E A F E BE B BE E D A E FE E F DA E B A D BE D BE DE ADA E E DE B DA E D E D EB E D AE EA BE D A BE E DA F E E DE D A E E E DE DBB E E DE D A E E D E E E F EF E A EA A D D E E AF E BEDA BE B BE E D E E E B AD AEF E ED A D E BE DA BEDFA F E F E A EA A D D E E E D A E F EDA E A E E E DA BEDF F BE E BAE E F E D F E E BE B E FB F BE DBB BE F AE BA E DE DBB E B E D E DFAE F D AED BE A E A E D E EF E A E D EA A D D E B AE B F EB BE EDA BE B BEB AE AB E DE A E B A E D BE DE ADA E E D EF E EBF E BEDA BE B BE D BE E D E FE D E E F D AE E EBF E BEDA BE B BE D BE ED A D E E E E FAE E DBE A E F E AA E DBB E AD AE DE DBB E D E DFAE F E BE A BE DF E E E

26 BE BE E ADA E B F E AA AE EB E A AE FE E DE D A E E A E F EBA F AF E B E BE D B BE A BE E AE E F AE A EFA B BE F E F E DE A E DA E BEDA BE B BE F E F E D B E D B E EBDE D A F D A E E FA B E F F E E D E F E E DAE BAEA BEFA E F E EBA D E EA BE D BE D F BE E A A BE F B E BE DABE E E C C ABE DABE BA AE D AE BE DA BE AD BE A E DA E BE B DA D B E D E D A E F E E EBF EF E F E E D B E F EDA E D E E E FE E DAE EFA B EF E E D E F E DE A A E A EDA BE BAEDFBB E AD ED E EA E E D B E D B BEB B E F B EA B ED DA F B E AE BEDA BE F B E D E BE DABE BE FBE F D AEFA B B E EA F E E DAE C E FA B E F E BA F E DE A ECDADE D E E E DAE C E A F AF ECDADE DA E E FE E DAE E E DAE C E BAEBF A FAEFA B E F E BE D F B EA BE F E BE A B E C E F E D BE E B E AA AE E DBBD E D BE BBD E F E DAE EF EDFA E BE F E A E EE D E DE B E A E F E DABE CE BAE DA AED B E D A E BE FE DBBD E F E DAE E EF E DAE C E DA E CE AE A E E D EF E D E E B E E E E E DE FD A E E BE BE F E A AEBF E BE BF ADABE BE D F BE BE FA BD A BE ABE ADABE BDA E BE F ABEA A D BE F AE F EBF E F BE A BE B F BE AEA FBE BE D BE E D E E BE AE DBE E A E D F A E D F E ADAE BDA E FAED BE A E B AE D BE E E EBA F AF E E FE FD D E AA AE AE D A D BE B BE BBFBE BE A F BE F AE A E A BEB E F E E C A E AD E D BE E AEBF D A E F E E DBEA BA E D E D E EB E BE B BE F E F D AE BF A E A E D A BE E DFB E F E E C A E D FDA

27 E E C A E E C A E F E F E BAE B E BE D D A BE D D A BD AE BDE B A E D BE D B E BAE E E B ED B A E BDE BA FA E B E AD B EB E A E AEBDEA A E B F E DE E D BA DA E D E BAE B E D B A E FE D AEB E DE D E DE F F FB E A BA D EB AE D E FB E DBB EB AE D EA D B AED A E E BE D BE D B E DE F E DE A E D E E AD A B E E BBFBE BAE A D AEBF E FB F BE AB E BE E D BA DA E FE D A E E DE B E DE A BB E E D DA E FE B E A D AE E D A EBDE F E D BA DA BE A DA B E F BEDF E A BE A E BE AD B BE E F E D AB DEA A E E D AE F D AE A E FE FB E FE B EA F E E AD AE AD B DE D BBD E E DEA A E F E D AE BAE F D E E BAE E AE D E F F E D AED AEF E EA D FA F E E BAE E E F E DE A DA E DB DA F E A F EB AEB DA AE FBE E F E DE A DA E DB DA F EA D FA F DE DA BDA E FE D E EDE A D E DE BB A E E BE C A E F E E D AE BE B BED D AE BE A BE B F BE BE BEB D BE D E BE D ABE B BEBF E E D E F E EB AE DBE E F E E E F A EF E F E BAE B E F EB E E E EDFE BE E BE BE E B E E DBB E F D E F E FE D E E ECD E AE E D AD E A E DFE AEF EB D AE D F E E F E FE D E E E BE E E F BE F BE E D B BE E BE E E F BED A F BE E D B BE C F B E BE DA BE AE A ED A B E AD AE D E B E AED E F E B AE D E EF E E A E E AE EF E DBB E F D E B E A E E AE ECDE AEF E E DA BE A E E AE DE A E E C A E AEDFBB E A E BE D BB BE A A BE BEB BE B A BE D E D E E F E F DEB E D AE F E A BDA E FE AE E

28 A DA E EA A BE EB E E E C A E FAE A E A E EFA BD AE BE A BE AD BE AE E D E A DA BE F BE AE E E C A E BAEBDA B D BD AE F AE A EB F BEDFE D E E E DEBA F AF E E DE A E E BAE F EF ED E DB EBF E DEBA F AF E E AA E E BAE D AEFA B E E AA EBA F AF E BAE F E D BE F F E FE FB F BE F BEB AE F BE F E A ED A BEBF E AA E A EF ED E DB EBF E F BEBA F AF BE F DE A E BD E D A E BE F EA BE D BE F AE A E FA B BE D BEF E A E D F EF E AD E E E F BE AD AEB A BE E EF ED E DB EBF E DEBA F AF E F E D E E ED D AE A E BE D BE EB A E E D B E A A E BE F EA BE D BEB AE AD BE D BE BE F E D BE D A B E E D AE D E BE A BE DB BEBF E DEBA F AF E F E FE E FB F BE D BE E

29 E D E E A A BE F BE DB EBF E DEBA F AF E F E D E E E E D D B F E E E B ADA E FE D D E F E A A E F E E D E E E BED A F BE E D B E EB AE B A BE E A E BE BED DA F BE E D E BE F ABE BE EAF F B E AE DE D E DA E E F DE EFA BDA E FEA E D D E BAEA F E D BEF ED A E E AE E E EC F B E BE D D A BA F BEB F BE AD AE DBEBF BD A BE F E F E D A A E F E F E F E F E B B A E B DA D E DE A E D FA E AF A E E D D E BAE E E AD AE D AEA B E BE D D A BA F BE AAD AE EF E F E D EF ED A A E F EB F E E DFA BEA B EF E D D E BAE B E BE AB E A BE F B D A F BE E D B E

30 E B E F E F EB E DE DA EB DA D E BAD AE D AEBDE DA E D BE EB A E E D B E E DE E AA E A E BAE E DE DB E EB EFA BDA E E D E E EB D F D AEBF E E BAF DAEB E F E BE F BED D AE E E D D EDF AEF ED A A EB D EBF E DE E F D AE AA E EB D D E FE BB BE F D E E DE F EB D AE E E E F E BAE DBE E DB E D E E BE A BE EB D A E C E FD E FB F BE F BEB AE F BE E AD AE D A BEBF E DE E A A E BE ABE A BE F BE E B E FE E DE D A E A E BEB AE A FBE F E BA AF E E D D EBF E F EB AE BE BE F BE EA BA DE D A E BE A BE E BA F A E E D D BE A AEBF E E E E E E BE B BE FBE F EB E E D BE E EB A E E D B E E DE EB AE FA B BE F E BA F E E E EF E E DA E BAE D A EBF E BE B B ED E E BED E E DE F E D E F EB A E AA E AD E FAEDFBB EB E D E FBEAD E D AE A DA E FE A E D D F E E BE ABE A BE F B EB AE A D AB E E BE ABE D D F BE E D F E B E B AE B A BE D BE B D E D E BE AB E E BE A BE D D F BEB AE A BE E D A E BE D BDA BE DA BE E D F E AE D D F E AE EF EB E BAEDAA F E E D F E A E A DA E FE A E FAE A E D A EB AE EFA BD AE E D F E E ABE D D F BE F BE EA FBE BE D BE FE EB AE EFA BD AE AA E E E D E FBEB F E BAE E E F E AD BE ABE D D F BE F AE A E B BE EB BE EB AE DBE D AD BE D EA FA BE BE F BE FE DE BA F A E F E D D E BB A E DF F E DA BE E DE BEBF E DE BA F AF E E D BE E E AEBF E F ED A A ED E E A E F BEB AE BE D A BE F BE D BE AA ED A A E AE F EB AE E F E A E E F BE F BE D A B E D A BE A BEB A BB AE A AE E DEBA F AF E BE F BE E E AE BE B BE E E E F E BE D D B E EB FB D A E BAE F E BE F BE F E AEF EBA F AF EB D E AE D B D AE D AD E BE A BE

31 B D B E AF E E DEB D A E A E A BE F BEB E DB EBF E ABE B A F B E E FEA B B BE A BE EB D A E CEB AE DB BEBF E E D AD E E B A F BE CE A E BE BE AE BE B BE B E BE B A F BEB AE D AE BEB F BE D BE AD AE DE B E E FE D B E E EB FB BA F AF B E BE A BE EB D A E CEB AE EB F AE DB BEBF E BEBA F AF BE CE BE F B E D BEDFBB EBF E F BE DA B E D A E DE DA E D A E F E D E BA E E FBEB F A E F EB F AEB E DEBA F AF E AD E E DE A E E BAE F E D A E E FE FB F BE DA BEA B BE E AA E F E F AE A E BE D E E D E EB D AE A B BE F E E D E BE F BE A BA B E B F E FB F BE D BE B AE FB E F E E E DA E BAE D A ED E D EF ED AEF F E F BD AE EF E E A E F ED A A E F E DE E EB D A E CEDE F E BD A BE D BE E E DEB D A E E DA E BE F E F B E F E FAE A E B E E A E EA AD AE E B E BE BE A E BE F E BF D BE F D BE F B E FE E D B E E F AE FE F E BE F E F BE E E DEB D A E BE ABE D D F BE F E FAEB E A F E D BE FA BDA E E E F F BE E D E E D E ED A DA E E BE A BE BAE DEB D A E E D A A E F E BE A BE E E EB D A E A BEB D F AEBF E E EB E F E BE F BE E BA F AF BEB D BEDF AE FBE E D BE D E BE A BEB D B E AE EB E EDFE E F E E A B E D A E DEB D A E F EB F E E F E DBE F E D A A E F E B EBF EF E E EB D BDA E FEF E F E E BE EB D BDA E E A EF E AEB D E E AA E A B E BAE DE B E E A E E F BE A D D AE E E A E D BED D AE BEBA F AF BE B B E BE A BE E D AE DBE E A E D A A E F E DF D E AED EB EB AE DB BEBF E BE A D A BE B BE A EF E AD E E E B BE AE FB F BE A BE F E BA F E BE E A BE D A E E AE ED B E BED A BE E F DF E B B EC E DE E D E E AE D E E AE AE BE A D A BEB F AD BE E F BED E E A E E D E A BED BE E B A DE

32 EDFA ED A DA E A E FBE A E BAE FA BDA E BE B DF E D B BE F E E D E E A A BE F B ED BE F BE AE D E A E BE F E DE E E F AB BED ABE A E BE A BE F BE AE BF A E B E A E D F B E E BAE D AE E D E F E A F E EB D A E F E BB E F E BD ABE DF EB E D E AED E E E E F E BAEFA B E F E DE D D B E B ADA E BE F B E B A F B E AE E E AE EB D A E F E AE FD A E E E E BB D E A E F E B ADA B E D A E E F E E E E ABE EB D A E AE E BAD EB AE D E DF F E A E F EB AE BE BEF BEDF EDFA B E E F E F E DE B E E D E D E BE A BE A F B E AAD AE E BE F E E A BE DBB B E BA EA BE DF EA BE E A F BE EB D A E E BE ABE E BAD E BAD E E D E DBB DA E D D D A E B E AE E DA E D B E A B BE ABE E BAD E AE AA E E FD A E E E E E A E F E B B E D E E DE BAD E E D E E B A E E E E BE A E BE B BE E AE ED EB E E E A BE D BE E B E E EC E E E A BE D BE E B E E AE E E E A BE F ED D D BB AE D BE BE F E B B E BE ABEB AE D AE BE AD BE E ABE DBB DA E FBE F D AEFA B BE BE ABE DBB DA E A E FD AE E F E FD A E E E EB D A E A E F E B B E E AE E D A E BAE E FBEB F AEFA B E D BE BE AF BE D AE E D F E DEB D A E A E ABE B B E A A EA B EA E E ABE E BE ABE E DA E F E BF AE E E E DA E A E A BE D F B E D F E A BE D D A BD AEF E D E AC E B B E D E E E AE E D B E E B A A C C D E E E A BE F E D D D BB AE EDF F E BE F E B B ED E

33 E D E E A A BE F BE DB EBF E DEBA F AF E E DE A E E E A DA E FEB A E E D B A DA E FEB A E E D B E BAEF E AD E F D E BE A BE E D E EB E DB BEBF E DEBA F AF E F E D F E E E AED E BA AE F E F E D F E A E E E E E D A D BE A D A E BA A E FA BD AE DBE BBD AE BE BEB A BE E D B E E BA E F E BB A BE F E A E EB A E E D B E E E E A E BE DA BE B BE E EB EF EBA F AF E E DE A E E ED EF E F ED A E BA EB EB A E E D B E FAE A EFA B E EB E DEBA F AF E BAD A E BAE DBE ED EF E D E AD BE A BE F AED E E DE E EB A BE E D B BE A A BE E DEBF D E E DE A E F DE AED E AE DBB E BE A BE EA BE F BE E E BE A BE DB BEBF E DE A E F E E BE A BE A F B E E BE A BE BE E D BBD BE F B BE A BE A F BE AE BE D A BE FE BE BE E DEBF D E E DE A E E EB E D BD AE F EBF E DE E E DE A E E D BBD AE D BE E A E BE A BE B F BE BE B FB E BE A BE F AEFA B EF E E A E E E FE BEB BE E F E A E BE D A BE B A BE E DE BF D E E DE A E E BAEF E D E B E E E D F D AE BE F BE AE BF D BE BE BE E D B E E D E E BE EBF D ED BB EDFEB D AE BF D E E D E AE EBF D E F D E BF D E E E DE E DE FBE D E BAE D AE B E ED D AE E FBE E D E A E DE E E D B BE A BE A F BE F AE DEBF D E A F E EB BE AE D F AE BF A E BE A D A BE E D E E DD BE AE A BADA F BE A E DE A E AE BEB B E BE BE D BE B F BE BE A D A BE A E BEB BE AE DE A EB AE A F AE D D BE B A AE BE BE A B E BE BEB AE BF A E DBB BE E A E E DEB E BE BE A D A E E D F EB E DE A E A E D AE D A E E AA E

34 DA E AEFA B E BE F ABE A E E F B E EB B E E BAE E A E F E DE E B AD AE E DE FBE D D E BAE DBE AE DE FBE D E A F A BE A BE BE BE D BBD BE F BE AE B E A BE BADA BA F B E BE B DF E F DF E BE D BE E A F BE EBF A E FE DE DBB DA E D E D B E AE BE A BE EB D A ED E BE BE FB E BE BE F AE E E A E BA E EB D A E EBA F AF E D E A E DE A E AF E AE BEB A BE E D B E FB E FE DE B DA E E B FBE D BE BEB A BE E D B C BE A BE BE F AE D AE A EFA B B E D E ECD E AED EFA B AE D EF ED A E A F E F E E BE A BE BE A A BE E DE BF D E E DE A E F BEB E DB AEBF E DE B DA E EB F E E BE BE F E A E DE E DE FBE AA FB EC D AE DE D B E E D E AE EB A E E D B E E DE D F EB D D A AE F E E DFA E F E A E F E E E FBE FBA AE BB E AA E D B E BE A BE E E D AE E E B D E DA E E BE F E A A B E D A E E A FA BE BE D B BE BB BE BE DA BE BE B FBE E DE A E EB D A E F E F ED D A D AEDFEB A E E D B E BAE F E BAD AEA BE A F E EA BE E D F E E D A E E D BE AE E AA E DA E AE AE F E B D E DA E E DE A A E F E EA BA F E E B ADA E BE A D A BE A E EB A E E D B E FE D A F E DE AEB E D E

35 EC ED E D E E D E AE DE A EB AEA FBE BE F E B BE E AD AE B E F BE BE E BE E A E EA D B DA E AE ADA EB AE BE F E E D E BAE E E F E E D E FAE BF E BEA D B DA BE AE BE ADA BE D BE D F E BEA BE B B E D E E E D E C E A E EB A E E D B E E D E F E A E AE B A E E D E E A F E E E DE E DA E E E F D AEA FA BE BE A D B DA BE AE ADA BE BB B ED EDFE F E E E AE E E E C E B A A E A E D F E D A E D F E B EBD BE DB E BAE A E E D E F E A E EB E BE B BED D AE BE F BEB BEB AED BE D E E D E F EA BA E BE ADA BE AE A D B DA BE BE CE BF F E E E E AE E E B A A EC DFA BE D B EA BE F EC E AEDFBB E FE E E DE B E E BE A BE FAE A ED E E D AE FB F BE BE F E D F E D E BE BE F AE A E BEDFE D D E E D E E D BE E E E E FE E FA BDA E E A F BE E DA BE BE F E D E E E F E BE B BE F E BE A BE E E B E F E D A E AE F E DA E D A E BE AE A BBD A BE F E BE A BE E D E D F E BE F AE BA AF EF E E A E D AE E DE A F E BE F BEA E D BE FE B AD AEF E DF D B E AD A ED E EB A E E D B EC ED E D E B F E BE A BE E E AE E A E DE A E FE D E F E AD BEB AE AF BEBF BB AE D AEA FA E DE F E FE E DE E AD E B E E F E DA E E B D E DA E E D E E A F E D E BE DA BE B B E DE DA E D A E D AE DE F E AD EF E A E EB E D F E BE DA B E E BA E FB F BE A BE D A BE E E DA E DBB BE D E D BE AE F A E E E E EB BA DA F E D ADA BA F A E ED DA E A E D ED A BE A F B E A BE EB F DA E D F E F D

36 E EB BA DA F BED A BE E EB BA DA F E BBD AE EA FBE BE BE E A E F E F E A D B DA B E ADA B ED BE EA B E D B EA BE D A EB E B E E E E B E DA E E A E E E E DA BE F EF E D E BAE A E FE E D BE BE E E D E AE E F E D F E E E E F D E E E E D BE BE AE E DE D F E E D E E E AE E DE D B ED D A D AE EF E BE D BE F B E F E D E E E E BE A BE E E B BA DA F EFA B AE EB F AEF ED A E E D ADA BA F A E F E B BA E E B E E D E E FB F BE D ABE F E E BA F E D BE EB A E E D B E E D E E E D E BAE B E E D ABE BE AE B E FBE BE D ABE BEB AE D BE D BE EB A E E D B E AE BE D ABE BEB AED FA BE E D E A E EC E E F E ED DA BE A BE AE BE D ABE D DA B EBF E DE DA E FE DE B A E A D B DA ADA E F E D E FE F E F DA E E D B E E F DFE D E BAE BF A E D F E D EF E A E EB EC F ED BE F D BEB AE BED A BE E A E D E AE A F B E D A E E A E D E EF E DA E A D E F E D E D BE EB A E E D B E E D ED DA E AA E DA E BAE D F E D EF E A E EB E AE AE DE DA E E E E F E DA E BAE BF A E E AE D F E E AA E DA EDEF EB E FBE D D E F E DE A E E AE DE F E DA E E ECD BE E DBE A D E E FAE E DE DA E E EB EF E A E D ADA E D E E E A E E A BE E D E DE D A E E A D E D ED E E AE DE BE BE E DE F E DA E AE E E E E B A A E E DE BAD A E E A D E AE E DE A DAF E E DEB F DA E BE AD BEB AE A BEF E AD E E E B E E F E

37 B EDFE E E DBE E A E D E DE E B E FA C E AE E B AE F E A BE DB BEBF E D A E E A E D E BE D A BE A F BED D A AE BE BE E A A E F E AE E D F E BE F DA B E FB F BE DA BE B D AE EF E F DA E FB EB AE B E D F E B D AE EF E B E E D D BE D E E D BE B E F BE E D B BE ED A F BE E D B BE E FE B B E BE DA BEB AE D F BE D EF E A E EB E A E EBF E AE BE F BEB AE B B E F BE D D BEB AED BE BED DA A EB AE E D E AF E FADA EB AE E D D AE F E F BE E D D BE BBE E D AEF E F E DA E E DA B E DE A E C E AE BE B BE FBE A BE FA C EFA B AE BED A BE A F B EC D F E F D A D AEDF E A BEBA DBA F B E DE D F E F D E E FAE DBE DBB E BE DFA BE D BE E F E BEB F DA BE F E F E D B D E B E B F E E D BD E E A A E BAE D A E DE B F DA E FAE BA E B E F E F E D E C A BED BE AE A E BE F E AA EF E F E DA E FE D BD E E A A E E F A E DEA DAF E E DEB F DA E F E DE D BB E D E DEBF A E F AEB F E D E E E C E D F E E D BD E A F E F E E E FBE BB E AE E BB EB E DA E A E F E BE BE E A E E DE F E E A BEA DAF B E D AE DE B F DA E BEA DAF BEDBB BEDF E BE E A E B BD BE E DE A E FE D EB AE D B E AA E AD ED E D A E E E DBE BA E B E F E F E D E E E E D A E E FB F BE DA BE E D A E D F ED FA BBD AE EF E F E D E A

