SUPPORTING INFORMATION. Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside

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1 SUPPORTING INFORMATION Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside Bisubstrate Analogues: SAR of the Nucleobase Domain of 5 -O-[N- (Salicyl)sulfamoyl]adenosine João Neres, a Nicholas Labello, a Ravindranadh V. Somu, a Helena I. Boshoff, b Daniel J. Wilson, a Jagadeshwar Vannada, a Liqiang Chen, a Eric M. Bennett, a Clifton E. Barry III, b Courtney C. Aldrich a * a Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota b Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Table of Contents I. Complete Citation for Reference 58 and 88 S2 II. Molecular Modeling S3 A. Molecular Dynamics (MD) Simulations. S3 Figure S1. MD simulation: MbtA backbone atom movement S3 Figure S2. MD simulation: Distance between the Lys332 Nζ and Val448 S4 Figure S3. MD simulation: Distance between the Lys332 Nζ and Glu470 S5 Figure S4. MD simulation: Distance between the Lys332 Nζ and Ser466 S5 B. QM/MM Studies S6 Figure S5. QM/MM optimization of 4, 24, and 26 in the MbtA homology model S6 III. Chemistry A. HPLC purity of the final compounds S7 B. NMR spectra S8 IV. In Vitro Translation Assay. S43 Figure S6. Inhibition of Luciferase Translation by Sal-AMS (6) and AMS. S43 S1

2 I. Complete Citation for References 58 and 88 (58) Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.; Harris, D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.; Barry, C. E., 3rd; Tekaia, F.; Badcock, K.; Basham, D.; Brown, D.; Chillingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.; Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.; Krogh, A.; McLean, J.; Moule, S.; Murphy, L.; Oliver, K.; Osborne, J.; Quail, M. A.; Rajandream, M. A.; Rogers, J.; Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.; Taylor, K.; Whitehead, S.; Barrell, B. G. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393, (88) MacKerell, A. D. J.; Bashford, D.; Bellott, M.; Dunbrack, R. L., Jr.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E. I.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 1998, 102, S2

3 II. Molecular Modeling. A. Molecular Dynamics. The RMSd of the heavy backbone atoms over the 32 ns simulation is presented in Figure S1. The RMSd equilibrated during the first 2-5 ns and remained relatively constant thereafter. Figure S1. The RMSd of CA, C, N, and O backbone atoms relative to the structure after 60 ps of warming during a 32 ns molecular dynamics simulation. Figure S2 tracks the distance between the N terminal domain residue Lys332 N(ζ) and the interdomain hinge residue Val448(O). A hydrogen bond present in the DhbE crystal structure breaks and reforms several times during the dynamics run with no ligand present to provoke the change. This conformational shift is indicative of a large amount of flexibility in the region which is a crevice at the joining of the two domains and hinge. S3

4 Figure S2. The distance between the Lys332 Nζ and Val448 O during a 32 ns molecular dynamics simulation. A hydrogen bond between the two atoms breaks and reforms several times. Figures S3 and S4 present the distance between the Lys332 Nζ and (a) one of the Glu470 sidechain oxygens, and (b) the Ser466 backbone oxygen, respectively. The lysine sidechain spends several nanoseconds hydrogen bonded to each oxygen suggesting that there may be no significant energetic penalty for the noted rearrangement. This hydrogen bond breaking along with the usual perturbations and relaxation of the protein tertiary structure allows C2 substituents access to the large cavity vicinal to the purine N3 position. S4

5 Figure S3. The distance between the Lys332 Nζ and one of the Glu470 sidechain oxygens during a 32 ns molecular dynamics simulation. A stable hydrogen bond is noted between nanoseconds 6 and 13. Figure S4. The distance between Lys332 Nζ and Ser466 backbone O during a 32 ns molecular dynamics simulation. A hydrogen bond is noted between nanoseconds 6 and 13. S5

