Differential binding of tenofovir and adefovir to reverse transcriptase of hepatitis B virus

Abstract

Introduction: Resistance of the reverse transcriptase (RT) of hepatitis B virus (HBV) to the tenofovir nucleotide drug has not been observed since its introduction for treatment of hepatitis B virus (HBV) infection in 2008. In contrast, frequent viral breakthrough and resistance has been documented for adefovir. Our computational study addresses an inventory of the structural differences between these two nucleotide analogues and their binding sites and affinities to wildtype (wt) and mutant RT enzyme structures based on in silico modeling, in comparison with the natural nucleotide substrates.

Results: Tenofovir and adefovir only differ by an extra CH3-moiety in tenofovir, introducing a center of chirality at the carbon atom linking the purine group with the phosphates. (R)-Tenofovir (and not (S)-tenofovir) binds significantly better to HBV-RT than adefovir. "Single hit" mutations in HBV-RT associated with adefovir resistance may affect the affinity for tenofovir, but to a level that is insufficient for tenofovir resistance. The RT-Surface protein gene overlap in the HBV genome provides an additional genetic constraint that limits the mutational freedom required to generate drug-resistance. Different pockets near the nucleotide binding motif (YMDD) in HBV-RT can bind nucleotides and nucleotide analogues with different affinities and specificities.

Conclusion: The difference in binding affinity of tenofovir (more than two orders of magnitude in terms of local concentration), a 30x higher dosage of the (R)-tenofovir enantiomer as compared to conformational isomeric or rotameric adefovir, and the constrained mutational space due to gene overlap in HBV may explain the absence of resistance mutations after 6 years of tenofovir monotherapy. In addition, the computational methodology applied here may guide the development of antiviral drugs with better resistance profiles.

Figure 1. Structures of adefovir, (R)-tenofovir and (S)-tenofovir.

PDB coordinates of Mg-adefovir and Mg-(R)-tenofovir were taken from the PDBids 1ZOT and 3FKB, respectively. (S)-tenofovir was created via CORINA. Mg-ions near the red phosphate moieties are in green. Arrows indicate the carbon atom linking the purine group with the phosphates (a center of asymmetry in tenofovir). Numbers in white are the total protein-ligand energy interaction values indicating the affinity to wt HBV reverse transcriptase.

Figure 2. Clustering analysis of in silico modeled HBV RT with crystal models of polymerase.

The 3D-structure of HBV-RT and the PDB coordinate files of the other polymerases were aligned and the pairwise RMSD values were put into a distance matrix for tree construction. HBV-RT is indicated in bold-face. The scale bar represents a RMSD value of 1. The date is added to anticipate future additions to the PDB database.

Figure 3. Binding of deoxyribonucleotidetriphosphates to wt HBV reverse transcriptase.

The Mg-dNTPs are in red-colored, space-filled format. Other space-filled residues indicate the YMDD motif sequence marking the HBV-RT nucleotide binding site. Numbers in white are the total protein-ligand energy interaction values indicating the Mg-dNTP affinity to wt HBV-RT.

Figure 4. Binding of tenofovir and adefovir to wt HBV reverse transcriptase.

Mg-adefovir (left panel, red cartoon) binds close to YMDD (space-filled format) in the large central pocket of HBV-RT. Mg-tenofovir (right panel, red cartoon) binds in a small pocket at the backside of YMDD (space-filled format).

Figure 5. Sequential docking of dATP, dGTP, and adefovir (left panel) or tenofovir (right panel) into the HBV-RT structure carrying S78T & A181V amino acid replacements.

T78 and V181 are in hot-pink. YMDD is indicated by space-filling. Mg-adefovir and Mg-tenofovir are in red cartoon format. Mg-dATP is white and Mg-dGTP is blue colored. Total protein-ligand energy interaction values (Kcal/mol) are −9.38 (dATP), −7.65 (dGTP), −7.55 (tenofovir) and −5.07 (adefovir).