38 E B E E A E E DE A E FEB A E E D B FE E A BE E E AE E A E DE A E FEB A E E D B E F AD A E E F EF E E D F E D BE DE DA E FE E A D E BE D BE DA D BE BE B FBE FEB A E E D B E D D AE E DA E F EB D D A EDFE D E F E A E FB F BED BE AE A E B BEDFE AE F E E E A E DE A E DFE BEF E D A E E DE E E E BED A BE E A E D E E E BE A F BE E AD B E E E BE BE B BE E A B BE A F BE E AD BE B AE B D E DA E E DE A EBF E DE DB E BE DA BE AD BE BE D BE DA D BE BE FBE F AE B B E BEB AE F BE EF ED A E DA E BE DBB BE D E DA E F E E BE DA BE BE D BE DA D BE F E D FA BB AE DBE E F E F E E E AE E A E E E E DA BE B BE E A BE BE B BE E A BE F AEDFBB E A EFA B BE F E E E A E DE A E FEB A E BE B BE F AE A E B A BEBF EF E E E A BE D B E DE E B BA E E EF E E E A A E E BE DA BE FE A F E D E EFA B ED EC E E EDFA E BB A E B BA E EDBB E E D F E F E E DE E BE BE A A BE E FB F BE DA B E EB E BAE BF A E DAA F E DF E D BE D E D AE E D F E FE E A A BE A F BE FE A F E A BE EB CD BEF EB D A E DE D B E F E A A E F ED EBDE E BAE F A E D EF E D B E ABE A D F BE AE A F BEBF E DE A A E F E DE E AE BE F BE FEB D A E FBE BE D D A BE AE A E BE E A E F EB D E E FBE BB E E E E E D B BE DB BE E DE D BBD E F D E AE A E AD BE D E B E E E D BEB E E B F ECD BE E E E D EB E E EBDE EB E F BE BEB AE AD B E E E DF E AE C E D AE E E E BF D AE DE BAD BDA E BE BE F D BE D E BE A D A BE EA E D E E DD B E

39 A BADA F BE AE D E DE DA E E D B BE E E E DF D E A E E E BE A D A BE BE D BE BE A D A B D AE DE D B E BEB FA BEB AE E A D A ED E EB D A E BE E DE DA E FE E E EDE B DADA E BE D A BE BE F E F BE F EB AE E AD A E E AE E E E BAE BBD E E DE B DADA E BE D A BE D BE AE D BE E BE F B E E AE BAE E D A E A D D E D E BE A D A BE A BADA F BE AE BE D B BE E BE D BE E E D BE DBE EA AD A E D E BEB AE BE D D BE F E BE A D A BE DA BE D A BED E BE F BE DF E DE B DADA E BE D A BED D BE E BE F BE DF E E F EDF A E A E FEB D A E E E BAED E AE E AE BAE B E E DE E D F E F EBAD B E BE B E E D E BE A D A BE A BADA F BE AE BE D B BE EB D AE FA AE B BD BE E DEB A E BE A D A BE A E BEB FA B E BE BE E BE A D A BE BE AE A BADA F B E AE A E ABE D ECD BE AED E E E D AE F E A E FEB D AEDF A E A E BEB FA BE F E E D B E BE A D A BE B BE D E DE DA E FE E AE E DE A E E BE E A E DFAE EDFBB E E E A E E A E BE F EB FA B E F E FAE A E FBE E B F BEB AE D BEF E E F E ADAE E E D BE EB D A E AA E E BAD B E E DEBAD A E FE E E E DE BAD A E F E E F E B A E E D AE A E DE BAD A E DBB DA E B E AE DE BAD A E E BB DA E D E DE BAD A E F E BAE A AE F E D BE E E E D B E C D E C D B D E E E D B E BAEDFBB E DEB E BE A FA BE A F BE AE A D F BE FE E AE BE F EB FA BE C D AA E E BAE D A E E FB BE A BE EA DAF E BB E E F E E A DA E BEB FA B E E BE BE BA AE D BE BE A BE A E F E

40 ADA B E BE BF ADABEB AE AE D D B E E E E D B E BAE E A F F BE A E D E D AE DF E A BE BAD D BE A DA E E A DAF E E F EB AE BB E E D F E A AE E E E D B E BE A BEB AE A E F BE F E BE A BE DB BEBF E DEBA F AF E E DE E E BA DA E BAE E D A E D E D FDA E BE A FA BE A F BE AE A D F BE E DEBAD A E FE E BE BE BE E D F EB FA E AE FE EB AE D F BEB D AED D AE E D E DE E C D A A A B D F E D F E E E F EB BA E BED BE BE FBE F FB BEB AE BE A BE E AF DA E E E E AE A DA EA D F ECD BE BE F E DB E BE DBB AE D EF EB F DA E F EA BE A E FA E FEB BA E F E ADAE BEF EDFA E D E E E D D AE E D E EBDE E BE F EA BE F EB F DA E E D F E F D EC DFA BE A BEFA B AE BE D BE E E E D F E F D ED E BA E DE A FA E A D F E E E E D B E BE ABE A F BEB AE D F BEB D AE F E EB D AE AE BEB FA B E BE D BE E E E D F E F D EB AE D AE E DE EBF D A E B A A A A F AB C C C A F A B C D B D DAB D A B E F AB D A E BEA BE BEA BE E FDA E E D B AE BE D ABE E F F E AE D E BE D B BE D E D AEDF E D F BE E E E EA E BA E BE A FA BE A D F BE E E A A E FEB BA E A E BEDA BE FE D E AE F E FE A F E D E E A A E E D B E F E BE A D A BE E D E E DD B E AE E A A E E F E F E DE E BE A D A BE A BADA F B E E E EB DADA E BAE B E E F EA BE EF EA ED D E AEF EA E D E EA ED D E B A E E AE A F EDBB E E DE DA E F E D A E F D AED F E EB FA E AE E BE A D A BE E D E E DD BE A E EB D AE AE E

41 B FA E EA E A AE D BE DEBAD A E FE E DE E A E E E AE B BE BD ABE AD AEB F AE D D E EA E D E B E E DE E E A BADA F E A E EB FA E D BEF EB D AE FE D BD E AE EB FA E D BEF EB D AE A AE D BD E AE D E EB DADA EBF E DEBAD A E FE E BAE D F E D BE E E DBE A E BE A D A BEB F BE F E F AE D E BE F BE DFE D E EB FA BE BE B F BE AA AE E FDA E E E E A D A EBF D A E C D A B D FDA E BE B B ED FA BBD AE E F E DBB A E A BE D E BE A BE E E BAEF E AD E F D E BE A BE E D E E E B F E E E E E D B E BAE AE BE E DE E AD E FE E DE A E EB E AE BF A E E BE B BE A BE BE B BE A B E DE B ED D AE E F EB E BAE B E F E B A E D F E F E AE BE E D BE E D BE AED EF E F EB E F E BE DFA BE B B E BE A BE D F D AE E E E D B E E FBE F FB AEB AE A E A FB BE E D F E F E E D E E D BE A F B E BE A BE EB BE FA B BE D E BE A BE E E AE BEB DA BE D BE D FDA E BE BE AE E AE DBE BBD AE E A EA FBE BE D D A BE E DE D BBD E F D E BE DBB BE E A BE EB E F AE A E A BE E E BE A BE F B E E BE A BE DB BEBF E BE D BE E E E D F E F D E AE E BE A BE E DB E E A BE F B BE BE AD BE D A E AE E AD ABE E D ABE A EF E D E AEB E A F EB AEFA B BE F E AA EDFE AE BEA BE BE E BE A BE EB E D BE E FAE D F EDFE F E E E FE E E AE F E DE E BE D E ED E C E E E E E BE A BE BAE F E D EDF E F E E BE F D BEFA B BE F E F E BA F A E FB F BE A BE EB E F B E E C E E FE F E A AE BEA BE D AE E A E E E E D B BE AD BE F ED E BE D A BE F BE E DE A E A E BE E DE DA EC DFA BE A BE EB EA BE F E B E D BEDFBB E F E

42 A AEDFBB E BEA BE F E D F E DE B DADA E E D E AE FE A F E BE E DE DA E FE E A BE DB BEBF E BE D BE E E D E E E E D F E F D E E E F EB BA E EAD AE F E B E E ABEA B EA BE F E BE D ABE D E D AEDF E D F BE E BE F E BE A BE BE AE BE A D A BE BE AE A BADA F BE A EF E A E AEB E D E F E BEA BE E B E E BAE DB EBF E E D E E E B E E AE FA C E BF E BE A D A BE A E E D E AE E A F EB AEB F AE B A BE D E BE BE E D E E DD BE AE A BADA F BE FDA E E E E D E E DD BE BAE BA E D EF E A A E E D B E BE D D A BE E E A A E D AEB EBDE E F A E C EFA B EF E A A EBAD D E EAD BE F E EFA B EF E A A E FBE DAA F E E BEA BE A BADA F BEB AE BA BE D EF E A A E F B D C D B A B D A E E AE E A E FEA E DA BE E A D A E E DE BAD E A E BE A BE BE DA B E E DE D E D A EDBB E E D F EDA E AE E DE BAD A E A F E FE F E DE E A E E E A E FE D E BAEB D E E E E E A D A E A D E FD AEDFBB E BE BE E D E E DD BE AE A BADA F B E BE A BEBAD D BE DB BEBF E BE D BE E E AE A E F BE AE F E B E BE A FA BE A D F BEBF E DEBA F AF E AE BE B E BE E A D AE E DBE EA BE A F BE AE EB DADA EC BE A BED D BE AE BF A E A E BEA BE F BE A E F E BED BE BE E FE BEA BE F BE D FD AE E D E A E E BE D B BE E C E AE FA C E E EA E AE DF A E E BED E E DEB A E BE A D A BE D A

43 E A BE E DB E BE A BEB AE BA F A BEB FBE A B E F F E E BAD D F E B E B A E E D AE A D E BEDA BE FE D E DA AEDF EDA BE E DE A E B E E D F E BE A A BE BE D BE DA BE D E E A EB E D AE E D A E BE BE BA F AF D BEF F A E D E E BE A A BE E E E EB AE BA F ABE E D A E BE BE E DE C E AE BE A A BEBADA BA F BEB AE BE D EA BE B D BE DA B E E AEC F EB AE DFA BE A BE E DB E E F D B E

44 E D FDA E E A B E B E FE B A A E D E E BAEF E D E E E D B E AE E E E A BE B B E F BEC EDF A E D A E BE D BE E BE A BE AE E D E D A E BE BE D E E A F EFA B BE F E BE AA EDFE A E D E E BE A BE EB E F BE E A AE E F EB E BE F BE AE BE A D A BEB D BE E E F E BAE B E D BE BE BEFA B BE ED A BBD E E BAE E BBD E E BE D F E F EBD E F BE A BEFA B E E A E E AE E AF D FDA EF A E BAE E E E D E B A E E F DF E D B E BAE E E D F E DE D D A E F E A E E E E E D A E E D B E F E D E F E B E A F E BE A BED AF BE E E AAD AE DB E D FDA EB E D AE E D E A B A EC F E A BEB AE AF BE E E DE D D A E E DE A E E A F E E E E D B E AE FE D E E DE D D A E E B E BED A BE BE D A B F E D D A E E A F E E E E D B E AD E BAE D AE BF E D E D AE FD DA F E E AE D EB FD E DA E E C E B E A E DEBA F AF E AD E FE D E AE DE B E A E E CE BF E DE BAD E A E BE D BE DA BE B D ABE F E F E DA BE A B E BE F E DA BEB AE B BE A F BE B F BEB BEB D BE D EF E CE F E FE D E E E E AE DE B E BAE DBE B E E A E B F BEB AEB D BE D EF E CEBF F E E E E E E E CE BAEFA B E E D B E EB E A A E AEBDEB A E DFA DA BDA E D BE E A E DBEA F F BE DE D A E E D EBDA B D BD A E E E AED E E E E DBE A E BE A D A BE D A BE A E E D E AE E A F E E F E A D E EF EA E D EB B A EDF E D DA BE BE B BE A B E E B F E E E E D B E BAE DBE AE

45 EF EDF ADA E FE CED BE F E E E E D B E BA E A EB EF E D A E E DE F E E D BD AE DBE A D A BE BED E E A F E BAEA BE EF EDF ADA E FE CED EF E E E D B E B F E A E B F F E D E F E BAE B F EB A F E CE FBE D E BAE D AE FBE D AE B E F E BE A A BE F B C DFA BE A F BEB AE B B EA BE F E F E E D AE DA E C E FE DE DBB DA E DB EBF E DE B E BE A D A BE C E AE E F E D AE BE D BE BEBF EBDE D F E E D BE BE DFA BE BE FE CE BA AE B AB E E BAEF E A E D AE FE E E D B E B A E E D E BAE B E E F E D A BE D F BE E EF E DFE D AE BE D D A BA F BE BB A BE EBDE D B ED E DE A E EF ED E F E BE AD A E F E DE D B E E E DFE BAE B A E B AE D BE F E D E F E E A E DE B E BAE B E E AD AE A E BB AE F E A F E F E A E D FDA E BAE DE D D A E F E A E EB E E B E BED A BE BE D A B E BAE BB A E D E E D E E AE AA E EA BA E AD AE AD BE F BE D E BED D AE BE F BEB B E AE E D BB E DFA BE E A E BE BE E DBB B E FB F BE D F BE D FDA E AE A E B BE E D BE F E D F E E A E BBD A E AED E AE BA AF E F E D F ED E ED A BEBF E DEA E DB E AE ED A BEBF E E A F EDF E BA B E D BEBDE DA ED AD BA E E F BE B BED DA AE AEB E D A BEBF B B E D E DEBF A E D E AE D E AED D E DE D F E A E E ED FAD AE E B BE F E AE ED A BE F E E B E BE E F BE E DE C EC F ECDADE AE CC E AEDFBB E A EFA B BE E B F E D A BE AE D A B E BE D A BE D D AE DBE A EB A BE E A E E F BE B D A E B F BED E BED A B E DE B DA E FAE A E D A EBF E EB E A E E D BE BAE BE AD AE F E BE D F BEFA BD AE DE CC E D BE F E BA EA FAE E EB DA E FD E AED E AE E DEC CE C A E E B F EC B E D AE EB BA B E D F EB BA E BAE B E E DEBA F AF E F E A E E D A BE A E

46 E AE EBF E AA E E AE D A B E E D ED A B E BE D A BE AE A EB A BE EB A E F BED AE BE A BE B F BEB D BEDF ED A B EA FAE E AD AEBA F AF D AE AB E DEC CEDE F BE A E B E E F E C C E AE E D A D AE E A BE AE BE D A BE BE AD A E E FBE BE D A BEBF B B DE D A E BE B A F BE E BB AEB AE DB BEBF E F E D F BE E E DEB B A E FDA E E E DEB A E FDA E E E BE E BE F BE F E A F E DBB E D EF E A E EB EB AEB A B EA FBE BE B BE F AE A E DBB BE E AA E D E BA E E E BED A BED D A D AEDF E E F BEB A BE E D BE B A BE E BED A BE EB A BE E DF E DA BE E E BE D A BEB A BE E DF E B A BE E BE D A BE EB A BE E D BE DA BE E F E E B ADA E E DEB A E E E F BE D BEF E A F E EEE B B DEB B A E E AE E DA E FE E D A BE A F BE A E E E D A BE B ABE D BE DE A F E B F E B F B A E

47 DEB A E E AE E DA E FE E D A BE EB A BE A E E E D A BE B ABE D BE DE A F E B F E E B F B EA BEF E A E E D E EB EF E D A ED D AE E E E E BE B BEB AE A BA BE AD A E E A E AE E EB F AE EF E EB EDF E D A B E D BE AEDFBB E F E EF E F EB E F DF E D A B E AE E B A F E E BB AE AEA E A E E A DA E E BED A B F E E DA BE A E BE D BE B A BE E BE DF E B A BE E BE D BE DA BE E AE BE DF E DA BE E BE D F BE B A AE DE D E A E BE BF ADABEDAA FBE AE BE BF ADABE B BE F EF EB F E E BE AA AE E D F E DEB B A E E DEB A E E DE D F E A E B A E E AE DE D F E A E DA E E D A F E BB A E D A F E BB AE E E A E E D DA E FEADF E D A BE D BE EB FBE B E E B BEB A BE D E D AE EF EB A ED DA E

48 E BAE E F AEFA B E E F E A B E E E BE ABE ABE D E BE F E AF BE BAEBDE D E D EDFE DA E D A BE B ABE D BE DE A F E E BAE E AD AE FA B E AA E A F E B F E F E A BEB AE D BEBF EF E E A F E FE F E A F BED D AEF E E DA E D A B E EDFA E E BAE DE A FA E D E E D F ED A E E DE D F E FE E B E B E F E A BE E AE E A F AE E E E D A BE D BE BE E BE B BE E DE D F E D BED E BE BA FA BE A BE A BA E BEB AE B BE EDFBB E D B ED BE F E DE A E E BAE FBE D E F E DE A E E AA E A F E E AE E DBE E E E A E A DA E E BED A B E D A F E BB AE EF ED FE AF E E DE D E F E A E BE F BE BB AE B A AE E F AD E D A BE A F BE F E A E F E F AD E E DE D F E DBB E D EF E A E EB E BE AA AED B E D EF E F E D E E DE D E E DE A E E D F E E BE E BA FA E BED A BE D BE BE E BE F BE F E D F E DBB E E D A E F E A E EB E E DA E D D A BA E E F DE F E E E B A E FA E E DEB B A E E F E A E F E D AE BB E E E E FAEDFBB E A E E E AD AE EADF E E D BE B A BE F E A E F E D F E FEADF E E DF E B A B E B E F EF E BA FA E D EBD BE DF AE A E BEB BE BED A BE AE BE D A B E DEB B A E AE DE

49 B A E AEA FA BE BE F E DE D F E E E BF A EB F BE BE D A BE F AE A E A F BE AE DE F EBF AEF E E AD E DEB A E D AE DE D F E E E DE F E E E B AD AEF E BA FA E E BED A BE D E BE D A B E BAE DE D D E D AE E AE E AE B AD AEF EB B A E E E AEF EB A E E E F ED DA E F E BE A BE D D BE E EF EB E EBF F EDF E D A B E DE F E E EB EB AF E A E BE F BE D E AED DA E F E F BE E E EB E B AE DB E DE F E DE FBE E FE AE E E E B A EF E A E FBB BBD AE F E E B E BED A BE BE D A B E E F E AE F E D D B E BF E A E BE A B E BF ADAE FAEDFBB E A E A A E E D E E D EB FBE DE F E E E E F EF E BA FA E D E E E DE D F E E E AE E F EF E BA FA ED DA E F E BE D BE A D AE F E F E D DA E E E EF E D F E B E A E E AE E E E BAE F E E EF E D A EB A EDFE DBD EDE FBE F E D EBF E F E A E F E DBB E F F ED A E B EDFE DBD E D E E F EF E E D E E E F E D A E B EDFE DBD EDF DEF EB EBF F E EF ED A E B EDFE DBD E E BEBF E F E E B ADA E E E F BE E E F E D E F E F E BA FA E E A E F E A D E D E F E E DE BA FA E E BAE E D E FDA E

50 D D AD E E AA E A F E BAEB E D E D E D AEDFE DA E D A B E A D AE DFE D A F E BB A E D A E E E AE DBE DE B DA E E F E A BE E A E E F E D D A E E A F E BED A BEDFE FAE F E A F E DBB E E A E F EB E B E F E BA FA BE A BE D A BE D E BE D A BE F AED E DE E E BA E F AD A E DE F E E A F E FBE D A BEDFE FAE E DE A F E F E DE F E E E D AE D EF E E FBE D E F F EDFA E E B E DBE E FBEBF E E E BA F E E BE E E E E DA B E D E DE F E E BAE F E F E DE F E E F EF EB A E E D BE BE EB AE A F B E E DE F E E BAE F E F E DE F E E F EF EB A E E D BE E E DE F E E BAE F E FBAE A D E D AE AA E A F EDE A E E D E D E AED E E E D BE E FAE D EF E A F E F E EB F E DBE E D BE D DA BE B F E FE D A BEB AE B BE AE F E AE E D B E BE A BE A D AE E D E E BED A B DE A F E E BAE B A E D E DE A E BB FBE

