The HIVIIIB strain of virus was provided by the EU programmes EVA Centre for
AIDS Reagents, National Institute for Biological Standards and Control (NIBSC, South Mimms, Hertfordshire, UK). MT4 cells and viral isolates (multiplicity of infection, 0.01) were dispensed into a 50 mL Falcon tube and centrifuged (2000 × g,
15 min, 25 °C) in a Galaxy 170R incubator (Sanyo, Japan). The Falcon tube containing the MT4/HIVIIIB pellet was then incubated at 37 °C and 5% CO2. After
2 h, the supernatant was aspirated and the pellet resuspended in RPMI-1640 supplemented with 10% sterile filtered FBS. MT4 cells containing virus particles were then seeded at a density of 1 × 104 cells / 80 µL into each well of a 96-well
plate also containing 20 µL of a range of concentrations (5 × concentrate) of ABC or
D2-ABC dissolved in 0.5% MeOH and RPMI-1640 supplemented with 10% sterile
filtered FBS resulting in final concentrations 0.0128, 0.064, 0.32, 1.6, 8, 40, 200, 1000 or 5000 µM. The plates were then incubated for 5 days at 37 °C and 5% CO2
prior to the MTT assay as described above. Inhibitory effects of drugs on HIV replication can be assessed indirectly through the inhibition of virus-induced cytotoxicity to the MT4 cells, estimated using an MTT assay.39,52
3.6 Supplementary Information
Figure S1A: MRM chromatograms for the formation of ABC-COOH 17 from ABC (10 µM) in human liver cytosol at the following hours: 1 h (A); 4 h (B); 8 h (C); 16 h (D); 20 h (E) and 24 h (F). Protein and NAD+ concentrations were 0.75 mg/mL and 7.5 mM, respectively.
ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) D C B A F E
Figure S1B: MRM chromatograms for the formation of ABC-COOH 17 from D2-ABC (10 µM) in
human liver cytosol at the following hours: 1 h (A); 4 h (B); 8 h (C); 16 h (D); 20 h (E) and 24 h (F). Protein and NAD+ concentrations were 0.75 mg/mL and 7.5 mM, respectively.
ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) E F D C B A
Figure S2A: MRM chromatograms representing experiments measuring COOH formation from ABC (10 µM) in human liver cytosol under a variety of compromised metabolic conditions: Without NAD (Experiment 1) (A); magnified image of the formation of 3 COOH isomers (Experiment 1) (B); with 4-MP (600 µM) and DSF (500 µM) (Experiment 2) (C); with NAD (Experiment 3) (D); with 4- MP (600 µM) (Experiment 5) (E); with DSF (500 µM) (Experiment 6) (F). Protein and NAD+ concentrations were 0.75 mg/mL and 7.5 mM, respectively. Comparing experiments 3 (D) and 6 (F) there is a marked increase in the proportions of the lesser carboxylic acids 17b and 17c. Under the circumstances of ALDH inhibition, by DSF, when aldehyde oxidation might be catalysed principally by ADH1A, the selectivity of oxidation of the isomeric aldehydes is altered.
D C B A ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) E F
Figure S2B: MRM chromatograms representing experiments measuring COOH formation from D2-
ABC (10 µM) in human liver cytosol under a variety of compromised metabolic conditions: Without NAD (Experiment 1) (A); magnified image of the formation of 3 COOH isomers (Experiment 1) (B); with 4-MP (600 µM) and DSF (500 µM) (Experiment 2) (C); with NAD (Experiment 3) (D); with 4- MP (600 µM) (Experiment 5) (E); with DSF (500 µM) (Experiment 6) (F). Protein and NAD+
concentrations were 0.75 mg/mL and 7.5 mM, respectively. Comparing experiments 3 (D) and 6 (F)
B A ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) D C F E
there is a marked increase in the proportions of the lesser carboxylic acids 17b and 17c. Under the circumstances of ALDH inhibition, by DSF, when aldehyde oxidation might be catalysed principally by ADH1A, the selectivity of oxidation of the isomeric aldehydes is altered.
