In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

Abstract

Several mutations (V521L, P525L, L528M, T532S, and V555I) in the gene for hepatitis B virus (HBV) polymerase have been identified in HBV isolated from patients that displayed break-through viremia during famciclovir treatment. To determine whether these mutations cause phenotypic resistance to famciclovir, we compared the inhibition constants (K i ) of penciclovir triphosphate (PCVTP, the active metabolite of famciclovir) for recombinant wild-type and mutant HBV polymerases containing these mutations. In in vitro enzymatic assays, the V555I mutation displayed the most resistance (with K i increased by 6.2-fold) to PCVTP. The V521L and L528M mutations showed moderately decreased sensitivity to PCVTP (K i increased by G3-fold). We also analyzed the cross-resistance profiles of these variants for adefovir and lamivudine, two other antiviral agents that also inhibit DNA replication by HBV polymerase. All 5 famciclovir-associated mutations were sensitive to adefovir diphosphate (ADVDP) in in vitro enzymatic assays (F2.3-fold decreased sensitivity). The V521L, L528M, and T532S mutations were also sensitive to lamivudine triphosphate (LAMTP); however, the P525L and V555I mutations displayed moderately decreased sensitivity to LAMTP in enzymatic assays (3.6-fold decreased sensitivity). The lamivudine-resistant mutations M552I, M552V, and L528M؉M552V, which were previously shown to display 8-to 25-fold resistance to LAMTP, were less resistant (I3.1-fold) to PCVTP. (HEPATOLOGY 2000;31:219-224.)Hepatitis B viral infection is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and it is one of the 10 most common causes of death worldwide. 1 Current treatment regimens rely primarily on interferon alfa therapy. However, interferon has limited efficacy, is expensive, requires parenteral administration, and is associated with frequent and unpleasant side effects. Nucleoside or nucleotide analogs, which inhibit DNA replication through hepatitis B virus (HBV) polymerase, represent a new class of promising anti-HBV therapeutics. In addition to lamivudine, 2-9 which was approved recently for the treatment of hepatitis B infection in the United States, several nucleoside and nucleotide analogs including famciclovir 5,10,11 and adefovir dipivoxil 12-14 are currently being evaluated in late stage clinical trials. These compounds have been shown to be well tolerated and can produce rapid and marked decreases in serum HBV-DNA levels.Famciclovir is the oral prodrug of penciclovir, which has to be converted to penciclovir triphosphate by cellular enzymes to become an active inhibitor against HBV polymerase by competing with the natural substrate deoxyguanosine triphosphate (dGTP). 15 Famciclovir was originally approved for the treatment of herpes simplex virus and herpes zoster virus. 16,17 Later, famciclovir was shown to have activity against human and duck hepatitis B viruses in enzymatic and cell culture assays and in animal models of infection. 15,[18][19][20][21] Clinical studies subsequently showed...

Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir

“…Interestingly, antiviral studies of the nucleoside analogs lamivudine (3TC) and PCV, two potent inhibitors of hepatitis B virus, (HBV), also suggest that both the fine molecular interaction of these inhibitors with viral polymerases and their basepairing interactions may directly influence the type of drugresistant variants selected. A substantial difference in the K i for inhibition of recombinant HBV polymerase by PCV-TP and 3TC-TP is apparent (4.8 and 0.25 M, respectively), similar to the differences in the PCV-TP and ACV-TP affinities for inhibition of the HSV polymerase (46,47). Although mutations within the catalytic site of the HBV reverse transcriptase have been found to occur following treatment of chronic HBV infection with either 3TC or FCV, variants containing a point mutation in the YMDD motif are most readily found after 3TC treatment (27,43).…”

Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient

“…This mutant harboring five closed mutations in RT protein represented approximatively 15% of the population at the end of entecavir therapy, and its proportion slightly increased after the switch from entecavir to lamivudine+adefovir. A mutation at this position in leucine, but not serine, has already been described for resistance to famciclovir [30]. This proline at the 177 position is conserved in HIV RT (P157), and seems to directly interact with M184 in HIV RT or M204 in HBV RT [28].…”

Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region

“…64 Adefovir dipivoxil (bis-POM-PMEA), a drug in clinical evaluations for HBV 66 and HIV-1, 67 selects for a codon K70E mutation in HIV-1, but has not been linked to any domain F changes in HBV polymerase. 68 Huang et al 69 described the interaction between the HIV-1 rt enzyme and its substrate (rt ⅐ primer:template ⅐ dNTP complex). Several of the amino acids involved with these interactions are located in the conserved regions (Table 2).…”