title: Efficiency of incorporation and chain termination determines the inhibition potency of 2'-modified nucleotide analogs against hepatitis C virus polymerase.
abstract: Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2'F-2'C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2'F-2'C-Me-UTP was efficiently incorporated by HCV polymerase with apparent Kd (equilibrium constant) and kpol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s(-1), respectively, giving an overall substrate efficiency (kpol/Kd) of 0.0059 ± 0.0015 μM(-1) s(-1). We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2' position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH2 > F-C-Me > C-Me > N3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2'F-2'C-Me-, 2'C-Me-, and 2'ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2'-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2'-modified NTP analogs against HCV polymerase.
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Fung
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA zjin@aliosbiopharma.com jdeval@aliosbiopharma.com.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Dyatkina
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Guangyi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA.
authors.forename: Leo
authors.initials: L
authors.lastname: Beigelman
authors.affiliation: Alios BioPharma Inc., South San Francisco, California, USA zjin@aliosbiopharma.com jdeval@aliosbiopharma.com.
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
mesh_terms: D000465:Algorithms; D000998:Antiviral Agents / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; D014544:Uridine Triphosphate
references.citation: J Virol. 2012 Jul;86(13):7107-17
references.pmid: 22532694
references.citation: Antimicrob Agents Chemother. 2005 May;49(5):2050-8
references.pmid: 15855531
references.citation: Antivir Chem Chemother. 2006;17(2):79-87
references.pmid: 17042329
references.citation: J Gen Virol. 2000 Mar;81(Pt 3):759-67
references.pmid: 10675414
references.citation: Antimicrob Agents Chemother. 2008 Feb;52(2):458-64
references.pmid: 17999967
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):E348-57
references.pmid: 23307808
references.citation: Structure. 1999 Nov 15;7(11):1417-26
references.pmid: 10574802
references.citation: Hepatology. 2005 Oct;42(4):962-73
references.pmid: 16149085
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22505-10
references.pmid: 21148772
references.citation: Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20509-13
references.pmid: 22135458
references.citation: Bioorg Med Chem Lett. 2010 Dec 15;20(24):7376-80
references.pmid: 21050754
references.citation: J Med Chem. 2010 Oct 14;53(19):7202-18
references.pmid: 20845908
references.citation: J Biol Chem. 2004 Nov 5;279(45):47212-21
references.pmid: 15294895
references.citation: J Virol. 2012 Jun;86(12):6503-11
references.pmid: 22496223
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13034-9
references.pmid: 10557268
references.citation: PLoS One. 2013 Jul 10;8(7):e68347
references.pmid: 23874596
references.citation: J Biol Chem. 2003 Apr 4;278(14):11979-84
references.pmid: 12554735
references.citation: Antimicrob Agents Chemother. 2007 Feb;51(2):503-9
references.pmid: 17101674
references.citation: J Biol Chem. 2008 Mar 21;283(12):7705-12
references.pmid: 18184655
references.citation: Biochem Pharmacol. 2000 May 1;59(9):1045-52
references.pmid: 10704933
references.citation: Antimicrob Agents Chemother. 2004 Feb;48(2):651-4
references.pmid: 14742230
references.citation: J Biol Chem. 2007 Oct 12;282(41):29812-20
references.pmid: 17698842
references.citation: J Biol Chem. 2012 Mar 23;287(13):10674-10683
references.pmid: 22303022
references.citation: J Med Chem. 2005 Aug 25;48(17):5504-8
references.pmid: 16107149
references.citation: Antimicrob Agents Chemother. 1995 Nov;39(11):2454-8
references.pmid: 8585725
references.citation: J Virol. 2002 Apr;76(7):3482-92
references.pmid: 11884572
references.citation: Nat Struct Biol. 1999 Oct;6(10):937-43
references.pmid: 10504728
references.citation: J Biol Chem. 2006 Feb 17;281(7):3793-9
references.pmid: 16316989
references.citation: Nat Rev Microbiol. 2007 Jun;5(6):453-63
references.pmid: 17487147
references.citation: Bioorg Med Chem Lett. 2007 May 1;17(9):2570-6
references.pmid: 17317178
references.citation: J Biol Chem. 2008 Jan 25;283(4):2167-75
references.pmid: 18003608
references.citation: Nucleosides Nucleotides Nucleic Acids. 2005;24(5-7):767-70
references.pmid: 16248033
references.citation: Virology. 1998 Sep 15;249(1):108-18
references.pmid: 9740782
references.citation: Nat Med. 2000 Oct;6(10):1082-6
references.pmid: 11017126
references.citation: J Mol Biol. 2011 Dec 2;414(3):370-84
references.pmid: 22008450
references.citation: Anal Biochem. 2009 Apr 1;387(1):20-9
references.pmid: 19154726