51 B D E B AB B E E DE DA E A E E D E FE ED A E AE E E E B BEB A B E E A E A E BAEFA B E F ED EF E BE E E B F E D A E BAE E B E E F E DBB E AEF E DA E BBD A E BE FE D F BE E DE D F E E BAE DEB E E B F E BED A BEB AE DBB BED DA A E FD A E D DA E E DE BA FA E BED A BE D E D AE EF E BA FA E D DA E E BAEBF F E E E B F E DE A E E D E BAE D D E E EF E F EB E E FBE D A BE F E D DA E B F E DE BA FA E BED A BE BAED DA E E BAE D E E E D A E E E D A F E BB A E AA E A F E BAE D A E FE DA E D A B E DE D D B E A E F E D F BE E E E FAE E A E D A E F EB E BE A F BE BB AEF E DA E D A BEB D B E E A D D E B DA E E E C CD BE E FAE E E BED D AD BE E E A DA E E BED A B E AE E E D F BE B BE A E E AE E A F E D E F E AED E AE E C E AA E A F E BAE E E E AE FAEF E A E E DA E BBD A E D AED B E FBE E BEDF E B BE BE F E A BE D E DE A E EB E E B A E DE D A E F EF ED A EB A ED DA AE A E A F ED D AEF E B EB A ED DA AE E D A E F E BA FA E E D A E A A F E C E D E E DE BA FA E AF ED EF E BA FA E A A F E B F E BE F E BA FA BEB AE A F B E C E E DE D F E E E AEF E D F E E C E A E E AE EB E F E DE BA FA E AF E BAE F E F E DE BA FA E E

52 E A BE EA B EA D D EDE A E AF EDFA F E E F ED BE E D FDA E E A BE E D E EB E AE F EFA BDA E D BE DE E E A A BE F BE E A A EA D FA F E D AE E D A F E E B EAF D ED DE D FDA E E A BE E D E DE A E D AE D BE FB F BE FABE B EDFE AE AE A BDA E E A BE F EE D A E BE BE F E B F BE BE A BE E A E B B A E A A E AE F BEB AE BE D D A BA F BE E BE B D E DE FE BEBA F AF BE E E B F E FB F BEBA F AF BE F E E EB AE B B E F E A E BE F BE BF ADAB E BE A F BE D FDA BE F E D A E BE F EB AE BE FBED D A BE E BE A B BE D BBD BED B ED F B BEBF E BE A BE E D E EB E AE A ED F BE F E A F EF E F E A A AE A E FE

53 C F E D A E BF ADAB

54

55 E D FDA E E A B E D FDA E D DA E E A BE CE E D E E E D AE E D AE F D EBF E BB A ED D D AE F E DE D E BE A BE E D E E E E FB F BE D A F B E A BE F E DEB A E E DEBA F AF E E DE E AEBDE DA E DE DA E A D E BE B BEA BA B E AE DE FD A E E D AE F A FB F BE A BE EFA B AE BED A BE E E DA E D AD E D E BE A BE E E B D A B E D E E E F E E F B E ED A E E D ADA E E BA F A E AE A E E B E F E E E E ED A E E D ADA E E BA F A E AE A E EB E F E E E B E ED A E E A E D ED E A E E A BE AE A E E B E F E E E C E E D E B E E E A E E D ADA E E BA F A E DE E AE A E EB E DB EBF E DE AD A E BA F E F E C EBA F E AE F E F E BE A BE AE A E B BE D BE E D E F E D DA ED E F E FE BB F E BE E DBA F EC E FB E BE AA AE D F E ABEA BE D A BE E E DA DEC A E E B F EC BE C C EDE A EFA B E F E D F E BE E A B E EDE A E B E D E E AD A EDFE AE AF E DE D F E DE FBE E F E BE A BE E E FE D AE E DE BA F A E DA E EB E FE D A BE AEDFE DA E D A BE ED A BE EC E FB E BE DA BED A BE D E E DE AEDFBB ED D A BE E

56 D FDA E BE A BE E D E BE EB BA BE B B E E AE A E B BE E D B E BD DB E C E DE DB E E D A E C E DE DA E F ADB E C E DE ED AD BA E FE A F EDF E BA BE E E D A F E DE E DEA D B ADB E B E FE E E DE F D DB E E E DB E E E DEA E E DEA E DB E E AE DEA B E E BEB BA BE AE A E B BE D E BE AD AE B ADA BE BE ABEB BA BE B ABE D BE DEC CE E E E D A BE D EB BA E A E E F E E AE E F E E F AF E FEB A E E D B E A AE E F E E AEA BE F AE F E E DE B A E BED A BE B F AE E F BE F E ED A BE F E E E F BE F E E D A BE F E E D F E E D E E E D F E E B A F BE F BE E DE BAD E E D A E A E BE F BED E E E BE F B E DEAD E AE A E BED A BE A ABE AE BE F E E D BE AE BE F E C E FB E BED D D BB AE F AE D BE BE AF BE D FDA BE E A B D F E A EDE A E D F EBF E D F EB BA EB EA BE A BE E DE FD A E E D AE BED A BEA BA BED E DE F EFA B E E E DE B DA E D A E A E BED A BE AE BE D A B E DE B A E BED A BED D AE BE F BEB B E BB D AE DE B E AD E F E F E D E F E D F E BE B BE AED ABE E A FBE BED A B EF EB EDE A EFA B E E D F E FE CE BAE DE A F E E E F E D F E DE FD A E F ED A E D BE E BAE DBED D A E E D AE A E F E F BE A B E EB EFA B E D F E E BF A E DE B DA E BE A D A BE E FE BE ABE A BE B D A E C E FA AE F E DE B A E EA FBE BEDA B E F E D F E B E EB E BAE BE A E E AE E E E E B F E DE B E FE D AE FE B ED E DE E AD AE BE AE F E BE A D A BE FE BE ABE D D F BEDAA FBE AD AE A F B E E B F E ADA E FE B E AD AEB D E E E E DE E AE F E E BE A D A BE FE ABE D D F BE AD AE A F B

57 E E F E BEDFA BE B B DE B DA E A E BED A BE AE BE D A BEDE A E FD A E D E BE D A F BE BB AE E E E DE D F E DBB E D EF E A E EB E AEAD E E E EB F EDE A E B E D E E A EF E D A E AD E EB F BE BE B BE D D AE E F EB EDF AEF E D E A EA BA BE AD A E E BAEDFBB E A BBD AE EBD EB E DE A E AF E A F E BED A BE BAD ABE D A E E F E BAE E D A E D F E D E BB AEAD ECD BE E DBE A D EF E B A E BF E FBE D EDE A E D A ED E E A E BE DFB BE E E DF D BE DBB A E BE F BE BB AE AE A EA D B E D BE E FAE D EF E B ADA E D E AE AF A E E DE D E E D F E A E BE F BE E E AE D AE A EA D BE AE BE E B D A BE D F B AF E E DE B A EBA F AF D E BED A BED D AE BE F BEB BE AE EBD EB E BE A BE E AE E F AEB D E FE A E E DE F EFA B E E E F E D F EB BA E BED A BE AE A E F BE E A E E F E B D A E EFA BD AE BE B A F BE A BE F BE AE DE BAD E EB D A E E D A E E E BE F BE BB AE AE E E AE A EA D BE EFA BD AE D A E E F E DBB E E D F E F E E B AD AE E F

58 992 J. Chem. Inf. Model. 2010, 50, Comparative Evaluation of 3D Virtual Ligand Screening Methods: Impact of the Molecular Alignment on Enrichment David Giganti,, Hélène Guillemain,, Jean-Louis Spadoni, Michael Nilges, Jean-François Zagury, and Matthieu Montes*, Unité de Bioinformatique Structurale, Institut Pasteur, 26 rue du Dr Roux, Paris, France, and Chaire de Bioinformatique, Conservatoire National des Arts et Métiers, 292 rue Saint Martin, Paris, France Received December 26, 2009 In the early stage of drug discovery programs, when the structure of a complex involving a target and a small molecule is available, structure-based virtual ligand screening methods are generally preferred. However, ligand-based strategies like shape-similarity search methods can also be applied. Shape-similarity search methods consist in exploring a pseudo-binding-site derived from the known small molecule used as a reference. Several of these methods use conformational sampling algorithms which are also shared by corresponding docking methods: for example Surflex-dock/Surflex-sim, FlexX/FlexS, ICM, and OMEGA-FRED/OMEGA- ROCS. Using 11 systems issued from the challenging own subsets of the Directory of Useful Decoys (DUD-own), we evaluated and compared the performance of the above-cited programs in terms of molecular alignment accuracy, enrichment in active compounds, and enrichment in different chemotypes (scaffoldhopping). Since molecular alignment is a crucial aspect of performance for the different methods, we have assessed its impact on enrichment. We have also illustrated the paradox of retrieving active compounds with good scores even if they are inaccurately positioned. Finally, we have highlighted possible positive aspects of using shape-based approaches in drug-discovery protocols when the structure of the target in complex with a small molecule is known. INTRODUCTION In the early stage of research of drug discovery programs, high-throughput screening (HTS) procedures can be applied for hit identification in large small molecule databases. In the past decade, in silico screening has been extensively used to reduce the number of compounds going into HTS, reducing time and costs for hit finding. 1 The classical straightforward concept aiming at identifying analogues by comparing the physicochemical, structural, or pharmacophoric properties of a known active molecule with that of compounds in a collection has been massively applied during the last decades. 2,3 Initially, these ligand-based virtual ligand screening (LBVLS) methods were based on simple 2D descriptors or fingerprints 4 derived from the structure of the reference active compound and compared to the corresponding descriptors of database compounds using a similarity metric, such as the Tanimoto coefficient (Tc). These methods were generally efficient, very fast, and provided as a result hits sharing a common chemotype with the active molecule used as the reference. 5 To increase the structural diversity of the hits provided by LBVLS methods and thus to perform scaffold-hopping (i.e., change the chemotype, keep the activity 6 ), different methods using more sophisticated 3D descriptors have later been developed, such as pharmacophore screening 7 or shape similarity searching. 8,9 * To whom correspondence should be addressed: Matthieu Montes, PhD, matthieu.montes@cnam.fr. These authors contributed equally to this work. Unité de Bioinformatique Structurale, Institut Pasteur. Chaire de Bioinformatique, Conservatoire National des Arts et Métiers. In pharmacophore screening, the knowledge of a set of aligned known active compounds is required, in contrast to shape similarity search methods that only require the structure of a single active compound. Shape similarity search methods thus appear as the LBVLS methods of choice when the structure of only a single active compound is available. When the structure of the target in complex with a ligand is available, structure-based virtual ligand screening (SBVLS) methods like docking/scoring 10 or structure-based pharmacophore screening 11,12 are generally preferred. However, the coordinates of a bound ligand can also be used as a reference for shape similarity search methods. Using such methods, virtual screening success stories have recently been reported It appears that the performance of virtual ligand screening methods depends on many factors, such as the selection of the query structure and its conformation, 17 the conformational search of the compounds, 18 and the quality of the alignment produced. 19 Recently published shape/volume similarity searches, that is, 3D-ligand-based virtual ligand screening (3D-LBVLS) methods have been developed by the same research groups that had previously developed docking-based virtual ligand screening (docking-bvls) methods such as Surflex-Sim/ Surflex-dock, the ICM package, FlexX-FlexS, and OMEGA- FRED/OMEGA-ROCS. All of these 3D-LBVLS methods share their conformation generator with their respective docking-bvls method counterpart. We thus decided in the present work to evaluate these four different 3D-LBVLS methods and their docking-bvls methods counterparts. First, we wished to assess the ability of these four 3D-LBVLS methods and their docking-bvls /ci900507g 2010 American Chemical Society Published on Web 06/08/2010

59 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, methods counterparts to perform accurate molecular alignments using a large number of active compounds on 11 different targets issued from the DUD data set. Then, we wished to evaluate their performance in virtual screening and scaffold-hopping on the same challenging data sets. Recently, Kirchmair et al. 17 highlighted the importance of the query and its conformation for the quality of enrichment after virtual screening. We decided to proceed further in this direction by exploring the impact of molecular alignment on enrichment. This consisted in evaluating the impact of (1) the quality of the alignment of the query with the structure used as a reference on the enrichment obtained with 3D- LBVLS methods and (2) the docking accuracy on enrichment with docking-bvls methods. From the results of this study, we expect to outline the most favorable approach to be used when the structure of the target in a complex with a small molecule is available and to illustrate a currently unexplained paradox between the quality of molecular alignment and ranking, that is, enrichment. MATERIAL AND METHODS All the 3D-LBVLS methods used in this study have a docking-bvls counterpart with which they share an important part of the molecular alignment algorithm. For a given docking engine, the algorithm used to position, in a flexible manner, the query molecule into the binding site, according to putative ligand-receptor interaction points, is similar to the algorithm used to superimpose, in a flexible manner, the query molecule on the reference active compound, according to its shape and putative common interaction points. The biggest differences stem from the scoring functions used and from the volume to be explored as a protein binding site is generally wider than the pseudosite defined around a given known ligand. Hence, for each system, we carefully adjusted the binding site definition in all the programs to perform a realistic comparison of the methods in terms of volume to be explored. Keeping this idea of consistence between the different packages, we deliberately chose not to tune the other parameters of the different 3D-VLS programs for the difficult systems, in contrast to other studies. 20,21 Computational Methods. FlexX/FlexS. FlexX 22 and FlexS 23 are a docking/scoring method and a shape-similarity search method, respectively, based on a fragmentation/ reconstruction algorithm, to dock compounds flexibly into the binding site (FlexX) or to flexibly superimpose compounds on the reference active compound (FlexS). The query molecule is decomposed into fragments, whose physicochemical properties are represented by a set of spheric (FlexX) or Gaussian (FlexS) functions. The fragment defined as the base fragment is aligned with the reference active compound using the RigFit algorithm 23 (FlexS) or positioned into the binding site via a hashing-technique (FlexX). In FlexX the scoring is made using a modified Bohm empirical scoring function. 24,25 In FlexS, the scoring is made using a 3D similarity metric. 23 FlexX version and FlexS were used for all calculations. Surflex-dock/Surflex-sim. Surflex-dock 26 and Surflex-sim 27 use a modified Hammerhead fragmentation/reconstruction algorithm 28 to dock compounds flexibly into the binding site (Surflex-dock) and to superimpose flexibly onto the reference active compound (Surflex-sim), respectively. The query molecule is decomposed into rigid fragments that are superimposed to the Surflex-protomol, that is, molecular fragments covering the entire binding site (Surflex-dock) or to the molecular surface points of the reference active compounds 29 (Surflex-sim). In Surflex-dock, the molecules are evaluated by an empirical scoring function. In Surflexsim, the similarity of each fragment with the reference active compound is evaluated according to the alignment of their respective surface points. In this study, Surflex-dock and Surflex-sim, version 2.2, were used for all calculations. ICM. ICM is based on Monte Carlo simulations in internal coordinates to optimize the position of molecules using a stochastic global optimization procedure combined with pseudo-brownian positional/torsional steps and fast local gradient minimization. 30 The docking poses were evaluated using ICM-VLS empirical scoring function. 31 Chemical superposition with ICM-sim were performed using the atomic property fields method (APF 32 ). ICM, version 3.4, was used for all calculations. OMEGA/FRED/ROCS. FRED 33 and ROCS use atomcentered Gaussian functions parametrized to provide close approximations to hard sphere volumes. In FRED, version 2.2.3, used in this study, orientations of a single conformer are compared with a bump-grid defined using the active site during the rigid body docking procedure. Poses clashing with the bump-grid are eliminated and the remaining poses are evaluated using ShapeGauss. 34 In ROCS, 33 shape-similarity is evaluated by maximizing the volume overlap between the reference active compound and a single conformation of a query molecule using the Tanimoto coefficient. In version 2.3.1, used in this study, a color force field represents physicochemical properties in addition to the shape component. The conformational search of the different query compounds (up to 100 conformers per compound) has been carried out prior to all the calculations using OMEGA, version A summary of the characteristics of the different programs evaluated in this study is available in Table 1. DUD Database. To evaluate the ability of the methods to discriminate between active molecules and decoys and thus their enrichment performance, we decided to use the directory of useful decoys database (DUD). 21 DUD is a benchmarking data set designed for docking method evaluation containing annotated active compounds (from 30 to 120) for 40 targets, including 36 decoys for each active molecule issued from the ZINC database. 35 In the DUD, the decoys have been chosen for their similar physicochemical properties (molecular weight, logp, number of hydrogen bond donors/ acceptors, etc.) with known active molecules, which renders them harder to discriminate compared to random decoys. To be as challenging as possible, the DUD-own set was selected for each target. Each DUD-own set contains only active and decoy compounds designed for the corresponding target making it more challenging than the DUD-all set. 36,37 Because of its careful construction and its high rate of decoys (36 decoys for one active compound), it appears today as the most challenging available benchmarking database. 17 Moreover, as stated recently by Hawkins et al, 37 the recent modifications of DUD 2.0 (including new active compounds and structure clustering by Good 38 ) make it more suitable for ligand-based method evaluation.

60 994 J. Chem. Inf. Model., Vol. 50, No. 6, 2010 GIGANTI ET AL. Table 1. Main Characteristics of the Programs Evaluated in This Study: Flexibility Algorithm, Energy Terms Used in the Scoring Function, and Average Computational Cost in Seconds Per Molecule a program conformational search algorithm scoring terms average time per compound (s) Surflex-dock fragmentation/incremental construction steric, hydrogen bonds, hydrophobic, pseudoentropic (rotatable bonds, size) 14.6 Surflex-sim fragmentation/incremental construction steric, polar (ionic + hydrogen bonds), hydrophobic 6.7 FRED via OMEGA (fragmentation/ steric, shape complementarity 1.0 incremental construction) ROCS via OMEGA (fragmentation/ shape, hydrogen bonds, hydrophobic, aromatic 0.5 incremental construction) FlexX fragmentation/incremental construction hydrogen bonds, ionic, hydrophobic, aromatic, pseudoentropic (rotatable 15.6 bonds) FlexS fragmentation/incremental construction steric, hydrogen bonds, ionic, hydrophobic 6.9 ICM metropolis Monte Carlo van der Waals, electrostatic, hydrophobic, hydrogen bonds desolvation, 17.7 pseudoentropic (rotatable bond), size correction ICMsim metropolis Monte Carlo hydrogen bonds, ionic, hydrophobic, aromatic, pseudoentropic (rotatable bonds), atomic fields (electrostatic, size) 2.4 a All calculations have been performed on Intel Xeon 2.4 GHz processors with 2Go of RAM. Table 2. Description of the 11 Systems a target PDB accessible surface (Å 2 ) alternate PDB problematic side chains no. rot. actives (min-max) MW actives (min-max) (g/mol) no. rot. ref MW ref (g/mol) ADA 1NDW Z7G L62, F CDK2 1CKP DHFR 3DFR ER 3ERT FXA 1F0R LPG Y100, R HIVRT 1RT NA 1A4G P38 1KV OUK, 1BL6, 1BL7, 2EWA F THR 1BA TK 1KIM QHI, 2KI5 Q TRP 1BJU a The accessible surface of the binding sites for each system has been computed using StrucTools. Minimum and maximum molecular weight (MW), number of rotatable bonds (no. rot) for the set of DUD-own active compounds and DUD-own reference compound are also included. We downloaded DUD release 2 from the Web site dud.docking.org and carefully selected 11 out of the 40 targets available (ADA, CDK2, DHFR, ER, FXA, HIVRT, NA, P38, THR, TK, TRP) according to their presence in the literature for benchmarking studies and their diversity in binding site properties and in active compounds according to ICM. Hydrogen atoms were added in the DUD protein structures using ICM. Performance Metrics. All enrichment graphics were produced with the statistical and graphical tool R ( To complete the information of enrichment graphics, receiver operating characteristics curves were plotted with the ROCR package. The area under the ROC curve (AUC) was calculated on the base of the Wilcoxon- Mann-Whitney algorithm. 39 Ligand Cluster Definition. We classified consequently the DUD-own active compounds by ligand similarity using chemical descriptor fingerprints and Tanimoto similarity distance (Td) as implemented in ICM. We examined the resulting trees and visually inspected all the DUD-own active compounds to select a harmonized Td cutoff that resulted into at least 2 equilibrated clusters in each DUD-own data set. After multiple tests, Td was defined at RESULTS Presentation of the 11 Systems. The diversity of the binding site properties of the targets selected for this study is presented in Table 2. Buried or partially buried binding sites are the most frequent (i.e., ADA, CDK2, DHFR, ER, HIVRT, NA and TK), but 2 proteins display a binding site more accessible to solvent (i.e., FXA and TRP). Some binding sites are essentially hydrophobic (i.e., CDK2, ER, HIVRT and P38), whereas others should display mainly polar interactions with a potential ligand (i.e., ADA, DHFR, NA, TK, and TRP). We also provide the number of active compounds and decoys for each system, which range respectively from 22 for TK to 454 for P38 and from 891 for TK to 9141 for P38. Positioning: Molecular Alignment and Docking Accuracy. For all 3D-VLS methods, the conformational search is a crucial point for (1) producing a correct alignment of the known active compounds on the reference for 3D-LBVLS methods 19 and (2) producing accurate poses for docking- BVLS methods. 40 Molecular alignment evaluation is in general performed through cognate ligand docking with one active compound per system, using a database of known protein/small molecule complexes derived from the Protein Data Bank, such as the Fisher set. 41 Since one of the goals of the present work was to evaluate the impact of molecular alignment on enrichment, we decided to use a large number of active compounds issued from the DUD data set. Since very limited structural information was known for all these compounds, we assumed their binding mode to be somewhat similar to the one provided as a reference for each system (considering its scaffold). We thus defined an expert knowledge-based visual

