Pharmacological chaperones stabilize retromer to limit APP processing
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1 SUPPLEMENTARY INFORMATION Pharmacological chaperones stabilize retromer to limit APP processing Vincent J. Mecozzi 1,3, Diego E. Berman 2,3, Sabrina Simoes 2, Chris Vetanovetz 2, Mehraj R. Awal 1, Vivek M. Patel 2, Remy T. Schneider 2, Gregory A. Petsko 1,4, Dagmar Ringe 1,4, Scott A. Small 2,4 1 Department of Biochemistry and Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, 02454, 2 The Taub Institute for Research on Alzheimer s Disease and the Aging Brain, Department of Neurology and Pathology, Columbia University College of Physicians and Surgeons, New York NY Helen and Robert Appel Alzheimer s Disease Research Institute, Weill Cornell Medical College, New York NY These authors contributed equally to this work 4 Correspondence should be addressed to Greg Petsko (gpetsko@med.cornell.edu), Scott A. Small (sas68@columbia.edu), or Dagmar Ringe (ringe@brandeis.edu)
2 SUPPLEMENTARY RESULTS
3
4 *
5 Aβ42 Aβ40 fg/μg fg/μg VEHICLE 5uM R53 VEHICLE 5uM R53 sappα sappβ α-secretase β-secretase Aβ FL-APP βctf
6 Vps35 Vps26 actin
7 sappα in media FL-APP βctf
8 VEHICLE-Δ VEHICLE-CRE NT R55-Δ R55-CRE NT Vps35 actin Vps26
9 R55-Compound1 Certificate of Analysis Product Name: MedKoo Cat#: TPT-260 (Thiophene thiourea derivative) Chemical name Thiophene-2,5-diylbis(methylene) dicarbamimidothioate dihydrochloride N/A Synonyms Chemical structure CAS# and Theoretical analysis Name: TPT-260 2HCl MedKoo Code# CAS# Lot#ZY Chemical Formula: C 8 H 12 N 4 S 3 2HCl Molecular Weight: R55-Compound1 TPT-260 (free base): Chemical Formula: C8H12N4S3 Exact Mass: Molecular Weight: Batch No.: Mfg. Date: Test Date: Retest date: ZY September 15, 2012 September 15, 2012 September 14, 2014 Analysis item Specification Results Appearance White solid powder Complies Structure H-NMR and MS spectrum of the sample Complies correspond to the data reported in the literature Purity (HPLC) 97% 98.0% Solubility Soluble in DMF,DMSO Complies Additional information: N/A Conclusion This product conforms with MedKoo s quality s standards Shipping condition Ambient temperature Storage condition Short term storage (weeks): 0 4 o C under dry condition Long term storage (months): -20 o C under dry condition Self life At least 2 years if properly stored. CAUTION: NOT FULLY TESTED. FOR RESEARCH ONLY, NOT FOR HUMAN USE MedKoo Biosciences sales@medkoo.com P O Box 16222, Chapel Hill, NC , USA Tel: Fax:
10 R55-Compound1 TPT-260.2HCl C13-NMR analysis solvent: DMSO-d6 Normalized Intensity ThipheneBisuraC MedKoo Code#: Name: TPT-260: CAS#: (TPT-260, HCl salt) Chemical Formula: C8H14Cl2N4S3 Exact Mass: Molecular Weight: Chemical Shift (ppm) MedKoo Biosciences sales@medkoo.com
11 R55-Compound1 thiophene-bisuh TPT-260.2HCl H1-NMR analysis Solvent: DMSO-d MedKoo Code#: Name: TPT-260: CAS#: (TPT-260, HCl salt) Chemical Formula: C8H14Cl2N4S3 Exact Mass: Molecular Weight: Normalized Intensity Chemical Shift (ppm) MedKoo Biosciences sales@medkoo.com
