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Publications (10)37.46 Total impact

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    ABSTRACT: GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.
    Biochimica et Biophysica Acta (BBA) - General Subjects 08/2014; · 3.85 Impact Factor
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    ABSTRACT: Production of high-quality, well-characterized recombinant proteins facilitates screening of compound libraries. The protocols detailed in this unit are used to purify three recombinant enzymes that are widely used in HCV research: the HCV NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B RNA-dependent RNA polymerase. The active enzymes are purified to homogeneity by two-column chromatography to support a screening program for HCV inhibitors.
    Current protocols in pharmacology 09/2011; Chapter 13:Unit13B.6.
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    ABSTRACT: The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3' end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a "spring-loading" mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.
    Journal of Molecular Biology 02/2011; 405(5):1139-53. · 3.91 Impact Factor
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    ABSTRACT: Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.
    Journal of Medicinal Chemistry 05/2010; 53(10):4295-9. · 5.61 Impact Factor
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    ABSTRACT: The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s(-1) (oligo(dT)), 35 nt s(-1) (oligo(dU)), and 42 nt s(-1) (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K(0.5) for NS3h translocation at a saturating ATP concentration are as follows: 3.3+/-0.4 microM nucleotide (poly(dT)), 27+/-2 microM nucleotide (poly(dU)), and 36+/-2 microM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TDeltaS(0) are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (approximately 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.
    Journal of Molecular Biology 05/2010; 400(3):354-78. · 3.91 Impact Factor
  • Biophysical Journal - BIOPHYS J. 01/2010; 98(3).
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    ABSTRACT: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.
    JAIDS Journal of Acquired Immune Deficiency Syndromes 09/2008; 48(4):428-36. · 4.65 Impact Factor
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    ABSTRACT: The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3'-azido-3'-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.
    Antimicrobial Agents and Chemotherapy 09/2007; 51(8):2911-9. · 4.57 Impact Factor
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    ABSTRACT: GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type 1 strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with 14C-labeled GS-7340 or [3H]difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.
    Antimicrobial Agents and Chemotherapy 03/2007; 51(2):543-50. · 4.57 Impact Factor
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    ABSTRACT: To determine the mechanisms of resistance of K65R mutant reverse transcriptase (RT) to the currently approved nucleoside and nucleotide RT inhibitors (NRTI). Susceptibilities of K65R mutant HIV-1 to NRTI were determined in cell culture. The Ki/Km values were measured to determine the relative binding or incorporation of the NRTI, and ATP-mediated excision of incorporated NRTI was measured to determine NRTI stability as chain terminators. K65R HIV-1 had decreased susceptibility to most NRTI, but increased susceptibility to zidovudine (ZDV). Ki/Km values were increased 2- to 13-fold for K65R compared to wild-type RT for all NRTI, indicating decreased binding or incorporation. However, K65R also showed decreased excision of all NRTI compared to wild-type, indicating greater stability once incorporated. At physiological nucleotide concentrations, excision of ZDV, carbovir (the active metabolite of abacavir; ABC), stavudine (d4T), and tenofovir was further decreased, while excision of didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), and emtricitabine (FTC) was unchanged. The decreased binding or incorporation of ZDV by K65R appeared counteracted by decreased excision resulting in overall increased susceptibility to ZDV in cell culture. For ABC, tenofovir, and d4T, despite having decreased excision, decreased binding or incorporation resulted in reduced susceptibilities to K65R. For ddI, ddC, 3TC, and FTC, decreased binding or incorporation by K65R appeared responsible for the decreased susceptibilities in cell culture. NRTI resistance in cells can consist of both altered binding or incorporation and altered excision of the NRTI. For K65R, the combination of these opposing mechanisms results in decreased susceptibility to most NRTI but increased susceptibility to ZDV.
    AIDS 12/2005; 19(16):1751-60. · 6.41 Impact Factor