61 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, Figure 1. Examples of 0, 1, and 2 poses. The quality of molecular alignment with representative CDK2 active compounds is illustrated for docking-bvls methods in a, b, and c and for 3D- LBVLS methods in d, e, and f. The illustration a represents a 0 pose for the ZINC compound; b represents a 1 pose for ZINC , and c represents a 2 pose for ZINC Similarly, d represents a 0 pose for ZINC ; e represents a 1 pose for ZINC , and f represents a 2 pose for ZINC score for assessing the superimposition accuracy and the docking accuracy of the 3D-VLS methods evaluated in this study. For 3D-LBVLS methods, we used 2 for an accurate superimposition of most of the pharmacophoric interaction points, 1 for an orientation of the compound similar to the reference and at least 50% of well-superimposed pharmacophoric points, and 0 for the others. Similarly, we assessed the docking accuracy using a visual score evaluating the binding mode of the reference active compound provided by the DUD. We used 2 for an accurate pose displaying most of the expected interactions with the residues of the binding site, 1 for an orientation of the compound similar to the reference displaying 50% of the expected interactions with the residues of the binding site, and 0 for the others. We defined acceptable poses as poses and unacceptable poses as 0 poses. Examples of 0, 1, and 2 poses for both 3D-LBVLS and docking-bvls methods are presented in Figure 1. All compound positioning results are presented in Table 3. Overall, all the 3D-LBVLS methods are able to superimpose most of the active compounds on the corresponding reference provided by the DUD as the rate of acceptable poses (visual score >0) is above 68% for all the programs. ROCS and FlexS display a very similar performance with 68.46% and 69.25%, respectively, Surflex and ICM-sim being the most efficient with an overall acceptable pose rate of 82.22% and 90.97%, respectively. As expected, the performance of the methods depends on the structure of the active compounds for each target, ADA-compounds displaying a high global rate of unacceptable poses for all the methods (mean rate of 53.21%). Some other systems seem to cause problems to 3D-LBVLS methods such as HIVRTactives (more than 34% of unacceptable poses for all the methods but Surflex-sim) and FXA-actives (more than 50% of unacceptable poses for all the methods but ICM-sim). Concerning docking-bvls methods, similar trends were observed. In general, the methods provided acceptable binding modes, the most efficient being ICM-dock with an overall acceptable pose rate of 82.01%. FRED displays 79.64% of acceptable pose rate (very close to ICM-dock) and is even the best method on DHFR, ER, and P38. Surflexdock exhibited the best performance for TK, NA, and TRP despite displaying the least overall docking accuracy with 59.93% of acceptable poses. As expected, the global performance of all the methods were correct and depended on the structure of the target. Overall, the methods performed accurately on FXA, NA, TK, TRP (except for FRED), and DHFR (except for FlexX). Some targets such as THR (except for ICM-dock), P38, HIVRT, and ER (except for FRED) seemed to cause recurrent problems. Enrichment in Active Compounds on the DUD-own Databases. Because the 3D-VLS methods evaluated in this study were able to provide accurate molecular alignments, we assessed their performance on enrichment of the DUDown databases corresponding to the 11 targets we selected for this study using as a reference the compound provided by the DUD. Through the analysis of the corresponding enrichment graphs, we both evaluated their performance in early enrichment (within the top 1% of the database) and in late enrichment (within the top 10% of the database). Results are presented in Figure 2 and Table 4. Overall, three out of the four 3D-LBVLS methods evaluated in this study (ROCS, FlexS, ICM-sim) display an early enrichment rate of 13%, ROCS displaying the best average performance with 14.12% of active compounds retrieved within the first percent of the corresponding DUD-own database and the best early enrichment rate with 34.69% observed on NA. ICM-sim seems to perform better on late enrichment with 57.25% of actives retrieved in average within the first 10% of the corresponding DUD-own database. In similarity with the quality of superimposition, we observed a dependence of the enrichment gained by the different methods on the system tested. All the shape similarity search programs evaluated in this study displayed a good performance on ADA, ER, NA, and TK and performed poorly on P38, the best average performance being observed on ER. For THR, FXA, DHFR, and CDK2, only ICM-sim and ROCS for the two latter systems provided acceptable enrichment rates. Concerning the individual performance on each target, ICM-sim appears to be the best method on early and late enrichment on 4 out of the 11 targets (DHFR, FXA, THR, TRP) and displays the best late enrichment rate with 95.92% on TRP. ROCS and FlexS both perform best on 2 systems out of the 11: HIVRT/P38 for FlexS and CDK2/NA for ROCS. Concerning the docking-bvls methods, we compared the results with DOCK enrichments published on the same DUDown databases (the only ones published with these databases to our knowledge). Overall, Surflex-dock displayed the best early and late enrichment rates with 12.36% and 45.36%, respectively, of the active compounds retrieved in the 1% and 10% of the DUD-own database. It appears as the best method for early and late enrichment rates for ADA (7.69%-48.72%), FXA(17.12%-59.59%) andtrp(18.37%- 81.6%), followed by ICM-dock, which is the best for DHFR (19.02%-66.83%), ER (17.95%-53.85%), and NA (34.69% %). FRED and FlexX follow, being the best method respectively for TK (18.18%-22.73%) and THR (6.94% %). Compared to the methods evaluated herein, DOCK was the best on early enrichment for ADA and DHFR.

62 996 J. Chem. Inf. Model., Vol. 50, No. 6, 2010 GIGANTI ET AL. Table 3. Molecular Alignment of the DUD-own Active Compounds a ADA (39) CDK2 (72) DHFR (410) ER (39) FXA (146) HIVRT (43) Surflex-sim ROCS FlexS ICMsim ADA (39) CDK2 (72) DHFR (410) ER (39) FXA (146) HIVRT (43) Surflex-dock FRED FlexX ICM NA (49) NA (49) P38 (454) P38 (454) THR (72) THR (72) TK (22) TK (22) TRP (49) TRP (49) mean (1100) mean (1100) a The part of 0, 1, and 2 poses are presented in the table as percentage of all the active compounds. Between brackets is the number of active compounds for each system. Overall, global enrichment was in the favor of 3D-LBVLS methods for 8 out of the 11 systems evaluated in this study. This is particularly striking for ER and TK, where all the 3D-LBVLS methods outperform all the docking-bvls methods. We observed a mixed performance for both types of methods, with a slightly better outcome of the 3D-LBVLS methods for NA, TRP, HIVRT, and DHFR. There were exceptions to this statement: for CDK2 and FXA, docking-bvls methods globally outperformed the 3D- LBVLS methods and for P38, all the methods exhibited a poor enrichment. Chemotype Enrichment Analysis by 3D-VLS Methods. A critical assessment of the robustness of the methods could be performed by evaluating their ability to retrieve structures with different chemotypes and thus to perform scaffold-hopping. 42,43 Similarly to the work published by Good, 38 we thus assessed the structural diversity of the different DUD-own active compounds by assigning them to their respective clusters according to a defined Tanimoto similarity distance descriptor threshold (Td < 0.55). We defined cluster enrichment via a simple procedure: a cluster is counted as present if at least one of its members is retrieved within the subset sampled. Results are presented on Figure 3 using cluster enrichment graphs. Overall, the enrichment in active compounds and in clusters follows a similar trend for 3D-LBVLS methods. Slight differences can be noticed in the individual performance of the methods for some systems. Surflex-sim performed efficiently in cluster enrichment for CDK2, P38, NA, and TK and was one of the best methods for these systems. Similarly, FlexS displayed a good performance on cluster enrichment for TRP, HIVRT, P38, NA, and FXA. Regarding docking-bvls methods, their global performance was good for cluster enrichment. This was particularly striking on ER, FXA, and CDK2, where all docking-bvls methods displayed a very good performance. By comparing the relative individual performance of docking-bvls methods, it seems that the global trend was conserved for most of the systems used in this study. For 8 out of 11 systems, a method that displayed the best performance in active compounds enrichment on a defined target also displayed the best performance in cluster enrichment. Striking differences in cluster enrichment could be observed with FlexX that displayed much better performance for DHFR and with ICM that displayed much better performance for THR and TRP. Overall, both 3D-LBVLS and docking-bvls methods retrieved more than 50% of the clusters in the top 10% for 6 out of the 11 systems. To highlight global trends between 3D-LBVLS methods and docking-bvls methods in enrichment, we created mean enrichment graphs using the means of the ranks obtained after screening the DUD-own databases. As the performance of each individual method varies depending on the target, an advantage of such representation is to enhance the differences in the global performance for 3D-LBVLS methods and docking-bvls methods. Mean enrichment graphs for cluster enrichment (Td > 0.55) and active compounds enrichment are presented on Figure 4. As expected, global trends in enrichment of combined docking-bvls methods and combined 3D-LBVLS methods were similar to the trends of individual methods. The global performance of docking-bvls methods was relatively poor and similar to 3D-LBVLS methods in cluster enrichment for HIVRT and THR. Overall docking-bvls methods displayed the best performance on 5 out of the 11 targets, it was thus equivalent on this test to 3D-LBVLS methods. Impact of Molecular Alignment on Enrichment. It is obvious that, at least for 3D-LBVLS methods, there should

63 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, Figure 2. Enrichment graphs with docking-bvls methods (dotted lines) and 3D-LBVLS methods (plain lines). The gray line represents random enrichment. Each color is for a package: blue for Surflex-dock/Surflex-sim, green for ICM/ICM-sim, yellow for FlexX/FlexS, and red for FRED/ROCS. be a direct correlation between the quality of the alignment of the active compounds on the reference compound and their rank in the database. To highlight this, we plotted mean enrichment graphs for the acceptable and unacceptable poses of the active compounds of the corresponding DUD-own database provided by the methods evaluated in this study. These mean enrichment graphs are presented in Figure 5. It clearly appears from the analysis of these graphs that, for 3D-LBVLS methods, compounds that are accurately superimposed to the reference active compound provided by the DUD are present in the early ranks. For 8 out of the 11 systems (all but HIVRT, P38 and NA), more than 95% of the active compounds available in early enrichment (1% threshold) are accurately superimposed (visual score ) 2). At a 10% threshold, most of the active compounds retrieved are superimposed in an acceptable manner (visual score ) 1 and 2) but up to 50% of these actives are superimposed with a visual score of 1. This highlights that the less accurate the superposition provided by the methods the lower the score. It also shows that the scoring functions used for the shape-similarity search methods evaluated in this study are efficient and reliable. Concerning the docking-bvls methods, at a 1% threshold, more than 95% of the active compounds were accurately positioned in the binding site only for DHFR. For all systems but TK, CDK2, and TRP, a non-negligible part of the ligands that ranked in the top 10% of the corresponding DUD-own database displayed an inaccurate positioning, the most

64 998 J. Chem. Inf. Model., Vol. 50, No. 6, 2010 GIGANTI ET AL. Table 4. Early (1%) and Late (10%) Enrichments for the DUD-own Active Compounds a enrichment all actives Surflex-sim ROCS FlexS ICMsim DOCK 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% ADA CDK DHFR ER FXA HIVRT NA P thrombin TK trypsin mean st. dev median enrichment all actives Surflex-dock FRED FlexX ICM DOCK 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% 1.00% 10.00% ADA CDK DHFR ER FXA HIVRT NA P thrombin TK trypsin mean st. dev median a Corresponding results with DOCK from the DUD original paper (Huang et al. J. Med. Chem. 2006) are also presented for information. striking examples being for THR and ER where 10.45% and 6.45%, respectively, of the active compounds retrieved in the top 10% were docked inaccurately. Overall, using docking-bvls, 0.57% of the active compounds retrieved in the top 1% of the ranked database were inaccurately positioned. This rate reached 3.38% when considering the top 10% of the ranked database. Concerning 3D-LBVLS methods, the results were respectively 0.13% for 1% of the ranked database and 1.11% for 10% of the ranked database. We could also observe that out of the active compounds ranked late in enrichment (from 10% to 100%), 16.15% and 10.82% were positioned accurately by docking-bvls and 3D-LBVLS, respectively, methods. This highlights that even though 3D-VLS methods provide accurate molecular alignments, a non-negligible part of the active compounds can still be missed because of scoring. DISCUSSION Molecular Alignment and Docking Accuracy. From the observation of the results, we can conclude that both 3D- LBVLS and docking-bvls methods are able to produce conformations similar to the bioactive conformation. Docking-BVLS methods are more challenging since the conformational space of a binding pocket is more complex than the pseudoreceptor defined by the reference ligand in 3D- LBVLS methods. But they display a good performance in terms of docking accuracy even on difficult targets where artifacts because of the construction of the DUD may sometimes affect the results. In particular, when the protein is considered as rigid (as in all the methods evaluated in this study), it may be very difficult for some ligands to be docked into the binding site of the structure provided by the DUD. For example, in the P38 structure, there are 82 active compounds (i.e., 18% of the P38 active compounds) that cannot be docked into the binding site because of major clashes occurring in the current protein conformation. We retrieved from the PDB database structures corresponding to some of the 82 active compounds that caused problems with the P38 structure, and it seems that there is a major shift of F169 that caused this docking issue (see figure 6). Similar problems can be observed with the structures of FXA, ADA, and TK, where some of the active compounds cannot bind properly to the currently provided structure because of the orientation of some key side chains. Concerning the relative performance of the methods, it seems that for both docking-bvls and 3D-LBVLS, ICM displays the best performance in terms of molecular alignment and docking accuracy. This may be caused by a more effective treatment of the flexibility of the compounds in ICM via its biased probability Monte Carlo procedure. 44 FRED displays an overall very good performance despite being the fastest docking method. It is even the most effective docking method on DHFR, P38, and ER, which pose problems to the other docking methods. These targets display the largest binding sites, making the conformational search within these

65 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, Figure 3. Cluster enrichment graphs with docking-bvls methods (dotted lines) and 3D-LBVLS methods (plain lines). Each color is for a package: blue for Surflex-dock/Surflex-sim, green for ICM/ICM-sim, yellow for FlexX/FlexS, and red for FRED/ROCS. sites more difficult. One of the possible reasons for the good performance of FRED on these 3 systems could be that FRED focuses on the optimization of only translational and rotational degrees of freedom during the calculation, which seems more effective for exploring such large binding sites than using fully flexible ligands. For FRED calculation, the torsional degrees of freedom are estimated using multiconformer libraries generated by OMEGA. Also, FRED calculations were performed using shape-gauss to evaluate the ligand poses, which is known to be relatively clashtolerant when using default settings. 34 This could also be a possible reason for its success compared to the other methods for P38, taking into consideration that a non-negligible part of the DUD-active compounds cannot bind to the provided structure because of the conformation of F169. Surflex-dock appears to be the best on 3 systems (TK, NA, TRP) despite displaying the least overall docking accuracy (59.93% of acceptable poses), principally, because of its very poor performance on P38 and THR. Overall the different programs evaluated in this study are able to perform acceptable molecular alignments that lead to an accurate superimposition of the active compounds on the reference for 3D-LBVLS methods and acceptable docking poses for docking-bvls methods. Comparative Performance in Enrichment. The quality of virtual ligand screening methods is evaluated by their

66 1000 J. Chem. Inf. Model., Vol. 50, No. 6, 2010 GIGANTI ET AL. Figure 4. Mean enrichment graphs (gray) and mean cluster enrichment graphs (black) obtained by docking-bvls methods (dotted lines) and 3D-LBVLS methods (plain lines). ability to retrieve active compounds within the early ranked database, the so-called enrichment. Overall, mean early and late enrichments are clearly in favor of 3D-LBVLS methods (9.48%/34.91% for docking- BVLS versus 12.07%/42.05% for 3D-LBVLS). This is particularly striking for ER where systematically all the 3D- LBVLS methods outperform docking-bvls methods. Von Korff 45 suggested that this should not be surprising because of the construction of the DUD database as, decoys are mostly topologically dissimilar to the active compounds. 21 However, as shown by Cleves, 36 the impact of this bias is important principally for 2D similarity search methods. An opposite opinion is given by Kirchmair 17 when he suggests that docking-bvls methods are in fact favored in the DUD considering that docking has, more likely, data present on the dimensions of the active site. We believe that shapesimilarity search methods are less sensitive to the possible bias highlighted by Von Korff and Cleves as several features directly related to the topology such as the bond and atom types are not primarily considered in 3D-LBVLS methods and, as Kirchmair stated, 17 that ligand-based methods have to represent the binding site properties using a single active compound, which is already very challenging on its own. Even considering possible bias, docking-bvls methods are still very effective on 6 out of the 11 systems evaluated in this study (NA, TRP, HIVRT, DHFR, CDK2 and FXA), the most striking performance being with CDK2 and FXA.

67 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, Figure 5. Molecular alignment of the active compounds using 3D-LBVLS methods (plain lines) and docking-bvls methods (dotted lines). Light gray line shows the random enrichment. Black lines are for acceptable poses and gray lines for unacceptable poses. Surflex-dock appears to be the most effective docking- BVLS method on enrichment. It also outperforms most of the 3D-LBVLS methods in several systems. By comparing our results with Cross et al, 20 the Surflex-dock ringflex parameter seems clearly valuable at least for serine proteases where we could see a significative impact (data not shown). It improves the enrichment for 5 out of the 11 systems evaluated in this study (ADA, CDK2, ER, FXA, and THR). FRED appears to show its limits in enrichment except on the systems where the more complex docking methods fail, such as HIVRT, P38, and TK. This shows that in particularly difficult systems, multiconformer rigid-body docking methods can perform at least, as well as flexible docking. As for docking accuracy, one possible explanation can be the clash tolerance of the shape-gauss scoring function implemented in FRED for these targets where several clashes can occur

68 1002 J. Chem. Inf. Model., Vol. 50, No. 6, 2010 GIGANTI ET AL. Figure 6. Illustration of a clash between F169 of the P38 structure provided by the DUD (green) and one of the compounds from the P38 DUD-own active set, ZINC , positioned using ICM (pink). The blue structure represents an alternate PDB that could have accepted this compound. with a non-negligible part of the active compounds of the corresponding DUD-own data set, for example, in the currently provided structure for P38 (see the dockingaccuracy section), and in the small and buried binding site of TK and HIVRT. The variance observed between the results is important within the DUD targets and, as observed by von Korff, 45 highlights the importance of multiple systems for benchmarking studies. Comparison with Other Studies. On 2 out of the 11 targets evaluated in this study (ADA and DHFR), DOCK results from the original DUD paper 21 display a better performance in early enrichment compared to the other docking methods. For the other targets, its performance is comparable to FlexX. As expected, our results with ROCS are almost identical to those obtained by Kirchmair. 17 The slight differences we observe can be explained by the different settings used for the multiconformer generation of the DUD-own sets with OMEGA but seem to have a very low impact on the final performance of the program. It is reensuring to have similar results using the same data sets and encouraging for using standardized data sets, such as the DUD for benchmarking studies. As proposed by the creators of the DUD, 21 a broadened use of such data sets will be beneficiary for the whole community to compare their benchmarking results. By comparing the ROC AUC with the early and late enrichment rates, we can draw several observations. ROC AUC is a good measure of the global discriminating performance of the different methods since we use the same conditions for testing. In general, similar conclusions can be drawn from the analysis of ROC curves/roc AUC and enrichment graphs around 10% or 20% of the ranked database but in early enrichment it is not necessarily the case. 17 For real life project decisions, the important point about the methods is the definition of the selection threshold to use, that is, the number of compounds to test experimentally after a virtual screening. This measure, in our opinion, cannot be necessarily provided using a metric, such as ROC AUC or even the analysis of ROC curves but rather by classical enrichment values comparisons. For instance, in the case of ADA, by observing the ROC curves and AUC, the best method seems to be ROCS but in fact in early and late enrichment, it appears that Surflex-dock is the best method to use even if its ROC AUC is 10% lower. This highlights the point stated by Kirchmair 17 that both ROC AUC and enrichment rates need to be considered in method evaluation and thus in the definition of new drug-discovery protocols. Once more, the use of standardized metrics in benchmarking studies will benefit the whole community. Comparative Performance in Scaffold Enrichment. As expected, methods that display a good performance in enrichment also display a good performance in scaffoldenrichment. It is widely accepted that docking methods should be appropriate to retrieve compounds with different molecular scaffolds, as a protein s binding site is more permissive than a pseudoreceptor derived from ligand-based hypothesis. Indeed, docking-bvls methods perform well in scaffold enrichment, retrieving in average more than 50% of the different clusters in the top 10% for 7 out of the 11 systems studied here. However, for TK, NA, ADA, and P38, 3D-LBVLS methods outperform significantly docking-bvls methods, highlighting their good performance in scaffoldhopping. By analyzing the trends from the mean enrichment graphs, we can conclude that overall, 3D-VLS methods have a similar acceptable performance in scaffold enrichment, depending on the system. It is thus of importance to underscore that, in the systems tested in the present study, docking-bvls methods do not perform systematically better in scaffold-hopping than 3D-LBVLS methods. This makes 3D-LBVLS methods also reliable for retrieving diverse molecular scaffolds. As a confirmation, we also tested the ability to retrieve compounds within the same cluster as the reference active compound provided by the DUD (thus more similar compounds i.e., the inner ability of LBVLS methods). As expected, in this trivial test for ligand-based methods, 3D- LBVLS methods and ROCS in particular display a much better overall performance, outperforming systematically docking-bvls methods (11 out of the 11 systems studied, data not shown). Molecular Alignment and Enrichment. It should be obvious that, using 3D-VLS methods, compounds that are retrieved in the top scores should be positioned accurately in the binding site/superimposed accurately on the reference compound (i.e., accurate molecular alignment). We thus analyzed the molecular alignment of the different active compounds after 3D-VLS. As expected, for the 3D-LBVLS programs evaluated in this study, more than 80% of the active compounds retrieved in the top 10% are positioned in an acceptable manner, while a very low number display unacceptable poses: 19/338 for Surflex-sim, 8/500 for ROCS, 10/712 for ICM-sim, and 19/ 399 for FlexS. Only few exceptions occur for FXA and CDK2 with Surflex-sim and for ADA with ROCS. This is not surprising since the scoring in 3D-LBVLS methods is directly based on the molecular alignment and not on a (pseudo) binding energy evaluation as in docking-bvls methods. In the case of the docking-bvls methods, all the programs but FRED retrieve more than 80% of the active compounds in the top 10% with an acceptable positioning but we can

