Activity and the metabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661 |
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Authors: | Furman Phillip A Murakami Eisuke Niu Congrong Lam Angela M Espiritu Christine Bansal Shalini Bao Haiying Tolstykh Tatiana Micolochick Steuer Holly Keilman Meg Zennou Veronique Bourne Nigel Veselenak Ronald L Chang Wonsuk Ross Bruce S Du Jinfa Otto Michael J Sofia Michael J |
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Institution: | a Pharmasset, Inc., 303A College Road East Princeton, NJ 08540, USA b University of Texas Medical Branch, Galveston, TX 77555, USA |
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Abstract: | PSI-353661, a phosphoramidate prodrug of 2′-deoxy-2′-fluoro-2′-C-methylguanosine-5′-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5′-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O6-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2′-deoxy-2′-fluoro-2′-C-methylguanosine-5′-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration. |
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Keywords: | HCV hepatitis C virus HPLC high pressure liquid chromatography PCR polymerase chain reaction ddC dideoxycytidine GMP guanosine-5&prime -monophosphate GDP guanosine-5&prime -diphosphate RdRp HCV RNA-dependent RNA polymerase NS5B HCV non-structural protein 5B Cat A cathepsin A CES1 carboxylesterase 1 Hint 1 histidine triad nucleotide-binding protein 1 ADAL1 adenosine deaminase-like protein 1 hGUK1 human guanylate kinase NDPK human nucleoside diphosphate kinase PK pyruvate kinase 3-PGK 3-phosphoglycerate kinase Km Michaelis constant kcat turnover number kcat/Km catalytic efficiency |
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