Figure S2C: EPI scans for fragments m/z 315 ([M+H]+ for the oxime derivative of the D2-ABC
aldehyde intermediate) from experiments 4 (co-incubation with methoxylamine) and 7 (co-incubation with methoxylamine and DSF) for D2-ABC (A and B respectively). The corresponding spectra are
presented for experiments 4 and 7 (C and D respectively). The imine product 19b (m/z 315) was not detected and the sample may have been contaminated with ABC, hence the detection of the precursor ion for 19a. The slow dehydrogenation of D2-ABC might have precluded production of detectable
amounts of oxime 58.
B A
D C
FigureS3A: MRM chromatograms obtained from the rate-substrate concentration experiments when ABC (10 µM) was incubated with human liver cytosol. Diagrams show representative chromatograms at the following concentrations and times: 500 µM at 0 min (A), 500 µM at 120 min (B), 20 mM at 0 min (C) and 20 mM at 120 min (D). Protein and NAD+ concentrations were 0.75
mg/mL and 7.5 mM, respectively. ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) A B D C
FigureS3B: MRM chromatograms obtained from the rate-substrate concentration experiments when D2-ABC (10 µM) was incubated with human liver cytosol. Diagrams show representative
chromatograms at the following concentrations and times: 500 µM at 0 min (A), 500 µM at 120 min (B), 20 mM at 0 min (C) and 20 mM at 120 min (D). Protein and NAD+ concentrations were 0.75
mg/mL and 7.5 mM, respectively. ABC (m/z 287/191) D2-ABC (m/z 289/191) ABC-COOH (m/z 301/191) B A C D
3.7 References
(1) Masubuchi, N.; Makino, C.; Murayama, N. Chem. Res. Toxicol. 2007, 20, 455.
(2) Srivastava, A.; Maggs, J. L.; Antoine, D. J.; Williams, D. P.; Smith, D. A.; Park, B. K. Handb. Exp. Pharmacol.2010, 165.
(3) Sharma, A. M.; Uetrecht, J. Drug Metab. Rev.2014, 46, 1.
(4) Park, B. K.; Boobis, A.; Clarke, S.; Goldring, C. E. P.; Jones, D.; Kenna, J. G.; Lambert, C.; Laverty, H. G.; Naisbitt, D. J.; Nelson, S.; Nicoll-Griffith, D. A.; Obach, R. S.; Routledge, P.; Smith, D. A.; Tweedie, D. J.; Vermeulen, N.; Williams, D. P.; Wilson, I. D.; Baillie, T. A. Nature Reviews Drug Discovery
2011, 10, 292.
(5) Walsh, J. S.; Reese, M. J.; Thurmond, L. M. Chem. Biol. Interact.2002, 142, 135.
(6) McDowell, J. A.; Chittick, G. E.; Ravitch, J. R.; Polk, R. E.; Kerkering, T. M.; Stein, D. S. Antimicrob. Agents Chemother.1999, 43, 2855.
(7) Mabic, S.; Castagnoli, N. J. Med. Chem.1996, 39, 3694.
(8) Ferraboschi, P.; Grisenti, P.; Santaniello, E. Journal of Labelled Compounds & Radiopharmaceuticals1994, 34, 303.
(9) Avery, M. A.; Bonk, J. D.; Mehrotra, S. Journal of Labelled Compounds & Radiopharmaceuticals1996, 38, 249.
(10) Charneira, C.; Godinho, A. L. A.; Conceicao Oliveira, M.; Pereira, S. A.; Monteiro, E. C.; Matilde Marques, M.; Antunes, A. M. M. Chem. Res. Toxicol.2011, 24, 2129.
(11) Bell, C. Thesis. Title: Characterisation of HLA-restricted T-cell responses to abacavir using lymphocytes from drug-naive volunteers, University of Liverpool, Liverpool, 2012.