69 3D VIRTUAL LIGAND SCREENING METHODS J. Chem. Inf. Model., Vol. 50, No. 6, observe that a non-negligible part of the active compounds retrieved in the top 10% display unacceptable binding modes: 66/576 for Surflex-dock, 39/268 for FRED, 36/518 for ICM, 37/467 for FlexX, and particularly in the case of THR and P38. One possible reason might be that the active compounds of these systems, where the positioning is difficult, display high flexibility and are to be positioned in relatively large binding sites which makes the conformational search more difficult for docking-bvls methods. This illustrates one of the surprising current paradoxes in virtual screening that is retrieving active compounds in the top scores while displaying an inaccurate molecular alignment. This paradox is much more frequent with docking- BVLS methods compared to 3D-LBVLS methods as the conformational space to explore is wider in a real binding pocket relatively to a pseudobinding site derived from a ligand-based hypothesis and thus more susceptible to errors. It is difficult to pinpoint a specific cause for this paradox but there might be several factors including (1) the weight of the different parameters in scoring functions which generally do not take correctly into account solvation/ desolvation and entropy and (2) the rigid-body treatment of the atoms of the binding pocket or the limited handling of flexibility of the compounds during the simulation (especially for highly flexible compounds). CONCLUSION We have investigated the performance of 8 different 3D- VLS programs on 11 DUD systems using their corresponding DUD-own database. The use of the DUD-own database is more challenging compared to the general DUD data set and is more appropriate for benchmarking studies. The programs evaluated in this study are all able to perform acceptable molecular alignments with standard parameters. In this exercise, ICM appeared to be the most efficient underscoring the good performance of the probability biased Monte Carlo procedure used for simulating the compounds flexibility. We identified several problems with some of the structures provided by the DUD. In particular, for P38, a non-negligible part of the ligands provided as active compounds appeared unable to bind the provided structure because of major clashes biasing negatively the results for docking-bvls methods evaluation. Concerning the performance of retrieving active compounds among decoys, that is, the enrichment, it was overall acceptable but variable depending on the target and the program used. Surflex-dock and ICM had the best global performance in enrichment. In terms of scaffold enrichment, 3D-LBVLS methods showed a comparable performance to docking-bvls methods, which is quite surprising as docking-methods are generally considered to be the methods of reference for retrieving diverse molecular scaffolds. This is very interesting for the definition of new drug-discovery projects since 3D-LBVLS methods use very limited computational resources compared to the more sophisticated docking-bvls methods (excluding FRED) thus opening new questions about which method to use when the structure of a small-molecule/protein target complex is known. The variance observed between the results is important within the DUD targets and highlights the importance of multiple systems for benchmarking studies. Finally this study illustrates the molecular alignment paradox in enrichment that consists in finding a nonnegligible number of active compounds displaying good scores, and thus good ranks but with an inaccurate positioning into the binding pocket. Considering that docking methods are in most of the cases able to produce accurate binding modes, this highlights the current limits of the scoring functions used in docking-bvls methods that still need to be optimized to avoid such problems. Abbreviations. 3D-LBVLS, 3D ligand-based virtual ligand screening; docking-bvls, docking-based virtual screening; 3D-VLS, 3D virtual ligand screening; Tc, Tanimoto coefficient; ADA, adenosine deaminase; CDK2, cyclin dependent kinase 2; DHFR, dihydrofolate reductase; ER, antagonists for estrogen receptor; FXA, coagulation factor Xa; HIVRT, HIV reverse transcriptase; NA, neuraminidase; P38, mitogenactivated protein; THR, thrombin; TK, thymidine kinase; TRP, trypsin. 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72 F E E F BE E E D B E BE A BE EB AE B A BE D EF EA D AE E AE BE A BE E D E BE A B E BE A BE F F B E C E E AE B EB AE B A BE E F E F E DF E AE A E B A A

73 BE F BE E E AA AE B E BE BEA D BE F D E BE F BE BB A E BE AA AE E E BE BF ADABE B BEBF E BE F BE BB AE EB D D AE FE DA ED A BE E D A B E AA E B E A ADA E EB E D AE FBE E A E FE F AD E D F FE E DE D F E D BE FEADF E E DF E B A B C BE F EA BE E A BE F AE F EF E DA EB D E E DE DA E D A E E DE F E E E DE F A E BAE FBE AD A E F E BE A BE E B D E DA E FEB A E E D B E AD AE FBE E F E F E F E B F A F E E E D A E F E F E E EC DFAD AE FBE F E DE BA F A E E DEC CE FAE A D E DE B E D A D AB E D E E DE DA E FEB A E E D B E B A E D BE DEC CE BAE DBEA F F BED D A E EA FBE BED A B E AD AE E E F E E E FB F BE DA BE F AE A E B BE F E E B FE E FEB A E E D B E DE DA E A F E D BE DEC CE E E E B A AE E E BE B B E BE BB ABE D E BE A BE EB AE D F BE DA AE D E D AE DBEF E D F E E E E E AEF EB F E A E FBB BBD AE E D EF E BB AE E FBE E E E E B E F BE A BE E B A BE BEBF DBB AE FDB EB BA DA F AE B F E A E E CEBF DBB E E E DE E B E D BEF E E BF E FAE A E B E F E C E E AE E D A E E AE B EB AE BE A BE F E AE BE F BE B A AE AE D AB E B A A E FA A E E D B E F E F E DA E E B D E DA E D E D A E E A E D E D A EA FA BE BE A BEB AE D D BE E D E BE B A ABE FED ABE BE AE EB E BA F EBF EF E E D A F BE BB ABE BE E E AE E EB AE D AE E D F E BE A BE E E F E EB D AE DBEBF D AE F B F E BE D A BEB A E F E DE F D A EA F AE ABE BED A B E AE BE A BE EB E DBD AE E D E D A EBF E DEB D A E E ED E DE F E E E B E BE F BED D AEF EA E A E E DE F E E EB AE D F BE D D A E D A E AA E E F D AE BF A FAEF E E AD AE F E BE A BE EB D A E C E D E BF EDFE A D E F E E E BAE D B E D E E BB E BE DA BE FEB A E E D B E

74 A B E E D EB E A E DE FBED AD E BE A BE EB D A E CE E B D AE DBE A AE BE D B BE AEA BE DA BE BE E DEA E D BE D AE B A EF E B F B A E E D B E E F E BAE E EB E E A E BE A BE E AEF E F E BB AE F E BE A BE E F E EB BA BEBF E E F E BAE DE A E E DE FBE D E AE D AEF E F E BB AE F E BE A BE EBF E FB F BEB BA B E DE D D E B E A E BE BF ADABE BAE AD A E D E BE BE E DEC C E E F EB F E AD E FA B E FB F BE B BA BE F E D FDA E E A B E BE BF ADABEB AEB D BE E F E A FBE D E D DE B A E F E E BB AEDEDFBB E A E AF E EDAA F E BE A BE D BD AEF E E BB AE ED A BE AEDFBB EF E E BB AE E A B E E F E FB F BE FBA BED AE A E AD BE F E BED A BE E D F E E F E E A AE D AE DBEF E B A EA BE E BE A BE E EB AEBF B BE A E F B E EB A E E D B E AD AE FBE BB E F F E B F B A E E D B E E E D A E F E F E E E E F E BAE A AE E DBE F E DE D A E BE B E D A E BE A BE E E A AE F E F E E BE EB BA B E E F E BEB BA BE F E B F BE BED A BEB AEA BE D BE D E E ED E E F BE E A BE F E ED A B E BE A BE E A AE E E E A BE E E A AE F E F E F E A E E B A E E E AEF E D A E BAE B E E EC E D EBF D A E BE A BE E E AE DBEF E BB AE B AD F D E BED A BE E E E F BEB AE FE D BE E F BE F E ED A B E D BE BEBF DBB AE BE A BE E FDB EB BA DA F A E A E D A E E CE BBD AE F E F E E E DE D D A E BE A BE E A F E BE F BE BE FBEB D BEDFE D E E EDE D A E A E B E E DAA F E B E A B E E A AE B BA DA F AE F E F E BE A BE E AA E D EDEBD BE FA EF E D AE BF E BB AE D E B E BE ABE F BE E A BE AD AE DBE EAD E B D BEDFEB E F E EB BA E AE E D E E E D D A D AE DBEA F F BEDFE FBE D E F E D D AE AE F E BED A BED D AE BE F BEB BE D AE A E B A BE D BE EB A E E D B E FEBF B BEBF E E B E E E E D E B E F E BE A BE

75 E FBE E E BED A BE A F BE D BE EA E E E DE D F EB AED BE E D ED AD E E BAE ED ED E E D AE F E BE A F BE EB D A EB AE DB BE A AEBF E D AE F D E FA BE BE A BE E E D AE C E A F AE FBE E E BED A BE FEA E ED EF E B ED AD E D A E E E E B BE D AD BE BAE DBE D E AD AE F E E AE E BEB A BE E D B BE E BE A BEB AE D BE AE DA E E B D E DA E BAE E FBE E F E BE A BE E D A EF EB F E E D E EDE A EFA B E F E D F E DE FD A E FE B A A E E D AE DBE FE F E E A E BE BE E D B BE F E AEB E BED A B BE B DA BEB F AEF E BE D D BE BE A BE E D E E E AD BE D A BED D AE A FBE BE F BEB BEB AE D E B A BE FED B E E D D E BAE FBE F AE B ED E BE A BE E BE DA BE BB BE F AE A E D D BE E E BE A BE EB E E AE D AE DBE E A E DEB DADA E AE A E A A E EB A E E D B E BAE B E E D E E E D AED B E DBE E A EA FBE BE BE E D B BE A A B E AE DE BA E E DE A E BE D BE BAE DBEA F F BE BF BD A E AD AE F E BE D BEA BE B B D E DA E E B E BAE FBE D E F E BE A BE E F E F E BE A BE E FBE E B A ABE AD AE BB BE F E D F E B E CD BE AA E AF EF EB F EBA F AF EDE A EFA B E F E D F EB BA E FA BDA E E FB F BEBA F AF BE D EB BA E F D AE AA E E E DE A E FEB A E E D B E

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77 E A F AF BE F A BE F E E D E E E A A BE F BE E A E BE A BE E E E DE EBA F AF E E E D A E BE A BE BEB A BE E D B C BE D ABE DA BEB E F B AE BE E DE D B E FE D E D BE EB A E E D B E FE A F E F E A E D B E E D E DBB F E DEBA F AF E FEB A E E D B E BA E E D AE E E FB F BED BE AE A E BE F E E E A E DE A E FEB A E E D B E E D E D F E D AE E E DE A E FEB A E E D B E FAEDFBB E A E B A E ED A E EFA BD AE FB F BE BA F AF BED D AE BE DA BE A B E AA E A E BAED E E B E E E BEBA F AF BEFA B BE F AED E A E A F BE AD A E E FE BE E D A E EB F DA B E E BED BEB AE FBE A FB BE EA BE E D F E F E BED BE E E DBB F BE AE EADF E E DF E B A BE B EDF A E D B E AA EDF ADA E FAE A E F E D E BE F BE EB E D BEDFBB E D E E E BE BA F AF BE BA AFD AE BE B B E E E D A E DE F D AE BE A BE E E D AE E A E DE A E FE A F E E AE E D E A E CD BE DE AA DAF E A E FB F BE BED E BEBA F AF BEB A BE D BE DEC CE AE A E BE E E E D E E E B FE E E DE A E E E DB E BAE A E EA EB A E F F E E E D E D A BE E F AE DBE A E B A BE D AE D BE EB A E E D B ED FA BBD AE E BE BB ABED A AE D B E F B F E FB F BEBA F AF BE E AA E A EB AE B BE D BE DE C E E D BE DF D AE FE A E A E E B BBD AEF EBA F AF E FBED D A E FE E D BD AE E B E E E F E FB F BE AF BED AE A E D A BEBF E E E E EDEA F F BE DBE E B BFBE F EF ED E FEF EDFA E F E BE B E E BE A ABE B BE E A BE BE E EB AE A E E E E E E AA E AED E B E E A E E E F E F E E EBF EF EBA F AF E B F EC BE AF BE D BE D AE E A E D A BE F E A E BE F BE BA F AF BE EFA B E E F EF E E E E E DE DA E E D AE BAE D E E FBED BE E E E E BEA D BE D BE BE A BE BEB A BE E D B E F BD AEDF E

78 D BE A D BE BE AF BE E E BEB BA BE E DEC CE F E B F BE FB F BEBA F AF BE AD BE AD AE B A BE D BE DE C E AE A EB A BE A FBE BDF E C E FB F E EBA F AF BE AD BE AE A E B B E F E B F BE BE A BE F E F AF E BEB A BE E D B EB AEB DA AE A B E F E D F EBA F AF E BE D BE EA E BB AE F E E A BE E E F E AE E AE A E D F BE E D E BED A BE AE D A BEB F BE E D F E B BA E AA E AF EDE A E D A E D BE E FAE E E E D AE FE E F EBA F AF EBF E BE BF ADABE F E D E E A E BEA D BE A A BE E A BEBA F AF D BE D AD BE D E BEBA F AF BE D AE BE F BE FE BE BE BE BF ADAB E F E E D BE BB E BEA D BE F E F D AE A E B E EFA BD AEF EB F E BA F AF E F EF E E F B F E DE F BBD E E D F E B E BAE D A D AEBF BD A E BE D BE E F E AE E AE A E D F BEBF EA FBE BE B BE EBA F AF BE BB BE B A BE F E AA E AF E AA E D FDA EDE A E D A E F E A E BEBA F AF BE A D BE BD AE BE B BE AE D E F BE A BEBA F AF D B E E C E DEC A E E B F EC B E BAEF E D F E D FDA E A D AE EB A B E D F EB BA E BAE B E F EBA F AF E BAD D F E E DE A E E AE FE D E BAD B E D A BE FBE A E F E D F ED A E E E D A BEB A BE F E F BE A BE B F BEB D BEDF ED A B E BEB AE E D EBA F AF D AE AB E FBE BEB BA B E F E B F BEDFE BEF EBA F AF E AD E E DE E AD AE B E AE A E A FB AF D E D A E DA E E B D E DA E E F E BAE E E D A E E D ADA E E BA F A E E D D E EB A E E D B E BAE B A E D EF E A E BAE E E BE D ABE F D BE E E E F D AEA FAE EB A E E D B E BE B BEB AE D F BE D EF E A E EB E F E

79 F E D F EBA F AF E EB A E E D B EDE A E E E E EDFA F E FE D E BAD B E DE B E E E F EDE A EFA B E F E AA E AF E E BAE DB EBF E D A E E A BE A E D E F E A B E E B A AE BE F BE D BE EB A E E D B E BE B BEB AE D F BED E DE A E EB E F E E DE B E E EDE A EFA B E F E AA E AF C D E F E D F E EA FBE BE B BE BB BE FB F E E B E F EF EB BA E E AE A E BA F ABE E D A E BEBA F AF BE AD BE E DE C E BE BE E EBF E BE B BE AE A E D ABED E F E AE E F EF E B E EBF EF E E E E F EB E A FED E BE A BEBA F AF BE E B E BAE A F E B E F E D F E B E BE BF ADABE FE E B BA AE E F E BA ED E BEB BE BE F BE B B A D AF A B E FBE BE D BE AE A E D ABED E FA EBADA BA F E AE D F E E E F E EDE A EFA B E F EA D E BE F BE E E AE D A E D A E F E D F E D EB FBE DE F E E F E A E DE DA E D E E E D E D E E A A E E D F ED D A AE D F EB BA E BE BE FEADF E EBF D E D ED BB EDFEB D AE AE FE F E BEB A BE E D B E BA AFD AE D F EB BA E EB BA E BAE D BE DE DA E BE A ABE B A BE E D B E B F E E F E E BAE F E FE D E E E E B F E DE E BEADF E EBF D ED D ED BB EDFEB D AE BAE F E E E EB BA E BAE D BE DE DA E BEB A BE E D B E D B E EADF E BAE D F ED E A D E E D BD AE E DA E E D E E DEBF D E E D ED BB EDFEB D AE E AE E D E E DEBF D E D BB EDFEB D AE E EC F E BEB BA BE E DEC CE F E B F BEDFE BEF EBA F AF E AD E BAE B E A FBEBDF E C EA FA BE BEBA F AF BE B A BE D BE DE C E AE A E AF B E BAE E E A E E AE EBA F AF BE D EB BA E FB F E EBA F AF BE D EB BA E AE A E B BE D BE E FAE E F EA FA E DE A E AD E B E F E E F E AE F AF E E DE E BE D BE EA E BB AE E F E AE

80 E AE A E B BED E E A E BEA D BE D E BE A BEBA F AF D BE BE DA BE D AE BE F BE FE BE BB AB E F E EB BA BEBF E E DE DA E F E D F E DE F E D E BAE B E F E D F EB BA E BAE F E E F E AE E F E EB BA BEBF E E DE BE E D E BAEDFBB EDBB E E DE E DA E F E F E AE E DE DA E E DEBA F AF E E DE EB E ED EF E D AEBF E DE FD A E E BB A E E F E BAE ED ED E DFA BE AF B E E EC E FB E F E EB AED E F E F E EB BA BEBF E E FE E EBF E E DE F E D E BAE B E F E BE DA BE AE EB A E E D B EDE E FBE A AE F E E B E DE BE E D E BAEB F AE B E F E BE FBE D BEB A BE E D B E EBF E E F E F E AE EBF E E F E E F E F E D E BEB BA BED D AEF E B A E E D B ED EF E A AE F E E DE DA E DE FBE E BAE E FBEB F AE DBB E E DE F E D E EBF E EAD BE F E DE DA E DE FBE F A E B D AE E DE BE E D E EBF E E F E BEB BA BE AE EB A E E D B EDEF E D E F E E DE DA E DE FBE E A D E BE BE BB ABE F E EB BA BEBF E E F E EB AED E F E FE F E D F EB BA EA FBE BE B BE E E E EBA F AF BE AE A E BA F AB E F E D F E B E E F EB E A FE D E E DBB F EBF E D F EBA F AF E E B E BAE A F E D D B EDE A E D A EBF EA FBE BEB BA BE E FBE F E B E F D AAE A E BA F A EB AE EB BA B E F E EB AE F E F E FE E BE FBE A ABE B BE D FA BB AE FDB EB BA DA F AEDF E F BE BB ABE E AE EBF E EB BA B E B A A E E F E BAE ED ED E BE AF BE A B E E E DE F E BA F AF EB F E BAE D AE B A E D BE E F E B E B BA DA F AE F E F E AE F E EB BA BEBF E E F E E AE E FBE A AEB A E E D B E BAE B AE D BE AE B E F E E AE EB BA BEBF E E B A A EC E DE E D E DE BA F AF EB F EDBB E E DE FBE DF D B E D E BAEA F F BE FE B F E D BE E FBE DF D BE B E E AE EB BA BEBF E E B A A E E F E F E F E E B BA BEBF E E AE DE DA E DE FBE D E D BE E FBE DF D BE B EDF F E A D E E FAE A E A E F E FA BDA E B BE E FB F BEBA F AF BE AE E E E A E DE A E FE B A E E D B E A D D AEF E F ED D ADA E E EB A EDF E F BE B E F BE

81 B AED A BE FE D A B E E AD AE E BBD E E A E BE DA BE D BD AE D D ADA EDF ED A BEB EA EDF A E D D ADA EDF E D A B E B EF E B E E A E B A E E DE A E E D E F E A AE E E DA B E FAE D D A E FBED D A E F F E B E E A E A F E E D BD AE E DE D F E F D E D E F EBD EB E BAE BBD E FA B E FB F BEBA F AF B E BE D BE BE F BE B BE AE BE F BEBA F AF BEB F BE AE A E D B E F F EA D E EB E D E F E F E AE E DE F EBA F AF EB F ED FA BBD AE EF E F E BB AE F E EB BA BEBF E E AE B AE F E EB BA BEBF E E F F E E AD E DE A E B E F E EB BA B E D A E E F E A BE E E FB F BEA D BE AE A E B BE AAD AE E FE BE A BE BEB A BE E D B E E BEBA F AF BE A D D AE BE F BE AE BE BE BE BB ABEB AE D AE F BEDF E F E A B E BE F BE BB ABEB AE B BE F E DE DA E E FBE D E F E E B E BE BE BE D BEB AEDBB BEDF E FBE D BE DA B E BE B BE BE FBE A ABE A AE E F E AE A AE B F EA F F BE DE F EBA F AF EB F E E BE B BED E BE BE BB ABE A AE D AE DE EBA F AF E B F E D E E EB E DBE ED E EA D E AAD AE E B E FA AE E F E B E F E DE F EBA F AF EB F E E E EB BA BE E D F E E D F E F E AD AEB D