12 D:\DATA\2012\September2012\n120914\Pan\ _EJ_Pan_ZY-2_CHC_RP2465_ Sample _EJ_Pan_ZY-2_CHC_RP2465_suppOFF\0_C3\1 Comment 1 suppoff\0_c3\1 Pan Comment 2 ZY-2_CHC RP2465_suppressionOFF Intens. [a.u.] R55-Compound1 x TPT MS analysis (M + K)+ = TPT-260 (free base) Chemical Formula: C8H12N4S3 Exact Mass: Molecular Weight: m/z m/z SN Quality Fac. Res. Intens. Area Acquisition method name D:\Methods\flexControlMethods\Specification\RP_2465.par Number of shots 100 Voltage polarity POS Laser beam attenuation Date of acquisition T10:18: :00 printed: 9/14/ :38:16 AM MedKoo Biosciences sales@medkoo.com
13
14 R33-Compound3 Certificate of Analysis Product Name: MedKoo Cat#: TPT-172 HCl (Thiophene thiourea derivative) Chemical name thiophen-2-ylmethyl carbamimidothioate hydrochloride N/A Synonyms Chemical structure R33-Compound3 CAS# and Theoretical analysis Name: TPT-172 HCl MedKoo Code# CAS# Lot#ZY Chemical Formula: C 6 H 8 N 2 S 2 HCl Molecular Weight: TPT-172 (free base): Chemical Formula: C6H8N2S2 Exact Mass: Molecular Weight: Batch No.: Mfg. Date: Test Date: Retest date: ZY Sepetember 17, 2012 Sepetember 17, 2012 September 16, 2014 Analysis item Specification Results Appearance White solid powder Complies Structure H-NMR and MS spectrum of the sample Complies correspond to the data reported in the literature Purity (HPLC) 97% 98.0% Solubility Soluble in DMF,DMSO Complies Additional information: N/A Conclusion This product conforms with MedKoo s quality s standards Shipping condition Ambient temperature Storage condition Short term storage (weeks): 0 4 o C under dry condition Long term storage (months): -20 o C under dry condition Self life At least 2 years if properly stored. CAUTION: NOT FULLY TESTED. FOR RESEARCH ONLY, NOT FOR HUMAN USE MedKoo Biosciences sales@medkoo.com P O Box 16222, Chapel Hill, NC , USA Tel: Fax:
15 R33-Compound3 TPT-172.HCl H1-NMR analysis solvent: DMSO-d6 thiophene-monothiou MedKoo Code#: Name: TPT-172.HCl CAS#: (TPT-172 HCl salt) Lot#ZY Chemical Formula: C6H9ClN2S2 Molecular Weight: Normalized Intensity Chemical Shift (ppm) MedKoo Biosciences sales@medkoo.com
16 R33-Compound3 TPT-172.HCl ---- C13-NMR analysis solvent: DMSO-d6 Normalized Intensity Thiophene-monoUC MedKoo Code#: Name: TPT-172.HCl CAS#: (TPT-172 HCl salt) Lot#ZY Chemical Formula: C6H9ClN2S2 Molecular Weight: Chemical Shift (ppm) MedKoo Biosciences sales@medkoo.com
17 D:\DATA\2012\September2012\n120914\Pan\ _EJ_Pan_ZY-1_CHC_RP2465_ Sample _EJ_Pan_ZY-1_CHC_RP2465_suppOFF\0_C2\1 Comment 1 suppoff\0_c2\1 Pan Comment 2 ZY-1_CHC RP2465_suppressionOFF Intens. [a.u.] x R33-Compound TPT MS analysis 2.0 Name: TPT-172 Chemical Formula: C6H8N2S2 Exact Mass: Molecular Weight: m/z m/z SN Quality Fac. Res. Intens. Area Acquisition method name D:\Methods\flexControlMethods\Specification\RP_2465.par Number of shots 100 Voltage polarity POS Laser beam attenuation Date of acquisition T10:14: :00 printed: 9/14/ :32:19 AM MedKoo Biosciences sales@medkoo.com
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