6 B. QM/MM Studies Figure S6 overlays the conformation of ligands 6, 26, and 28 obtained in the homology model after QM/MM optimization. Large substituents located ortho to the base must sterically clash with the purine in any conformation, indicating that these substituents will likely result in a conformational change of the C2 group away from ideal coplanarity with the adenine. The ideal conformations (B3LYP/6-31+G(d) optimization of the biphenyls in gas phase, atoms in common with Sal-AMS frozen in the active conformation) of the meta and para compounds access the pocket between the N and C-terminal domains. Figure S5. Ligands 6 (colored by atom), 26 (red), and 28 (green) represented in a ball and stick model obtained through QM/MM optimization in the MbtA homology model. S6

7 III. Chemistry A. HPLC purity and conditions for the final compounds. Table S1. HPLC purity and conditions for the final compounds. Compound Conditions Retention time Purity (min) (%) 7 Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method Method S7

8 HPLC solvents: A: Triethylammonium bicarbonate 50 mm; B1: Acetonitrile:Water 9:1; B2: Acetonitrile B3: Methanol HPLC column: Analytical: Varian Microsorb MV100-5 C18, mm, operated at 0.5 ml/min Methods: Method 1: Isocratic elution with 70% A and 30% B1, 30 min.; Method 2: Isocratic elution with 60% A and 40% B3, 30 min.; Method 3: Isocratic elution with 80% A and 20% B1, 30 min.; Method 4: Isocratic elution with 60% A and 40% B1, 30 min.; Method 5: Isocratic elution with 60% A and 40% B2, 30 min.; Method 6: Elution with A and % linear gradient of B3 in 25 min.; Method 7: Elution with A and % linear gradient of B1 in 30 min.; Method 8: Elution with A and % linear gradient of B2 in 20 min.; Method 9: Elution with A and % linear gradient of B1 in 20 min.; Method 10: Elution with A and % linear gradient of B1 in 32 min.; Method 11: Elution with A and % linear gradient of B1 in 32 min.; Method 12: Elution with A and % linear gradient of B2 in 30 min.; Method 13: Elution with A and 20-70% linear gradient of B2 in 30 min.; B. NMR spectra S8

9 Compound 35 (CA-I-77) S9

10 Compound 36 (CA-I-79) S10

11 Compound 37 (CA-I-83) S11

12 Compound 38 (CA-I-89) S12

13 Compound 39 (CA-I-97_fr1) S13

14 Compound 40 (CA-I-97_fr.2) S14

15 Compound 41 (CA-I-121) S15

16 Compound 42 (CA-I-125) S16

17 Compound 43 (SVR-III-129) S17

18 Compound 44 (SVR-III-153) S18

19 Compound 7 (JV-I-219 / SVR-III-87) S19

20 Compound 8 (SVR-III-181) S20

21 Compound 9 (JV-I-175) S

22 Compound 10 (JV-II-117) S22

23 Compound 11 (JPN-I-169) S23

24 Compound 12 (JPN-I-193) S24

25 Compound 13 (SVR-III-215) S25

26 Compound 14 (JPN-I-195) S26

27 Compound 15 (JPN ) S27

28 Compound 16 (JV-II-125) S28

29 Compound 17 (JPN-I-119) f1 (ppm) S29

30 Compound 18 (JPN-I-137) S30

31 Compound 19 (JPN-I-121) S31

32 Compound 20 (JV.I.177 / SVR.II.187) S32

33 S33

34 Compound 21 (JV.II.165) S34

35 Compound 22 (JV.II.169) S35

36 Compound 23 (SVR-III-27) S36

37 Compound 24 (SVR-III-57) S

38 Compound 25 (SVR-III-55) S38

39 Compound 26 (SVR-III-23) S39

40 Compound 27 (JPN-I-211) S40

41 Compound 28 (CA-I-183) S41

42 Compound 29 (CA-I-181) S42

43 IV. In Vitro Translation Assay. In vitro translation of luciferase results are shown in Figure S6. SalAMS (6) did not exhibit any inhibition at 100 µm while 5 -O-(sulfamoyl)adenosine (AMS), which is a known potent inhibitor of protein synthesis, led to complete inhibition of protein synthesis. Figure S6. Inhibition of Luciferase Translation by SalAMS (6) and AMS. S43