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91 E BE F BE E A A E E A F E D FDA E BE A BE E D E E BA E DF F E E A BE E D E E E F EDE F AE EF EA D D E B D E F E A E BE F BE D BE E BE D F E E AD AE D AE D FDA E E F E D D A E EDF A E DE A DA E D A BE D BE BE BE F AD BE E DE D F E DBB E E D E F E A E EB DEB A E F EB F E A D E F E BEA BABE ADF E BAE D E AE BAE BA E E D D BD AE BE BF ADABE D FDA BE A B A BE BE A BE E D E E BE D FDA BE B BA AE EA BA E DE D D A E F E A E E B E F EF E E D A F E BE B BED A BE BE B BE D A B E DE A F E D FDA E AE E A E B ED EB E F E EB AE F E D FDA E F E A E E D E FE F E D E FB F BE A BE A E B EC A BE A F BEB AED D F BE E E D A F E BB A E DE F E BB A E DE F E E E AEB ED EB FBE DE F E E E D E BE FBEFA B B EC DFA BE A F BE AE A E B B E DB BEBF E BE A B E D BE BBD D AE E BED E E D E D A EB FBE DE F E E E E E C E FE E E FA BE BE A F BE AE A E F AE AF BE AE F BE BE AE D BB BE DAAF B E E D BEDF F E A F EBAD D E DE E A E E E D A F E AE DE F E BB AEB AEA BEFA B BE D E BE B A AE E FAE E FE D E E D BEB AE D ABE FEADF E D A BE D BE DE D F EC E FB E BE E AE E A E F F EB F EA E F E E DBB DA E DEB B A E ADF E E D BE B A B C F EDFA E A E BE F BE E E B A AE E DE BE DEB A E ADF E E D BE DA B E AE DEB B A EC E FB E BEB AE D A BE FEADF E D A BE B ABE D BE DE D F E D FDA E D A EA FA BE BE D F BE EB B A E AE E E B E F BE B AE EF EB A E D E FE CD BE AA E AF E DE F E E A A E BAEFA B E F EA A E E A E DE E E D BE DE D D B E BE D BE E A BE A B E EB D AE E AE A BBD AE D F E DE D D A E F E A E E E DE D A E F E F E A ED A E FA AE F D A E AA E F E B A E DE BA FA E F E F DA E E B F BE ABE D EF E D F F E FEF E E E A E E B F E E EB EDAA F E E D F E B E DE

92 A F E BAE A E D E FD E AE E E DE D EBF D A E F B BE F E E B A E D A A E F E B ED E E F EF ED A E AE E F EF E D A E E B A E DE A E E B E AF E A E E B F E D F E E D A E F E B E AE E EBF BD AE F E E B F E EDF A E E D E A E D E DE A E E DE F E E A A E F E E BAE ED E E BE A E E AE E B E E BAE E B F E B D AEDFE E A E E DE A E DE A E B E BAE DE A E E B BED D AEF E B F E A ED A BE F E FE D E E E E A E A BBD A E E AA E B ADA E BAE F E F AEF E E F EDF E A BE A B E AAD AE E D E BE D D B BE A E BE A BE F E EB D AE DBE AE D D BED E F BE BE B A B BE D D B BE F AE A E D A BE E D A E E EF E A E D BD AEF E F E A E E B F ED D AEA D E ED EF E FBE D E D D A E E D F E B E F E E DE A E EB E F E D F E E BAE E FBE EDEF E F E A E F E BE B F BE BE F E DFA EC F EB F BE F AEDFBB E A E B BE E E A FBE BE B BE B FBE EB F EB AE B BE E D A B E AE E A FBE BE B BEDF BBFBE E EB F E B AE B BED A B E DE A E EB ED D AE D AE DE F E A E BAE E F E D F E E BE E B BE AEF E B F E BE A E BE F EB F B E D EA AD EDE A E B E D E F DE AE DBA A E E E E BF E D E E DE F E E A A E D BAE E E D E B A E A E DE F E E A A E AF E AE DE F E B D AE E DE D E E ED A BE E DE D F E AA E F E B A E EF E A E EB E E DA EA FA BE BE F BED D AE DE E D A E A ED A B C F E A B E F E AE E AE A E D F BEBF E BE E BE AEF EBA F AF BE AD E AD AE B BE D BE DEC C E AE BE BF ADABEB AE B A BED E BE F BE E E AE E A A E F E D F E E AE D F E A E DE BA FA E BEB BE A FBEDE A E B A E D EF E F E E B A

93 E E C DEC A E E B F EC B E BAEF E D F E D FDA E A D AE EB A B E D F EB BA E BAE B E F EBA F AF E BAD D F E E DE A E E AE FE D E BAD B E D A BE FBE A E F E D F ED A E E E D A BEB A BE F E F BE A BE B F BEB D BEDF ED A B E BEB AE E D EBA F AF D AE AB E FBE BEB BA B E F E B F BEF EBA F AF E AD E E DE E AD AE B E AE A E A FB AF D E D A E DA E E B D E DA E E F E BAE E E D A E E D ADA E E BA F A E E D D E EB A E E D B E BAE B A E D EF E A E BAE E E BE D ABE F D BE E E E F D AEA FAE EB A E E D B E BE B BEB AE D F BE D EF E A E EB E F E F E D F EBA F AF E EB A E E D B EDE A E E E E EDFA F E FE D E BAD B E DE B E E E F EDE A EFA B E F E AA E AF E E BAE DB EBF E D A E E A BE A E D E F E A B E E B A AE BE F BE D BE EB A E E D B E BE B BEB AE D F BED E DE A E EB E F E E DE B E E EDE A EFA B E F E AA E AF A D AF A B E FBE BE D BE AE A E D ABED E FA EBADA BA F E AE D F E E E F E EDE A EFA B E F EA D E BE F BE E E BE F BE E A A E AE A EA D BED E E F E A E A D AEDF E F BE E E E BB AE F E B A AE DE D F E DBB E D EF E A E EB E E E E E FE E E E E DE F E E A A E B A E DE D F E E E E E E A EDFE DFE E DE D E BED A BEDE A EA D E E B E EF E BA FA ED DA E BED A BE D BE DE D F E E E D A F E F E DE D A E A EDE A EFA B E D E DE D E ED A BE BE D F BE BAEA E D E F E F E E B E D F E BE F B E BE A BE EB E BE A BE E D BE EB EB AE B BE E BEB BE FBE D D BEDF ED A BE F DF E D A B E F E F E BED A BEB AE B BED EF EB E FBE EAD BE F E DE A E EB E E E AE F E EF EB E FBE DA E BE

94 F BE E B A E BE BA FA BE EB BE AA AE FBA E AA E A B E AE E A E F E E BAE DBEB BA DA F E E A E BE F E BA FA BEB AE D BE E D F E BED A BE F EF E D E E EB BA B E F E EB AED E E E E E E E E B E FE F E E E E E E E E D D E E D E B EB E DF AE D E E AE F E FEB AE D BE B F AED E E E D F E BE D A BE F E E F EDFA BEB BA BE C E E E E E E F E E E E E E E E E F E F E FB F BE BEB AEDFBB E B BEBF E AD BE BA FA BE EB E D E E C E E E B E F E F D AE A E E BF ADAE E DE B E E FB F BE A BE D E BED A B E D A E BE BE EB AE DBEA F F BE B ABED E BE F E A BE E E BE BA FA BE EB E AE EF E ED FE E DE FD A E E BB AE F E D F E ED A B E D BE BE E AA AE DBE E D E BE D D B BE E D BE A E A B E BE BE BEB BE AD AE DBE BE B BE F BE E E AE BB AE AE A EA D BE EAD AE F E E BE F BE AA AE EB F AE E FD A E BB AE ED A BE F E D F E D EF E A E EB E D BE D AE E D E BE D BE E F E A BE EB E ED D D AE F E AD BEB BA BE F E BE F E A F BE AE BE BF ADABE A D A BE FE F EB AE E E A E F E A E A E E F EDFE FAE E DE D F E D E E F E E BE F BE E E AE BB AE A AE F E E A E F EBF E B E E DE D F E D BE E BAE E E A E F E A E A E E F E D BE E E F AD ED BE F E BB AE D BE E E F AD E E DE D F E BAE E F EB DEA BA E AD AE D E DEBF A E DE F E E A A E AE E A E F E EDE A AEF E FBE D E F E A EDFEA FAE FAE E DE D F E EDFA E E BAE C E E BE F BE BB AE A AE E F BE BF ADABE F E F EDFEA FAE FAE E DE D F EAD BE F E BE F BE E E AA AE E EF E F E B DA E E DE D AE E E BE F BE E A A ED F AE BE F BE E E ED D AEF E D D A E E A E FBE D E EDFE FAE E DE D F FA BDA E BE F BE E A A E F D AED A EF ED E F DFE B F E BE F BE E E AE BB AE E AE DBE E BF ADABE D BE FED D AE D BE E E

95 B B E E A E A D AEDF E F BE E E AE BB AE F E B A AE DE B B A E A FE DEB A E F E A E DE F E E A A E B A E DE D D A E F E A E E E E D D A ED A E F E F EC E FB E AA E A E E D A E AEDFBB E D EF E E F E F E F E A BE EB E AAD AEF E D D B E A E DB EBF E EB EDAA F E EF E F E AE E FBE B F AEBF EB E D E E E D EA AD E F D AE A EFA B E E DE E D E F E DE E E EAD AE F E BF E D E E DE F E E A A

96 AB C D E E FD E D E D C BF E D B D C D D C D F D B D A B D D D D E B E D C BF D F D D B D D B F E E B FF

97 D

98 F

99 AB C D E E FD E D E D C BF E D B D C D D C D F D B D EB B D D D D E B E D C BF D F D D B D D B F E E B FF

100 D

101 F

102 AB C D E F DD F F D B B EB F FB A B F D B D D D D D D E E D C BF B B F E D D D D F F D

103 D

104 F

105 AB C D E A B EB F FB A B F D B D D D D D D E E D C BF B B F E D D D D F F D

106 D

107 F

108 AB C D E E C B EB F FB A B F D B D F D B D D D D D E D D D F F DBF D F D F F E D B F D D F E F D F F F E D C BF B B F E D D E FD C F

109 D

110 F

111 E DA E E A A BE F BE A BE FE E D AED A BE E EDE A E BE E E E E D E D E AED E E A D AE F E EB F E E B F BE A BED E AAB B BA E AD AE D D E F E DE AE E F BE E BD B E EB F E A D AEF E D A F E F BD AE E DE BB EAF D E E E D A F E BD AE BEAF F B E BF A E D A B E E D A F E E B EAF D E E E AA E F EDE D AE A E F E BE D D BE E AD AE DEB F E D EC F B E EDE A E A E F E BAE F A E D E BEA BE F D BE D BE F D AE BE D D B E BE A BE E D BE D AE BE DBA B E BEB A BE FE E BE DA A B E BAEF E A E F E F EB BE A BED A A F BEBF E BE F BEAF D BED B E F E F EB E E D BE D DA E DE B E E A E A E BE A BE AE DE F DA E FEB BA E F AD E E DEBF F A EDE A EDBB ED E BE D D BE D DA BE F BEA BE F E DE D A A E F DA E DE D D E E E FE E B DB B E E D B E EB E E E D BE BE D D BE D DA B E E BAE ED E BF BE D E BE A D B E AD AED E BED A BE DF E D E D D F D E AEF E A F EB F E FE E AD A E D E BAE E ED A BE D AE E E ED E A E D B E E D E D E EAD AE F EA D A AE A E DE D A A E F DA E F BE A E DE D D E E E BAEF ED A BE F E E F D E E E AE F E E E E F E A D EF E A E D A E E EDFE E FEA B ED E D D A E D A BED A D E D D F D E FD AE E F E BAEF ED A BE A AE F D B E AEDFBB EFA B E D BEF E EA BE F EA D A E DE D A A E F DA E AD AE BAEF E A E FB E BA AF E E DE A E A D F D E FE A F E E FE E AE FE D AE E F E E AA E A E BAEF E A F EB F ED EF ED A E FBE D E F E BE BE D A BE D E E E D AE F E E EB F D A E BE A D BE B A AE F F BE ABE F F E A A E F E DF D AE DBE E ABEB D B E B BAD EDFEA D A A E AE A E FB F E E D BE D E

112 DA AE AE D ED E D A ED BE FB F BEA ADA B EDF F E A A E F E BAE D EBF E E D E F E BAD A E D E AED E AE A E F E DEBF D ED D AEF ED A E A E A EBF E E E E A D D AE DE BB DA E FEA E D E AED E AE BF A E BEDFE AE BE BE E DEBF D E ED D F BE E F ABE DAF B E BA F AF D AE B E AE D AE A E A BE D E E AED E AE F EDFA BE D E D E AE D E E AED E AE AF EDEB E E DB E F E A E B E AE A E E F AE E B A B E C E D D E E BB E E EA F E E E F E F AE D A E E F E AED E AEA BA E BE F BE E B A BEBF E E D E AE AE BE E E D A A E A E E F E F BEBF E E E E DE D D E AE A CD BE AA E AF EF E F E D E E A A BE F BE D AE E E A EDE A E A E BEB AEDFBB ED A BE E BE BED DF ED BA BE D E BE A D E AE D E BE F BE F D AED FA E E E F DF E B BE F D AEA D A E BE D D BE D DA BE F B E A D E C A E E D E E F B EDE A E D AEBF E DE E AAD AE E DBB E E E E E B BE F E E D E E D E EDE E A E D AE D E F E D BEF E EA B EBF E F E D E A EBF E BE B BE B A B E E F EDE BE A E BE BE EBF D E F E E D E E DEBA F AF E F E FE DE E C E E E BBF E E AF E E E AED EDE A E FA B E E E EB A E E D B E AD AE A E E E DFA F E FE D E BAD B E BE BE AE A ED FA BE E D E E D C BED D F BE BE B BE D AE BE F BE BF ADABE A E AE BF A E A E BE E D E E A BE E E EB D A E C C E B BEBF E EB F E E B D A EDE A E E E E E D F E AD E E D E E BE B BE AE A EB A BE F E BE BBD BE ADF ED BEF E B A E BF E BE F BED D AE A F BE BE F BE B B

113 In Vivo Orally Active Small-Molecule Inhibitors of TNFα. Matthieu Montes 1*, Hadley Mouhsine 1,2*, Hélène Guillemain 1,2, Bruno Baron 3, Lucille Desallais 1, Gabriel Moreau 1, Aurélien Latouche 4, Nesrine Ben Nasr 1, Rojo Ratsimandresy 1, Jean-Louis Spadoni 1, Hervé Do 1, Patrick England 3, Jean-François Zagury 1# 1 Chaire de Bioinformatique, Laboratoire Génomique, Bioinformatique et Applications, EA 4627, Conservatoire National des Arts et Métiers, 292 rue Saint Martin, Paris, FRANCE 2 Vaxconsulting SARL, 66 avenue des Champs-Elysées, 75008, Paris, France 3 Plate-forme de Biophysique des Macromolécules et de leurs Interactions, Proteopole Institut Pasteur, 25 rue du Dr Roux, Paris 4 Equipe MSDMA, Centre d études et de recherche en informatique et communications, EA 4629, Conservatoire National des Arts et Métiers, 292 rue Saint Martin, Paris, FRANCE *These authors contributed equally to this work #To whom correspondence should be addressed : Prof Jean-François Zagury, MD, PhD, e- mail : zagury@cnam.fr ** We thank Prof Jain for providing Surflex-dock. HM and HG are recipients of CIFRE fellowships from ANRT. NBN is a recipient of a fellowship from CNAM. LD is supported by a fellowship of the DGA. ABSTRACT TNFα is a key cytokine associated with chronic inflammatory diseases, whose direct targeting by protein biotherapies has been an undeniable success of biotech companies. In a quest for equally effective small molecule drugs, we identified by in silico screening a new family of compounds that bind TNFα and inhibit its activity in vitro and in vivo, orally and intraperitoneally. This breakthrough could open avenues for innovative TNFα-targeted therapeutics.

114 Tumor Necrosis Factor α (TNFα) is a key cytokine of the immune system mainly involved in inflammation, host defense against infections, regulation of the immune system and tumor regression [1, 2]. The overproduction of TNFα has been notably associated with chronic inflammatory diseases notably rheumatoid arthritis, Crohn s disease or Psoriasis [2]. Protein biotherapies targeting TNFα have been a revolution for the treatment of chronic inflammatory diseases with anti-tnfα monoclonal antibodies (infliximab, adalimumab) or soluble receptors of TNFα (etanercept). However, these biotherapies present several drawbacks including side-effects [3], treatment resistance, and prohibitive costs (up to $20,000 per patient per year) [4], that could be addressed by small molecule-based therapies. However, despite many efforts [5-10], no small molecule drug targeting TNF has been released so far. We report here a new family of small molecules that inhibit TNFα in vitro and are active intraperitoneally and per os in TNFα-linked animal models. These molecules could represent lead compounds to treat chronic inflammatory diseases. We have used high-throughput in silico screening approaches to identify small molecules targeting a binding pocket of the TNFα identified by He et al [9] in 2005 (Supplementary Figure 1). We carried out a hierarchical in silico and in vitro screening of a collection of 700,000 commercially available compounds using the TNFα structure as a template (Supplementary Figure 2): this lead to the identification of benzene sulfonamide derivatives represented by compound 1 (Figure 1). First, we demonstrated that compound 1 could enhance in a dose-dependent manner the intrinsic tryptophan fluorescence (ITF) of TNFα, confirming that it could bind directly to the TNFα trimer (Figure). Interestingly, the previously described compound SPD304, active only in vitro, induces an opposite ITF quenching [9], suggesting that the TNFα binding mode of both compounds are different. Second, compound 1 inhibited the induction of apoptosis by TNFα in the L929 cell line with an IC 50 of 12µM without exhibiting any cellular toxicity at 100µM (Figure 3). The targeted pocket is identical in both human and murine cytokines and accordingly, we found that compound 1 inhibited both human and murine TNFα with similar IC 50 s. This aspect is important to explain why our compound could be directly tested in mouse models. The in vivo activity of compound 1 was assessed using two different TNFα-linked murine disease models: the LipopolySaccharide-DGalactosamine (LPS-DGal) septic shock assay [11] and the Dextran Sulfate Sodium (DSS)-induced colitis assay [12]. As shown in figure 4, in the

115 LPS/D-Gal septic shock mouse model, compound 1 exhibited a fully protective effect with an intraperitoneal injection of 1 mg per mouse (p< ). In comparison, etanercept, one of the reference anti-tnf protein biotherapies, showed a similar protection at 600 µg per mouse. In addition, we observed a fully protective effect with a per os administration of 5 mg of compound 1 per mouse 8 hours before the induction of the shock (p<4.10-3, Figure 5). These results demonstrate that compound 1 has a highly protective activity in vivo and suggest that it has an acceptable level of oral bioavailability. In the DSS-induced colitis assay (Figure 6), compound 1 also exhibited a protective effect, as repeated intraperitoneal injections of compound 1 resulted in the maintenance of mice colon lengths compared to the controls (p< ). Using an in silico/in vitro screening approach, we have thus identified a novel compound that inhibits TNFα directly and is active both in vitro and in vivo. Other small molecules targeting TNFα have been described previously [9, 10] but, to our knowledge, this is the very first protein-protein interaction inhibitor that provides significant protection in animal models. In the septic shock assay, the effect of compound 1 was comparable with that of the reference anti-tnfα soluble receptor, etanercept. In the colitis model, our compound was efficient (no shortening of the colon) but less significantly than in the septic shock model. This can be explained by the two following reasons 1. the compound was administered only once every three days in the colitis test, and 2. unlike in the septic shock model, the TNFα pathway is not mandatory in the colitis model. The compound described in this report constitutes a considerable step forward for the development of alternative therapeutics to the biologics currently used to treat chronic inflammatory diseases, with its demonstrated oral availability that would make it a first-inclass. As shown by the undeniable success of anti-tnfα protein biotherapies, TNFα is an attractive drug target for the treatment of chronic inflammatory diseases. Small molecule drugs targeting directly TNFα would potentially provide several advantages over biologics: first of all, easier compliance and lower production costs, but also the possibility to stop treatment immediately in case of adverse side-effects and an additional class of therapeutics to fight resistance arising in patients. Our work shows it is feasible to develop bioavailable small molecule anti-cytokine inhibitors that could be efficient alternatives to the powerful biologics used since 15 years.

