Mechanism of entecavir resistance of hepatitis B virus with viral breakthrough as determined by long-term clinical assessment and molecular docking simulation

Abstract

The mechanism by which entecavir resistance (ETVr) substitutions of hepatitis B virus (HBV) can induce breakthrough (BT) during ETV therapy is largely unknown. We conducted a cross-sectional study of 49 lamivudine (LVD)-refractory patients and 59 naïve patients with chronic hepatitis B. BT was observed in 26.8% of the LVD-refractory group during weeks 60 to 144 of ETV therapy. A line probe assay revealed ETVr substitutions only in the LVD-refractory group, i.e., in 4.9% of patients at baseline, increasing to 14.6%, 24.4%, and 44.8% at weeks 48, 96, and 144, respectively. Multivariate logistic regression analysis adjusted for age, gender, HBV DNA levels, and LVD resistance (LVDr) (L180M and M204V, but not M204I) indicated that T184 substitutions and S202G (not S202C) were a significant factor for BT (adjusted odds ratio [OR], 141.12, and 95% confidence interval [CI], 6.94 to 2,870.20; OR, 201.25, and 95% CI, 11.22 to 3608.65, respectively). Modeling of HBV reverse transcriptase (RT) by docking simulation indicated that a combination of LVDr and ETVr (T184L or S202G) was characterized by a change in the direction of the D205 residue and steric conflict in the binding pocket of ETV triphosphate (ETV-TP), by significantly longer minimal distances (2.2 A and 2.1 A), and by higher potential energy (-117 and -99.8 Kcal/mol) for ETV-TP compared with the wild type (1.3 A; -178 Kcal/mol) and LVDr substitutions (1.5 A; -141 Kcal/mol). Our data suggest that the low binding affinity of ETV-TP for the HBV RT, involving conformational change of the binding pocket of HBV RT by L180M, M204V plus T184L, and S202G, could induce BT.

FIG. 1.

Flowchart of 100 naïve/LVD-refractory patients during ETV therapy.

FIG. 2.

Kaplan-Meier plot and tabulated data for time to ETVr and cumulative ETVr patterns over 144 weeks. Pretreatment variables (solid line, LVD refractory; broken line, naïve) were analyzed in relation to the occurrence of ETVr. A previous LVD treatment was associated with a more rapid occurrence of ETVr (Breslow analysis; P < 0.001). Among the patients examined at entry prior to treatment with ETV, for the 41 LVD-refractory patients, the cumulative ETVr substitutions were detected in 2/41 (4.9%) at baseline and increased to 6/41 (14.6%), 10/41 (24.4%), and 13/29 (44.8%) at weeks 48, 96, and 144, respectively. Neither the S202I nor the M250V/I/L substitution was detected in this population.

FIG. 3.

3D structures of the dNTP-binding domains of HBV RT of the wild type (A), an LVDr substitution (B), and ETVr substitutions (C and D). The molecular surfaces of the wild type and the LVDr mutant are drawn in green, those of LVDr plus ETVr mutants are drawn in blue, and that of ETV-TP is drawn in orange.