Development of Novel Agents Active against Hepatitis B Virus

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Since 4'-modified CAdA, CdG, and CMCdG potently blocked the replication of wild-type and various drug resistant variants, we designed and synthesized approximately 100 NRTIs containing a 4'-modification and ultimately identified E-CFCP. E-CFCP structurally bears resemblance to CAdA, CdG, and CMCdG in that all the four analogs have a 4'-cyano moiety and E-CFCP also resembles ETV and CMCdG in that ETV, CMCdG, and E-CFCP have a methylidene in the 4' position of their cyclopentyl moiety. ETV and E-CFCP were virtually equipotent. However, the inhibition of HBV-DNA production by high E-CFCP concentrations (100 and 1,000 nM) was virtually complete (by 100%), while that by ETV never reached 100% even the cells were cultured with its highest concentrations (100 and 1,000 nM). The inhibition of HBV-DNA production by high concentrations of TAF never reached 100%, either. We also examined the activity of ETV, TAF, and E-CFCP against HBVWTCe using Southern blot assay, employing HBVWTCe-plasmid-transfected Huh7 cells. The IC50 values of ETV, TAF, and E-CFCP were 7.0, 25.0, and 0.2 nM, respectively. The average IC50 values of ETV and E-CFCP against HBVWTCe were 16 and 0.7 nM. We also synthesized Z-CFCP that has a fluorine atom at the Z-position of the methylidene moiety. Z-CFCP showed much less activity against HBVWTD1 (IC50=414nM) and HBVWTC2 (IC50=938 nM) compared to E-CFCP. We examined whether E-CFCP exerted activity against HBVETV-RL180M/S202G/M204V and HBVADV-RA181T/N236T, employing HBVETV-RL180M/S202G/M204V- and HBVADV-RA181T/N236T-containing-plasmid-transfected Huh7 cells. In a representative data set, E-CFCP exerted potent activity against HBVETV-RL180M/S202G/M204V. The activity of TAF against HBVETV-RL180M/S202G/M204V appeared to be comparable to that of E-CFCP. As expected, ETV virtually failed to block HBVETV-RL180M/S202G/M204V replication. E-CFCP was also potent against HBVADV-RA181T/N236T, while IC50 values of ETV and TAF were 184 and 86.5 nM, respectively. The greater potency of E-CFCP against HBVETV-RL180M/S202G/M204V and HBVADV-RA181T/N236T compared to the activity of ETV and the comparable activity of E-CFCP against HBVADV-RA181T/N236T compared to the activity of TAF were well corroborated by Southern blot assays repeated on different occasions. However, the CC50 values of E-CFCP in MT-2 cells, HepG2 cells, Huh7 cells, and PXB-cells were 306, 115, 500, and 500 microM, although those of TAF were 21, 22, 34, and 8.2 microM, respectively. Currently available anti-HBV agents are orally administered once-daily (QD); however, if anti-HBV therapeutics are orally administered less often, such therapeutics would improve the adherence to the treatment and quality of life (QOL) of CHB patients, in particular, in countries and regions where QD dosing schedule is not well managed. Thus, we examined whether E-CFCP's activity persisted longer than ETV and TAF, by exposing HepG2.2.15 cells to 0.01-10 microM of each NRTI over various periods (for 3 to 25 days), removing the compounds from the culture, continuing the culture in compound-free medium for 22 to 0 days, harvesting the cells on day 25, and determining HBVWTD1-DNA. Culture medium was replaced with compound-containing or non-compound-containing fresh medium on days 3, 9 and 17. The acquisition of inhibitory activity of NRTIs against HIV-1 and HBV requires tri-phosphorylation mediated by cellular kinases. Therefore, we examined how efficiently E-CFCP is intracellularly converted to E-CFCP-TP compared to ETV by quantifying the intracellular E-CFCP-TP and ETV-TP levels. HepG2.2.15 cells were incubated with 20 micro M E-CFCP or ETV for 1-3 days and at the conclusion of each incubation, intracellular concentrations of E-CFCP-TP and ETV-TP were quantified. Additionally, after 3 day's pre-incubation of the cells, the culture was replenished with fresh medium containing no compound, the cells were further cultured for 1, 3, and 5 days, and intracellular E-CFCP-TP and ETV-TP concentrations were determined. Intracellular E-CFCP-TP concentrations were much higher than those of ETV-TP and progressively increased when the culture was continued over 3 days, while the ETV-TP concentrations were low and remained substantially the same over three days' culture. When E-CFCP was removed from the culture and the cells were further cultured over 1, 3, and 5 days, E-CFCP-TP concentrations gradually decreased, although ETV-TP concentrations became very low after one day and insignificant or undetected on day 3 and beyond. When we conducted similar assays using PXB-cells, intracellular E-CFCP-TP on day 3 after incubation of PXB-cells with 100 micrM E-CFCP was readily detectable and persisted by day 7 of culture in the absence of E-CFCP. However, intracellular ETV-TP concentrations were very low even on day 3 and became virtually insignificant by day 7 when cultured without ETV. These data should explain at least in part the reason the anti-HBV activity of E-CFCP persisted for extended periods of time compared to that of ETV. Z-CFCP did not well penetrate HepG2.2.15 cells and intracellular Z-CFCP-TP was not visibly identified, in line with its much less anti-HBV activity observed. We also examined the phosphorylation efficiency of TAF and its intracellular persistence in Hep2.2.15 cells and compared TFV-DP's profiles with those of E-CFCP-TP and ETV-TP. Intracellular TFV-DP concentration was the highest among the three agents tested when the cells were exposed to TAF, ETV, or E-CFCP. The reason why the TFV-DP levels were the highest among the three agents in the final active form is likely that TAF is already monophosphorylated and is readily phosphorylated to give TFV-DP. However, when TAF was removed from the culture and the cells were further cultured, TFV-DP concentrations rapidly downed and TFV-DP was not detected by day 3 and beyond, while ETV-TP was detected on day3 and good levels of E-CFCP-TP were detected on day 5 in the absence of E-CFCP in culture. These data should explain the relatively rapid loss of anti-HBV activity upon removal of TAF. E-CFCP-TP forms a halogen bond interaction with the backbone NH of Asp-205, whereas no such interaction with either Asp-205 or any other HBVWT-RT residues is possible for Z-CFCP. Not only do the cyano and fluorine groups of E-CFCP-TP contribute to additional polar interactions, but they contribute to better van der Waals interactions with HBVWT-RT over ETV-TP. Overall, these additional polar and better van der Waals interactions of E-CFCP-TP appear to be responsible for its potency against HBVWT over ETV. Apparently, HBVETV-RL180M/S202G/M204V-RT has subtle but important changes in van der Waals interactions. Notably, Leu-180 and Met-204 of HBVWT-RT are in close proximity and Leu-180 also has van der Waals interactions with Phe-88. All these residues are important for the shape of the binding site. In HBVETV-RL180M/S202G/M204V-RT, the nature of these interactions must be different. Also, the S202G substitution may introduce the so-called glycine kink and change the conformation around the active site. These changes in the active site conformation might be partially responsible for the loss of binding of ETV-TP to HBVETV-RL180M/S202G/M204V-RT. While these changes also affect E-CFCP-TP binding to HBVETV-RL180M/S202G/M204V-RT, the additional interactions derived from 4'-cyano and fluorine should be able to mitigate the effect of these changes.