116 EXPERIMENTAL SECTION Materials, cell line and mice Compounds were obtained from Chembridge (San Diego, CA, USA). Dimethyl Sulfoxide (DMSO), Lipopolysaccharide (LPS), Thiazolyl Blue Tetrazolium Bromide (MTT) and Cremophor EL were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Human TNFα was obtained from R&D Systems (Lille, France). Dulbecco s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin, L-Glutamine and Phosphate Buffered Saline were obtained from Pan Biotech (Brumath, France). Absolute ethanol was obtained from Prolabo (Strasbourg, France). Actinomycin D and D- Galactosamine were obtained from Fisher (Illkirch, France). Dextran Sodium Sulfate (36,000 50,000) was obtained from MP Biochemicals (Illkirch, France) L929 cell line has been grown in the Laboratory for years. 7 weeks-old female Balb/C mice and grs female C57BL/6 mice were obtained from Charles River Laboratories (L Arbresle, France). Mice used in all experiments were handled according to the guidelines and approved protocols of the Direction Départementale de Protection des Populations, Paris, France. In silico screening Structure preparation. The binding site has been defined at 4 Å around the co-crystallized SPD304 ligand in the structure of the TNFα dimer (PDB id: 2AZ5, Supplementary Figure 1). Hydrogen atoms were added using Chimera [13]. Compound collection. The compounds Chembridge screening compound collection was retrieved from After an ADME-tox filtering using FAF-drugs2 [14], compounds were selected to constitute our commercially available drug-like compound collection. Structure-based virtual screening. Molecular docking was performed using Surflex-dock version 2.5 [15]. Surflex-dock is based on a modified Hammerhead fragmentation / reconstruction algorithm to dock compounds flexibly into the binding site. The query molecule is decomposed into rigid fragments that are superimposed to the Surflex-protomol i.e., molecular fragments covering the entire binding site. The docking poses are evaluated by an empirical scoring function. Ligand-based virtual screening. Ligand-based virtual screening was performed using the 2D/3D similarity search methods implemented in the webservice provided by Analogues identifications were performed using 2D and 3D similarity

117 search with a 60% 2D or 3D similarity cut-off. Compounds were selected for experimental tests after a careful visual inspection of the retrieved analogues. Measurement of TNF- intrinsic fluorescence. All samples were brought to 10 mm phosphate buffer, 0.05% Tween20, 1% DMSO. Fluorescence readings were made with a Quantamaster QM4CW luminescence spectrometer, (Photon Technology International) by exciting TNF at 290 nm and measuring the emission peaks at 322 nm. Compound 1 inner filter effects were corrected using absorbance measurements of compound alone solutions at 290 and 322 nm. Neutralization of cellular TNFα induced apoptosis. The biological activity of TNFα was assessed by its apoptotic activity on L929 cells. The neutralization assay consisted in mixing TNFα with the potential inhibitor, incubating the mixture for 2 hours, and then testing its apoptotic activity. 80 % confluent L929 cells were plated in flat bottom plates at 4x10 5 cells per well in 100 µl of DMEM medium containing 10 % FBS, 2 mm L-Glutamine, 100 U/ml Penicillin µg/ml Streptomycin and incubated for one night at 37 C, 5 % CO 2. TNFα (150 pg/ml), Actinomycin D (4 µg/ml) and the compounds at different concentrations (ranging from 100 µm to 0,1 µm) were mixed in 150 µl complete DMEM medium in U-bottom plates. After two hours incubation at 37 C, 5 % CO 2, 100 µl of the mix was added to the plated cells and incubated at 37 C, 5 % CO 2 for 24 hours. A negative control was also used by incubating TNFα with no compound. Each point was done in duplicate (the test was highly reproducible). Supernatants were discarded and 100µl of MTT at 0,5 mg/ml were added to wells. After two hours, supernatants were discarded and 200 µl of DMSO were then added. Plates were read at 570 nm with a spectrophotometer providing the optical density (OD) of each well. The percentage of neutralization (%Neutra) by a compound was calculated as follows : %Neutra = ODcompound ODTNFα ODcells ODTNFα 100 An IC 50 could be computed from the percentage of neutralization for each compound.

118 LPS/D-Galactosamine induced lethal septic shock 7 weeks-old Balb/C mice were injected intraperitoneally with 100 µl of a solution containing A. 25 % Cremophor EL, 25 % absolute ethanol, 50 % PBS and various doses of compound 1 (1 mg to 0.25 mg) or B. 25 % Cremophor EL, 25 % absolute ethanol, 50 % PBS alone (compound 1 vehicle) or C. 0.6mg of etanercept or D. PBS alone (etanercept vehicle). Eight hours after, the mice received an intraperitoneal injection of 0,1 µg of LPS and 20 mg of D- Galactosamine in 200 µl of PBS. For each condition, groups of 8 mice were used. Mice survival was monitored during 48 hours after the injections. A similar test was done per os, by force-feeding 7 weeks-old mice with 100 µl of a solution of DMSO containing various doses of compound 1 (5 mg down to 0.5 mg) 8 hours before receiving an intraperitoneal injection of 200 µl of PBS containing 0,1 µg of LPS and 20 mg of D-Galactosamine. A control group (vehicle) was force-fed with 100 µl of a sonicated solution of DMSO alone 8 hours before to be injected with 200 µl of a LPS/D-Galactosamine solution. Mice survival was monitored during 48 hours after the injection of LPS/D-Galactosamine. Colitis induced by Dextran Sulfate Sodium (DSS) g female C57BL/6 mice received 3% DSS in water for 5 days followed by 7 days of water. At Day 1, Day 4, Day 7 and Day 11, mice received an intraperitoneal injection of 100 µl of A. 1 mg of compound 1 in 25 % Cremophor EL, 25 % absolute ethanol, 50 % PBS or B. 25 % Cremophor, 25 % absolute ethanol, 50 % PBS alone (compound 1 vehicle). A control group (water) did not drink any DSS and only received PBS i.p. At Day 13, mice were sacrified, and their colons were harvested and measured. For each condition, groups of 8 mice were used. Statistics For the LPS-DGal septic shock assay, one-tailed p-values were calculated using Fisher s exact test. For the DSS induced colitis assay, the equality of variances was investigated by a F-test and was rejected for compound 1 vs PBS at 0.02 level. Thus, to compare the difference between the means of each sample accounting for unequal variance, we used a two-sided Welch t-test at a 0.05 significance level. For Compound 1 vs PBS the t-test is statistically significant (observed value t=3.07, df=23.4) with p< (95% Confidence Interval [0.166; 0.848]).

119 REFERENCES [1] K. Pfeffer, Cytokine Growth Factor Rev 2003, 14, 185. [2] B. B. Aggarwal, Nat Rev Immunol 2003, 3, 745. [3] R. M. Fleischmann, I. Iqbal, R. L. Stern, Expert Opin Drug Saf 2004, 3, 391. [4] P. P. Sfikakis, G. C. Tsokos, Clin Immunol 2011, 141, 231. [5] H. Choi, Y. Lee, H. Park, D. S. Oh, Bioorg Med Chem Lett 2010, 20, [6] C. H. Leung, D. S. Chan, M. H. Kwan, Z. Cheng, C. Y. Wong, G. Y. Zhu, W. F. Fong, D. L. Ma, ChemMedChem 2011, 6, 765. [7] F. Mancini, C. M. Toro, M. Mabilia, M. Giannangeli, M. Pinza, C. Milanese, Biochem Pharmacol 1999, 58, 851. [8] R. Alzani, A. Corti, L. Grazioli, E. Cozzi, P. Ghezzi, F. Marcucci, J Biol Chem 1993, 268, [9] M. M. He, A. S. Smith, J. D. Oslob, W. M. Flanagan, A. C. Braisted, A. Whitty, M. T. Cancilla, J. Wang, A. A. Lugovskoy, J. C. Yoburn, A. D. Fung, G. Farrington, J. K. Eldredge, E. S. Day, L. A. Cruz, T. G. Cachero, S. K. Miller, J. E. Friedman, I. C. Choong, B. C. Cunningham, Science 2005, 310, [10] D. S. Chan, H. M. Lee, F. Yang, C. M. Che, C. C. Wong, R. Abagyan, C. H. Leung, D. L. Ma, Angew Chem Int Ed Engl 2010, 49, [11] M. A. Freudenberg, C. Galanos, Infect Immun 1991, 59, [12] S. Melgar, L. Karlsson, E. Rehnstrom, A. Karlsson, H. Utkovic, L. Jansson, E. Michaelsson, Int Immunopharmacol 2008, 8, 836. [13] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, J Comput Chem 2004, 25, [14] D. Lagorce, O. Sperandio, H. Galons, M. A. Miteva, B. O. Villoutreix, BMC Bioinformatics 2008, 9, 396. [15] A. N. Jain, J Med Chem 2003, 46, 499.

120 FIGURES Figure 1: Structure of compound 1.

121 Figure 2: Intrinsic Tryptophan Fluorescence of 0,5 µm TNFα diluted in Phosphate Buffered Saline in the presence of DMSO alone or compound 1 (5-100µM) in DMSO.

122 Figure 3: Compound 1 inhibition of TNFα induced apoptosis in L929 cell line. Data represent cell survival in presence of different concentrations of compound 1. IC 50 =12µM.

123 Figure 4: Effect of compound 1 in an in vivo murine model of LPS/D-Galactosamine induced septic shock. Mice survival after an intraperitoneal injection of LPS/D-Galactosamine and different doses of compound 1. Groups of eight mice were used. Values are mean ± s.e.m.

124 Figure 5: Effect of compound 1 in an in vivo murine model of LPS/D-Galactosamine induced septic shock. Mice survival after force-feeding with different doses of compound 1 and an intraperitoneal injection of LPS/D-Galactosamine. Groups of eight mice were used. Values are mean ± s.e.m.

125 Figure 6: Effect of compound 1 in an in vivo murine model of DSS induced acute colitis. Colon length after 5 days of 3% DSS followed by 7 days of water. Compound 1 was injected intraperitoneally every three days as control groups. Groups of eight mice were used. Values are mean ± s.e.m.

126 SUPPLEMENTARY ONLINE MATERIAL Supplementary Figure 1 : Binding pocket defined for the structure-basedvirtual screening procedure. The binding pocket was defined as the region 4 Å around the co-cristallized ligand SPD304 in the TNFα structure identified by He et al (PDBid =2AZ5). The «protomol» that constitutes the conformational space, that was explored with surflex-dock is displayed in magenta.

127 Supplementary Figure 2 : compound 1 identification protocol. 1st step : A collection of 700,000 drug-like commercially available compounds was screened in silico. Molecular docking was performed using Surflex-dock version 2.5. A visual inspection of the top scoring compounds was performed in order to select a 1000 compound hit list for experimental testing. 2 nd step: The compounds composing the hit list selected were purchased from the chemical supplier. Their inhibitory activity was evaluated in vitro on human TNFα. These in vitro experiments led to the identification of top hits: active compounds with an IC50 between 1 and 100 µm. 3 rd step: Using ligand-based virtual screening methods, we searched in our compound collection for analogues of the top hits identified after step 2. Up to 100 analogues were found per top hit. 4 th step: The analogues were purchased from the chemical supplier. As in step 2, their inhibitory activity was evaluated in vitro on human TNFα. Their activity was also confirmed on murine TNFα in view of a further evaluation on mice models. The 10 best compounds after these 4 steps were selected as drug candidates for in vivo evaluation on mice models. 5 th step: The in vivo evaluation of the drug candidates was performed in the TNFα-dependent septic shock model triggered with LPSgalactosamine using two different administration modes: intraperitoneal injection and force feeding. After this step, 2 compounds sharing the same structural scaffold were selected. 6 th Step: Using ligand-based screening methods, we searched in our large compound collection for new analogues of

128 the two best compounds identified after step 5. Up to 1000 analogues were identified and purchased from the chemical supplier. As in step 2, their inhibitory activity was evaluated in vitro on human and murine TNFα. The 9 best compounds were evaluated in vivo in our murine septic shock assay by force feeding as described in step 5. The best compound identified after the 6 th step is compound 1.

129 C E B E EA F ED BE E ED BE DE E D D F B E FAEB E E A AE DFE EB FBE EA F E E F EB E E E D B EB E A E AE E F E E C E E E E F B E A B F E FE E BA E E A E DBE E E D E F D A E D E BE F E B B E EDE A E A E D E BE BBD BE A E F E D AE E E F A E E D A B EBD BEA A E F D E F EF E E E E E D E E E F D E DE E B E BAE B ED E E E F E F EF E E B D E EF E A A E A EDFE EB A F EDE A E B E E DEB F BED BE A E E E D E E A D A D E A E E E F E AD A E AA E A A EDEDFBB E A E B ED E D BA DA E E E E D E E D E E B E EDE EF ED A A E DFA AE A A E AE EB E BB EF E E A A E E B A E D E E AE A A F E BA EDFBB EBF E BE BE E A E E DE E D D F B EF E F DFE B EDE A EA F E E F E DFA BE A A BE F BE D AE E DED AE A EA F BE FB F E D A D A E DEB E A E E E A F FE ED A E D E E D ED EF E AE D D E EF ED A E E E AD A E F E E EB A F E E BAEDFBB E D E D BE BE BE E A E E F E DEB AE BEB DA E F E F E E EB A F

130

131 FB

132

133 E FB E D FDA E E A BE E D E EB EA AEF E D E AD A E D BE DE B E E D E FE BBFBE E A E F E D A E E BAE E AE AD AE E D A E BE ABE ABE AE D BE BE A BE E D EFA B BED E A E BE BF ADABE A DF E BE E D DA EBF EF E E A A E E BAED D FE F EA FA BE BE A BE AF BE F D AE D E BED ABE FE BE B A ABE AB EC E FB E BE A BE DB BEBF E DEBA F AF E E DE A E E AA AE A EF E F E BB AE D E FBEBF D A E BE F EA BE E A B E F BEB AE DB BEBF E DEBA F AF E E DE E FE FE D E E E AA AE E A F E BED A BED D AE BE A BE BE E D E E D A E D E BE BE BF ADAB E AD BE BE B BA A E B E E D D E BED A BE D E B A BE D BE EB A E E D B E D BE A D AE BEB BE D E BE F BEDE A E FBA E AAD AE E E DE A E BE A BE EB E E A F E DE E B E E E E DE B E E A E FEB A E E D B E E A A E E D EF E D E D A E BE A BE E E BAEDFBB ED D F E AD AE E D B E FE E E BA F A E E DEC C E BE DA BE BEB A BE E D B E E AD BE A F BE AD AE DBED D A BE EA FBE BED A B E E BAE E BBD E E E E A E D D ADA E FE A F E D AE DE D B ED EB E D E E E DBB F EBF EF E B E EBA F AF BE F E E A F E BAE F AE FA B ECFE D AE FE AE EA BE E D F E F EA E A E AE FE E EBA F AF BE B BE F E AD BE B EA FBE BE B BE BB BE E F AE DBE A EA BA B E E AD AE E BBD E EA F E BE BA BE F E B E BEBA F AF BE E A E E B E BD BE E D E BE D BE D D B E BE BF ADABE D BE AE BEA D BE A BBD A B E E A E D E BEB BA BE AF B E BE DA BE AE EB A E E D B EDE E F E E FBE D EB AE D AEDBB BEDF E F BE BB A E AE B AE F E BEB A BE E D B ED D AE E FBE D E F E E E F E E BE B B E F E F E E A AE F E F EBA F AF BE F E A E BE AD AE B F E A F F BE DE F EBA F AF EB F EB AE E BE F BE BB AB E BE BF ADABE BA AEA FA BE ED EC E FB EF E F E B E E DEC C E DEC C E D AE A E F E F F E BE FB BE BA AE E A E BEBF E FA BDA E BE A F BE D FDA E BAEF EDFA EDB AE DA F E E D FDA E BE A BE E D E E D A F E BB AE BA EF E A F EA BEFA B E D A E F BE E DE F E E E D A E FE DA E D A BE D BE DE D F E D FDA E

134 A D AEDFE D A F E BB AE BAE D E E BAE DBEA F F BE D AE A AD E AE DFA BED A DA BE AE F BE A E B B EA BE F E C E FE E E B EDFBB E A BBD AE D F E DE D D A E F E A E EB E E E E B F E F E F E F E A ED A E E F E D A E FE DA E D A BE E DE D F E DE F E E A A EDE D D AD E E F EF E E F EDF E A BE A BE EB E AE E B A E BE D ABE EB E A E BE B B E E FBE E F BE D B E BE BF ADABE A FBEB AE E E F BE D D B BE F DF F E A A E F E A E FE E DE A E B EBF E E D E FB F E B A E D DA E E BE A BE E EDE BE DE B E E E F E B E A F ED D AEF ED A A E A E AE E D E E D E D BE BE BE E E B A F E AE E A E E DEB F B E E B E F D AE BA AF E BE DB BE E F DF E A D A ABE BE D D BE D DA BE F BED A BE D E E D E ED ED E DE AA DAF E AA ED DA E A E D AE F E D E E D AE F E BE A BE BB A AE A ED B E E D E E FAE AA E D A EF EB FA E AD AE E A DA E E F BE F BE E B EA D FA F

135 D E E E E C E E E E E E E E BAE E F E A EDEB BA DA E E B E E E E C E E E E E EC E E E E DA BE E DA D EA BEA E F E B E E DA D E D EB A B B E D EB EBA DA B ED E FAF E A B E A B F D B A E E E E E E E C C E E C E E F E B E A DA E E AD ED E FADA D E A B E A A DB E E E E E E E D F E E F E B A E EA E F AE D D FA D E D A E A E A E E E E E E E E E E C E E E E BE E D E F E B E A A B F BA B E E E E EC E E E E ED E FA E E EA F FAEB E A A B F BA B E E E E E E FA D E F E B ED E A E D BA F E E E E E E E CE B E A ED E AFD EB ED F D E A B F B F A B B D D E E E E E E E E E E E E E E E B A BE E DA E F E B ED E E A B F BA B BA B B B C B F B BD B F A BA B B B BD D BA B E E E E E EC F E A BED EA E DFB BE E EB F A ED E E D A E A B F BA B B B D B D E E E E E E E E E E C E E ED AED E D A E EBA F AF DB E F E B EDE F D E E B A E D B BA E E E E E E A B B D A B A A D B C B F B E E E DA F E AE F E E E E C E E E E EA E B A E B A E E D D D D E DAD DB B E A B F A D D BA E E E E E E E C E E E E E BB BB EA E D E E E A E B AEA E A E D A E DA B E A B F BA AB D E E E E E E FE ED AE E F D A B E E E FE E F AE E A B E E E FE E F AE E B A E D E BA D AE E E FE E F AE E DB E E BA E E F E E F A E E AA DB A B DB A A DAD D A E FB E E FE E F AE E AA DB A B DB A A DAD D A E E E FE E F AE E B B E C E D B E E F E E F A E E AA D B F AB DAD DB B B F D D D D B A A E D E E FE E F AE E AA B DD E B E E FE E F AE E AA B B A E C E C F E CDAD E A E E F E E F A E E AA D B F AB DAD DB B D A A A E C E E E E E E E E A E A BED E DAD DB BE A E DA B E DB BE E F BED E F E D DA B E F A B F AD B D E E E EC AE EC F E E E FE E F AE E AA D A F AB A E DA D E D E BA AFA E E FE E F AE E AA D AFB D

136 E E E FE E F AE E AA B D F E EC E E E E DAF D E F ABEDBEB F BE E E F BE EA E DBAE E D B E A B F EB AB A D E E E E E E DAF D E F ABEA E F B E DAF D E F A E F BE E D EA D B EEB AB A D A E E E E BE D E E FE E F AE E AA A B E E D E E F E E F A E E AA D B A B B A E E B A E E F E E F A E E B D B B A D B E E E FE E F AE E AA E E E EA EDAA FA ED ED A A E E E B E A D F EB B BD F F D B F A B E E E E E E E E E F DA BE E B E E A E A A B E A D BA B B E E E E E E E E E E E E F D E A ED E ABE D AE EA E D A E E E D BE E F E B E A B F B F A B B D A E E E E EC E E E E E E E E E D A DB E D E B EEB A A A E E E E E D AB E E F E E F A E E AA B D B D A D E E D AB E E FE E F AE E AA F B A B BA B D B F B D A E EC E EDE D E D FD ED E DA E BA E E A F A EA E A E D E E F B E A B F B F A B B D A E E E E ECE E E E E E E E E A E E DA E E F E E ADA E A B F B F A B B D A E E E E E E FE E F AE E AA D A D A A E C E E FE E F AE E AA E E E E E E E E E E E E A ECDADE D EDE FA DB ED D E E E D F D EBA F AF B E A B F BA E E E EC E E E E E E E CE E EC B A E E D E D E A F AF E E DABE B E E FA E D EC EDAE F D EC B E A E A B F B F A B B D A E E E E E E FE E F AE E AA A B DAD BF A E E E FE E F AE E B E FD D E E FE E F AE E AA B FD D E E E E E F D E C E F E A BE E F E B E A A DB E E E E C E E A D F A A A E D D E F E B ECDAD DB E E E A E E E FE E F AE E AA D B B F D A E E E E C E E EC E E E E AD ED E FADA D ED D BEA E BA DA E B F A ED E D A E E F E B ED E AEB AA B E D B D A A E E E E E E E C E E E C E E E DB ED D E E B E DA D E E D E BA E D BE E F E B E E E FD ADA ED E FD A ADA E D D A DA E E E F E DAD DB B E A B F C B A B A E E E EC E E E E E E E E E E A E F D E A D A BE EB E E E F EA E D D E A DA ED E ABED DA BEA E F E B E AA D B A E E E