(12) Lee, S.-L.; Shih, H.-T.; Chi, Y.-C.; Li, Y.-P.; Yin, S.-J. Chem. Biol. Interact.
2011, 191, 26.
(13) Brent, J. N. Engl. J. Med.2009, 360, 2216.
(14) Koppaka, V.; Thompson, D. C.; Chen, Y.; Ellermann, M.; Nicolaou, K. C.; Juvonen, R. O.; Petersen, D.; Deitrich, R. A.; Hurley, T. D.; Vasiliou, V.
Pharmacol. Rev.2012, 64, 520.
(15) Tamai, H.; Yokoyama, A.; Okuyama, K.; Takahashi, H.; Maruyama, K.; Suzuki, Y.; Ishii, H. Alcoholism-Clinical and Experimental Research 2000,
24, 97S.
(16) Shirota, F. N.; Demaster, E. G.; Nagasawa, H. T. Toxicol. Lett.1987, 37, 7. (17) Nagasawa, H. T.; Demaster, E. G.; Redfern, B.; Shirota, F. N.; Goon, J. W. J.
Med. Chem.1990, 33, 3120.
(18) DeMaster, E. G.; Redfern, B.; Nagasawa, H. T. Biochem. Pharmacol. 1998,
55, 2007.
(19) Moore, S. A.; Baker, H. M.; Blythe, T. J.; Kitson, K. E.; Kitson, T. M.; Baker, E. N. Structure with Folding & Design1998, 6, 1541.
(20) Lipsky, J. J.; Shen, M. L.; Naylor, S. Chem. Biol. Interact.2001, 130, 93. (21) Mays, D. C.; Nelson, A. N.; LamHolt, J.; Fauq, A. H.; Lipsky, J. J.
(22) Mays, D. C.; Ortiz-Bermudez, P.; Lam, J. P.; Tong, I. H.; Fauq, A. H.; Lipsky, J. J. Biochem. Pharmacol.1998, 55, 1099.
(23) Pike, M. G.; Mays, D. C.; Macomber, D. W.; Lipsky, J. J. Drug Metab. Disposition2001, 29, 127.
(24) Bell, R. P. Chem. Soc. Rev.1974, 3, 513.
(25) Kushner, D. J.; Baker, A.; Dunstall, T. G. Can. J. Physiol. Pharmacol.1999,
77, 79.
(26) Cavalieri, E.; Garcia, H.; Mailander, P.; Patil, K. Chem. Biol. Interact.1975,
11, 179.
(27) Foster, A. B. Trends Pharmacol. Sci.1984, 5, 524.
(28) Nelson, S. D.; Trager, W. F. Drug Metab. Disposition2003, 31, 1481. (29) Patanella, J. E.; Walsh, J. S. Drug Metab. Disposition1992, 20, 912.
(30) Bell, C. C.; Castelazo, A. S.; Yang, E. L.; Maggs, J. L.; Jeankins, R. E.; Tugwood, J.; O'Neill, P. M.; Naisbitt, D. J.; Park, B. K. Chem. Res. Toxicol.
2013, 26, 1064.
(31) Lenz, D.; Juebner, M.; Bender, K.; Wintermeyer, A.; Beike, J.; Rothschild, M. A.; Kaeferstein, H. Naunyn. Schmiedebergs Arch. Pharmacol.2011, 383, 647.
(32) Jelski, W.; Zalewski, B.; Szmitkowski, M. Dig. Dis. Sci.2008, 53, 2550. (33) Maeda, M.; Hasumura, Y.; Takeuchi, J. Lab. Invest.1988, 59, 75. (34) Wang, M.-F.; Han, C.-L.; Yin, S.-J. Chem. Biol. Interact.2009, 178, 36. (35) Xie, P. G. T.; Hurley, T. D. Protein Sci.1999, 8, 2639.