137 E E E E E E E E E E E F E D B E AFD EB E E B EBF E E B A ED DA B E A B F D B A E E E E E D D E E FE ED AE E AA B A F A E E E BA AC B A DA D E E E D E BB E E E E C E E B D E D D E DAA EB D E A A BA B D C BA E E E E E E E E E D A E E E AAE B E B A B B D B E DB A EC E E D E D E A E E E E E E E C E E EC E E D E D A E D E A B F B F A B B D A E E E E E E E E E B D B B F BA BA E E E E E E E BE E E E E E E E E E A D F A B E E F E D E F B BE E E E E E E E E E F A B C E E DBA E E E E B E E E E E E E C E E E C E E E E E A D BE E AFD E E E A D E E A B F B F A B B D D E E E CE E E E E E E E E E DA E A E ED A ED E D DA EFB E E FD A EBA F AF BE EA E A ECDAD D ED E D E A F AF D ECDAD DB E A B F B F A B B D D E E E E E E D DB EBA F AF D E A B BE E AFD EB E A B F D B A E E E E E E E E E E E E E E EB D DB E CEB D E E A ED E ABED DA E A EDE D A D E A A E A D A E A B F D B A E E E E E E FE E F AE E AA B B E E E E E E E E E E E DA E A E D D E E D E BA E EB A E E E E E E E E E E E E E E A E F D E D A E D E F BE E A E DB BE E D E A A E B E E E E E E E EC E E E E E E A E D E EA E A BE ED A E A E A E E E E E E E E E E C E E E E B A DED D B B E E A E D D A DA BE E E F D EBA F AF ED E D E B B E A B F D B A E E E E E E E E E E C E E E E D EB A DED D B B E E D ED A A E BEA E F D EBA F AF E A D F EB B BD F F D B F A B E E E E C E E E E E E C E E A E D E D E E D DB E AFD EB EFB E D B D E E A E A B F BA A FF B A B F F A BA A E E E E E E E E E D FDA E ED E E A DB E A D E E AB B F A B E E E E C E CE E E E E E E F E E ABE EA E D F DA E E A F D E B D A ED E BB D A EFB E CE D AE A BA B E C B A B A A A E E E E EC E E E E E E E D A E AB E DBF BE E F ED E BB DA E E E DBF BE E F E E A B EB AB E E E E E E E C EC E E E E E EE D A ED EC BB D A E A BE E BB E D E A F AF ECDAD DB B E A A B E E E E E E EC A A ED E E A E B E B A B E E E

138 E E E E E E E E E E E D A B D A DB E E E A E E D E D E A A E A B E B D A B BA E E E E E E E E E E E E E EC ED E DB A FB E A E ED DA E E F EAD A B E EB AB E F A B F AD B D E E E E E E E E E E E E E DBB D EBF BA F AF DB E AFD EB EA E D E A D DA E D B E A B F B F A B B D D E E E E E E E E E E E E E E E A E A D A E A BE E DBAEA E F D E AA A AB E E E E E E E C E E E E E DAD A E B F BE E DB B ED D B BE E A BE B E E D EB A E A E A B F A D D BA E E E E E E E E E E C E E A ED D B BE E D E B A BE A EB D E B A B E E E E E EC E E E E EDE FA E D BE A E E A ED E B D E A E D A BED EA EBF F ED ED B E A B F BA AB E E E E C E E E E E E E E EDFA DA ED E AE A A E E A A D EB D E F EB A BE E A B E A B F BA AB D E E E E E C E E E E E DBAE A ED E BFD DA E E A E E ABE A E E A B F A D D BA E E E E E E E C E E E E E E FA E A DBE E F D E D E E A B E E D BA E E E E E E E E E A ED E DB E A E EA E A E E A D E EB A B E F A B F AD B D E E E E ECE E E E E E E E E E F D E DB BE EA E A D A E A EA E E E A E E ED E BA E A ED D EE D BA E E E E E E E E E E C E E D E E A EBF D E D A BED E A E E E DBBE EDEB D E F D E A E A A E E E E E E E E E E E E E E A DA E E DAD A E B F BE E A E BA F AF EFB EBF AE A E D E A EB F ED EBA F AF D E DAF B E B B D B BA B E E E E E E E E E E E E E A E E E DAD A E B F BE DB E E D E D BE DBB DA E F A E E E EC E E E E E E E E E E E E A D E EB A BE A E A E DB E EA E A DA E E A EB F E B DA E DA E F A B E E E E E E E E E E E E EDE A E DB ED A E E E CE DA EA DA BE EB D EBA F AF D E DAD DB B E DA EA E ED A EB A B E A B C B F A E E E E E E E E E A E F A D E AD AE B F BE EB F E B DA E F A B F AD B D E E E E B E E FE ED AE E AA B E E E E C E E E E E E E DFBB D E E F A B E A E E E E E CE E E E E E E E E E A ED D EA E D F D E A D A B E A B F BA E E E E E E E E E E E E E E D AE E DA D E A E EA B D E B D A EB D EFB E DA E A B E A B F B F A B B D D E E E E E CE E E E E C C EC E E E EDEB BA EA EB AE FDB E D BE AD EA EDE A E E EA B D EBA F AF E A B F A D D BA E E E

139 E E E E E E E E E E E A DA E E E D E A BE EF F A E A D E DB E E AFD EB E A B D B A E E E E E E E C E E E ECE E E B E DB EB E DA E A BED E A A D ED A D E F E F BE B E E AFD ED E E A EB E E E E E E E E E E E E E E E E E F D E E A BE A EA F EBA F AF DB E AFD EB E A B D B A E E E E EC E EC E E E E E E EC ED EB E E AFD EB E E F E B E A BED ED DA B EEB A A A E E E E E E E E C E F D E A ED E ED A B E B F B D BA A A E E E E E E BA D A B D B E D E BB E E D B E DA E A E EE E E EC E E E C E E EC E E E EC E E D BEA E D F D E A BE E F D EB D E A B F D B A E E E E E E E E E E E E E E E DBAE E E A EFB ED E AD E BA F A ED A E A B F BA E E E E E E E E E E E E E EC E EB D EBA DA BE EDFA DA E F D E E E E F E DAD DB B E A B F A D D BA E E E E E E F E F EDFA DA E E F D E EFB EDE F D EB D A DB EB D E E A B F D B A E E E E C EC E E E E FA DA E E EBF BA DA BEA E A BE EB F DA ED D E A E E E E E E E E E E EC E ED E AEA F E EBA F AF E A BED E B E A B F B B A E E E E E E E E E E E E E EC AED E D DA E EDE A ED A E E E E A B F BA E E E E E E E C ECE E C E E E E A ED E D E E E E F A E A B F B B A E E E E E E E E E C EDE A EFA E BA E D EBA F AF BEA E E E D F D E A B F D B A E E E E E E E E E E C E E F A D D E B DA E E F D EC D BE F DA E E D E DA D E D E E A B E A B F B A E E E EC E E E E ECE E C E E F D E D BEB F DA E EA E E EBF BA DA BE A E A B E A E E E E E E E ECE EC E E EC E E E D BEA E E A BE EB FA E E F D E D BEB F DA E A E E E E C EC E DA E EDE E E E A EA E A E A B B E A D F EB B BD F F D B F A B E E E E E E E E E DA E A E A E E FADA D E F E B E BA BA B E E E E E E E C E E E F A BAD AE A E D E E A E A E E E E E E D E EA E A BE A E B A EB D E A E A B F BA E E E EC E E EC E E E E E E E E E D E DA EA ED E ABEFB E E A E B D E B A EEB A E E E E E E E E CE E E E F D E EA E B BE E A EBA F AF B E A B F BA E E E

140 E C C E E E FADA D E F E E A E A D E D D E EB A BE E D E AD AE D F B E A B F D B A E E E E E E FBBE E F DA EDF E E F E E E A D A BF E E E E E E EC E E E E E E E E E E E E E E E E E E E D A BE EA E A ADA E E A E DAF DA E D B A A E E E EC E E EC E E E E E E E E E E D E B F BE DA E ED A E A A E A EDAEA E EBF D E E C E A D F EB B BD F F D B F A B E E E E C C E E E E E E E E E E E E D F DA BED E D E E D B A A E E E E E E E C E E FADA D E EA E D F DA E B FA E A D E E E E A B F A B B E E E E E E E FA E D E C EDE E A E EA E E E B E E E A B E A B F A D D BA E E E E E E C E F DB EDFA DA E B E E EBF BA AF ABE E E A E D B E A B F A D D BA E E E E ECE E E E E E E E E A E ED A BE E A E D BE EA B D E B ED DA EA E A E EA E DBBE E D E BA DA A E A B E A B F D B A E E E E E E E E E E E E F D E A E E A EB A BEFB EDE A ED A E A E DE B A E E B DA E A B F BA E E E E E E E E E E EC E E E ED DEA E E E EB F DA BE E A BED E F ED B E A B F B B A E E E E E E E E E E E E D FDA E EA E E AD E BA F A E A E E A D EC E E A A B D E E E E C E E E E E E E E E E E A D E EFB E C E A E E E E E E E E E E D ED E DBAEB E F A E E A D E A D A B EDEB E A A D ED D E A B F D B A E E E EC E E E E E E E EC B E A E DB E E E DBAED E F DA E E E BA DA B E E A ED E F A E E A B F A B B E E E E E E E C E E E E E DB EB E F A EA E AE A D E A D A B E A B F BA E E E E E CE E E E E E E E E E A B D E F E E A B E A B F A D D BA E E E E E E E E E E E E E B E DA E EBA F AF DB ED E D DB E BA DA BE E AFD EB E A B F A D D BA E E E E E E EC ECE E E E E A D E E DB E E E E DA ED E D BA E D DA E D E A E E E E E E E E E EDE E B FA E D A E ED D EFB E A E A EBA F AF B E A E E E E E E F A E D BE E AFD EB E A B F A D D BA E E E E E E F EA E DA BE E A F E D B EDE A D E D FDA E EB D E F D E DA D EB D EA B E A B F A D D BA E E E

141 E C E E E C E E E E E E E AFD EB EFB E A D E E D ED A D E A E A B F B F A B B D A E E E E C E E E EC E DA D EBD E A EBA DBA E A E ED EB D EBF B A E BAD B E DB D E D D A BE EA E D A D EFB E A B F B F A B B D D E E E E E E E E E E C C E E E E BB BB AE E E B B E A D A B DB E D F D E DBB DA E E BFBE BAD EBA F AF E DA B E A B F B F A B B D A E E E E E E E E E E E E E E D E A D E E D BE BE F A E A E E E E E E E E E AD AED DE E C EDE FBAE DBF EA E D FDA ED F D E E A E B E A B F BA E E E E E E E E E EC E E E E D FDA E EA E D E E CE AFD EB E A B E CE D B B E AEDBB BB AB ED E EB A DAE D E E D E E D E BAD B E A B F A D D BA E E E E E E E E E E EC E A DB E AFD EB E E D E DAD DB B E E D FDA E E AE B E DA B E A B F D B A E E E E E E E E E D D A E BA DA E EB E A D E A D A BEFB E DA E A D E DAD E A B F D B A E E E E E E E E E DA E D E E F D E EB BED D BAE ED ED E E DA BE E B E A B F D B A E E E E E E E E E E E E D EB ABE E F D E E A B F D B A E E E E E E E E E E E E E EC A E E B F EC B E D E C C E AA E D BED EC BE E AA E D E A B F D B A E E E E E E E E E E E E E E AFD EB E E AE F E EA E E DA E D D A BA E F ED D E DA EA E A F FAE E E AD A E FAD DA E A EBF A E E A B F D B A E E E E E E E E CE E C E E E E E D FDA E F D E A BE E B E A ED E AE D A B E A B F B F A B B D D E E E E E E E E E D FDA E AFD EB E A B E ED E D E A BE EA E D E A E E A B F B F A B B D D E E E E E E E E ECE E E E D D DB E F D EC B E E E F E E E BA B A A B D A E DE D E D D E E E A DA D E B A E E E E E E E E C EDEBA F AF DB EB D E F E AFD EB ED D E A E D DA EA ED E A E F D E B E BA BA B E E E E E E E E E E E E E E D E E ED E AFD EB E D FDA E A E BA ED EC CEA BAEB AB E A B F A D D BA E E E E E E E E E E E E D DB E AFD E E D E B EDE E E F A E A B F B F A B B D D E E E E E E E E E E E E E DA E D D A BA E E F E D A D E E E D BAB E A B A B F BD FF B A B F A B BD B E E E E C E E E E E E C E E E E A BE E EA E A EA E A E D E E A D EB D A EB D B E A B F B F A B B D A E E E E E E EC E E E E E E E EA E A EB D DB E AFD EB E B EA E AE F ED E F E D E DA E A B F B F A B B D D E E E

142 E E E E E E E E E E E E D B E E DB EB D A EB D E A B E DA D E ED E E A B F B F A B B D D E E E E E E E E E FADA D E A BE EA EBA F AF D ED AE E F B E A B F A D D BA E E E E E E E E E E E E AE E E E D BE A ED A EB A BE E A B E A D A B B F A F A BA A B B F A F A BA E E E E E E E E E E E E EDE A E E DBAE E D EBF B A E A B F D B A E E E E E E A E A E B E D E CE D D E A A D E EDFA DA E D E BF B A E D D E F DA ED E CE E B A E E E E E E C E E FE E F AE E B E C E E E E E A DA E E CEA F BE E AFD EB E AEBAF B EDE E E D E EA EB A E A B F A D D BA E E E E E E E E E E E C E E D B E E D ED EBA F AF DB E AFD EB E EA E C CE DADEB A E A B F B F A B B D D E E E E E E E E E ABE E F A E DBE E FADA D E D FDA BE E D DB E E D E E F E B E A B F A D D BA E E E E E E E E C E EA E F E A A D E E E A E EFB E F A E BAD D EBA F AF B E A B F D B A E E E E C E CE E C E E E F BF AE E AFD E D E A DA E F E B BE E A E BA F AF BE E DB E ED ED F D E F E E A E E E E E E E A E E F E A E A E E A D E E EA BE E DAD DB E ED E AFD EB E A B F BA AB D E E E E E E E E E E E E E B A E A E A E E D E EA F E D AE D E B E A B F A B B E E E E E E E E E E D E EA E F A E A E DA B EDE D A D E D A DA E AA A AB E E E E E E E E E E E E E D FDA E E AE A E A E E F D E C E B E F D EC D BED E B E E F D EC D B E A E E E E E E E E E E E E E E AE ED E F A E A E DA BE E AFD EB E A B F D B A E E E E ECE E E E E C E E E E D DA E D FDA E E CE AFD E D EB E A B E D AE EA E F D ED AE E A E A B F B F A B B D D E E E E C E E E C E E E E E E E F BAD A D E ABE E D B D E ED E B EFB EA E E C EB E F A E A B F A D D BA E E E E E E E E E A DA E EDE D EBF B AE E A E DA BE E E F A E DA E ED EA BA EB E A B F B F A B B D A E E E E E E E E E E E E B E E A E F A E BAD D D E E A E BA F AF B ED E D FDA E DB E EA EBADA BA D ED D B BE E AB E A B F B F A B B D D E E E E C E E E E E E E E BE EA EB A E E AD E A E DA BE E AFD EB E A B F B F A B B D D E E E

143 E E E E E E E E E A EBA F AF BE E AFD EB EDE DB EBAF E A E C E A B F D B A E E E E E E E E E E C E E E E BA DA E ADA E E F A E A E DA BE E AFD E D EB E E E E E E E E E E E E C E E E E E DAD DB E AEA F E B E E A B F A D D BA E E E E C E E E E E E E E E E A D E ED D BAE DA E A E B E A B F B F A B B D D E E E E E E E C E E E E E E E E BFD E DBB E D E E E F A B F AD B D E E E E E E E E E D AED E AE D A D E D F DA E EA E BB E F D E DBE D EA E D ABE E D F B E A B F B B A E E E E E CE E E E E E E E E EA E ED E D FDA E A D BED EA D B E A B F A D D BA E E E E E E E E E DA BE E D FDA E E FADA D E A B E A B F A D D BA E E E E E E DB E A ED EB D E FADA D E D FDA BE E E A B E A B F A D D BA E E E E EC E D EDE A E EA E E F E D B B E E E E E CE E EC E D D E DBE E E F B E A B F A D D BA E E E E C EC E D FDA E D BA EA BAB E ED DEF EA E E F ED EA E D D E E B E B D E E E E E E E E E E D D A E E DB ED D E E D FDA EA E A BBE E A F FBE D BE E DB A EBAF B E A E E E E E E E E E E D E BB E FD A E A EDBB BB EA E AD E E A E E D E BB E BFD E A B A B E E E E E E E E E E E E A E E D A D E BD D E E F B E E F E A E E D DE ED E F E D A BB E E D DE B BE BD D E A B F EB B B A E E E E E E E C E E E E E E E E A F EB F E D A EA DAE DFB BE B BE E AF B E A D F EB B BD F F D B F A B E E E E E E D E F A BE EAF E B BE D A E A BED EA E A B E A B A E E E E E E D E DA D BE EA E EBF D EDE F EB EEB A E E E E E E E E E E E E B DA BE A EA EFB E E D EA D E EA E A DA AE E F DA ED A A B E A A BF E E E E E E E E E E E E E E F D E F BE DA E E F D EAF E B BE D A ED D E A B F B A E E E E E E E E E E E E E E A E EAF E B BE D A D DE D D D DE A E E EBA F AF D ED D F BE EBF D E B BA B E E E E E E E E E E E CE E E D F E A E E D D E E E E E E E E E E E E E E EC B E EA E A BE EAF E B BE D A ED DE A E BA F AF DB E AFD EB E A B D B A A E E E E EC E E E E E E E E E A F AF DB E B E E DAF D F A E E A B E B D A B B D E E E

144 E E E E EC E E E E E E A F AF DB E F B E E C D E F BEDBE E A B E D E E E E ECE E C E E E E E E E C F B E E C A E A EA EA EDBB BAE F E B ED E D E E AB E F A B E E E E E E E CC C E CE E E E E E E D DE BFD DA EB BA E E DA E B D ED ED D B B E A B F B B A E E E E A D E E FE ED AE E A D E E E E E E E E E E EC ED E DAD DB EB E D E E DBB BE E DB F E D D F E DA E F ADB E A B E A B F D B A E E E E E E E E E E E E E E E DA E F ADB E A B E A F AF DB E BFBE B A DB E D E B ED E A F FAEB A B BED EB E A B F D B A E E E E E E E E E B E E E A B BA B B BA B D EE E E E E B E E E

145 BA E BE F DA B E D A EC E BF A E D E E BE E D F E E A BE E D DA E D FA E E CE AFD E D E E A B E D AE EA E F D E AE E A E F D E E D E DA ED E E E E E E A FA E D E BE DFA F B E A BE E F B E E BF A E D E EC BD D BE E DFE E DA F E E E DB E E DAB D B E E D E EC E E D E E D F E E E E D E A E D F E A BE E E F B E E A FA E D E BE DFA F B E E DB E E BF A E D D E E D F E E A BE E F A EBA F AF BE E AFD E D E E E EB A E A B DB E A DEA E A EA EB A E EA E F E E D DA E E A FA E D E BE DFA F B E BE F BE E A A E E A F E D FDA E BE A BE E D E E E D DA BA E BE F DA BE D B E E F D E A DA E E D EB D E F BEAD AA E D DA E A B E A A B B A E E F D E A DA E E AFD E E A B E A A B B A E E F D E E F BE E A BBE F E A A B B A

146 B AB A CC D AEBAF EB A EBAD BA BF BE E DE A E E D AB E E D E EB E BAE E FBE E FBEFA B E AE BE A BE B BE BB A AE A E D F B E D FDA E E E A BEDE BE E E D A E BE A BE E D E EB E AE BE BE E BA F A E E DE D F E D FDA E E E C C E DE DA E B E F E BEB A BE E D B E AD AE DBEA F F BED D A E EA FBE BED A B E DE F BBD E DA F ED AF E E AAD A E FB F BEBA F AF BE AD BE AE A E B BE F EA A E E E DE A E BEB A BE E D B E EDFA E EDE A E BE E E E BE A F BE D FDA E BE A BEB F AE E D B EC E F BE A F BE AE E A E B B EA BE F E C E AE E EDFA ED A DA E BAE B E E BF D AE DE D D A E A E F E A E E D A B E EF E A A E F ED A EBF E E E E A E AE E EBF EB F BEDE A E A E D EF E A E E D E EB E B E D E E B E D DA E BE A B E E D E EB E FAE A E F E AD AEB FA E E A DA E E F BE F BEDE B EA D FA F ABE BE E D E EB E EB D A E C E A F BE D FDA E F E E A A E B E E BF E ED D B EA E A F A E E AFD EB E EA E F E B E BB EA E F E E AFD EB E A BE DBE E DB ED ED D D E A BE D EA E E D FDA E EA BE E AE AFD EB E A BE E D FDA E EA EC CE DAD DB EB ED FDA E E BED B E D EB EB A BE EA EC CE DAD DB EDBEA E EB A E DA EFB E EA EC CE DBE AE D AE ED EA ED A B E BE FADA D E E E ABEA ED BBEA BE BBF E DBB D E E F BE D E E E E B D E D EBA F AF B EFB EA E B AEA E EB A E A E BED B E D EA DAE D FDA E A BEB EB E DB B E E D FDA E A BE D EA FBE E B E E C ED E E E D A DA E A E DBED B E B EFB E A BBE F B E DB E E F ED A A E D A D EDE AFD EB E F E DBE ED EA E α E EB D E F E A EB E A ED E D A A E E E DBE E A E BE BA DA EA E D F E E AFD EB E EA E F E B E BB ED A F E AFD EB E A BE EA E E B E AFD EB E E CEB D A E D FDA E A B E A BBE F E B E E