(36) Chessman, D.; Kostenko, L.; Lethborg, T.; Purcell, A. W.; Williamson, N. A.; Chen, Z.; Kjer-Nielsen, L.; Mifsud, N. A.; Tait, B. D.; Holdsworth, R.; Almeida, C. A.; Nolan, D.; Macdonald, W. A.; Archbold, J. K.; Kellerher, A. D.; Marriott, D.; Mallal, S.; Bharadwaj, M.; Rossjohn, J.; McCluskey, J.
Immunity2008, 28, 822.
(37) Bell, C. C.; Faulkner, L.; Martinsson, K.; Farrell, J.; Alfirevic, A.; Tugwood, J.; Pirmohamed, M.; Naisbitt, D. J.; Park, B. K. Chem. Res. Toxicol.2013, 26, 759.
(38) Berridge, M. V.; Herst, P. M.; Tan, A. S. Biotechnol. Ann. Rev. 2005, 11, 127.
(39) Riss TL, M. R., Niles AL, Benink HA, Worzella TJ and Minor L In Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly and Company and the National Center for Advancing Translational Sciences; 2004-2013.; In: Sittampalam GS, G.-E. N., Arkin M, Auld D, Austin C, Bejcek B, Glicksman M, Inglese J, Lemmon V, Li Z, McGee J, McManus O, Minor L, Napper A, Riss T, Trask OJ and Weidner J, eds., Ed. 2001.
(40) Gyuris, A.; Szlavik, L.; Minarovits, J.; Vasas, A.; Molnar, J.; Hohmann, J. In Vivo2009, 23, 429.
(41) Cihlar, T.; Birkus, G.; Greenwalt, D. E.; Hitchcock, M. J. M. Antiviral Res.
2002, 54, 37.
(42) Melroy, J.; Nair, V. Curr. Pharm. Des.2005, 11, 3847.
(43) Gu, Z.; Allard, B.; de Muys, J. M.; Lippens, J.; Rando, R. F.; Nguyen-Ba, N.; Ren, C.; McKenna, P.; Taylor, D. L.; Bethell, R. C. Antimicrob. Agents Chemother.2006, 50, 625.
(44) Faletto, M. B.; Miller, W. H.; Garvey, E. P.; Clair, M. H. S.; Daluge, S. M.; Good, S. S. Antimicrob. Agents Chemother.1997, 41, 1099.
(46) Barratt, M. J.; Frail, D. Drug repositioning [electronic book]: Bringing New Life to Shelved Assets and Existing Drugs / edited by Michael J. Barratt, Donald E. Frail; Hoboken : John Wiley & Sons, c.2012, 2012.
(47) Charneira, C.; Grilo, N. M.; Pereira, S. A.; Godinho, A. L. A.; Monteiro, E. C.; Marques, M. M.; Antunes, A. M. M. Br. J. Pharmacol.2012, 167, 1353. (48) Giorgini, S.; Martinelli, C.; Tognetti, L.; Carocci, A.; Giuntini, R.;
Mastronardi, V.; Torricellio, F.; Leoncini, F.; Lotti, T. Dermatologic Therapy
2011, 24, 591.
(49) Stringer, R. A.; Williams, G.; Picard, F.; Sohal, B.; Kretz, O.; McKenna, J.; Krauser, J. Drug Metab. Disposition2014, 42.
(50) Alfirevic, A.; Gonzalez-Galarza, F.; Bell, C.; Martinsson, K.; Platt, V.; Bretland, G.; Evely, J.; Lichtenfels, M.; Cederbrant, K.; French, N.; Naisbitt, D.; Park, B. K.; Jones, A. R.; Pirmohamed, M. Genome Medicine2012, 4, 51. (51) Wu, Y.; Farrell, J.; Pirmohmed, M.; Park, B. K.; Naisbitt, D. J. J. Allergy
Clin. Immunol.2007, 119, 973.
(52) Selvam, P.; Murugesh, N.; Chandramohan, M.; Pannecouque, C.; De Clercq, E. Indian Journal of Pharmaceutical Sciences2010, 72, 806.