Mechanism-based Suppression of Dideoxynucleotide Resistance by K65R Human Immunodeficiency Virus Reverse Transcriptase Using an α-Boranophosphate Nucleoside Analogue

Abstract

The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2,3-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2,3-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k pol mostly accounts for the lower incorporation efficiency observed for 2,3-dideoxynucleotides. Examination of the crystal structure of the RT⅐DNA⅐dNTP complex suggested that both the charge at position 65 and the 3-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2,3-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. ␣-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

“…Other mutations, such as Q151M and L74V, decrease the k pol without affecting the K d (7,8). Consistent with prior reports, our data show that K65R predominantly exerts its discrimination effects by selectively decreasing the k pol (9,11,27), although small changes in K d were noted in this study and others (14,27). Our results show that the K70E mutation has the same mechanism of discrimination as the K65R, L74V, and Q151M mutations, in that it predominantly exerts its effects by decreasing the k pol for the NRTI-TP.…”

“…These resistance values correlate well with data from viral drug susceptibility assays (4, 4a). By comparison, the K65R mutation conferred greater resistance (12-to 13.1-fold) to the same nucleotide analogs, which is consistent with prior reports (7,9,14,27). The K65R mutation was selected in 53% of the patients receiving TNV/ABC/ 3TC in the ESS 30009 study, whereas the K70E mutation was selected in only 10% of the patients (25a).…”

“…By comparison, the catalytic efficiency values for K65R HIV-1 RT were decreased 1.4-to 2.8-fold, depending on the identity of the dNTP substrate. As noted by others (27), this decrease is primarily due to a lower maximum rate of dNTP incorporation (k pol ) by K65R and not decreased dNTP binding affinity (K d ). The selectivity of RT, which is defined with the equation (k pol /K d ) dNTP /(k pol /K d ) analog , is an indication of the ability of the WT or mutant RT to discriminate between the natural dNTP substrate and the NRTI-TP.…”

“…In the crystal structure of the ternary HIV-1 RT-T/P-TTP and RT-T/P-TNV-DP complexes, the -amino group of K65 interacts with the ␥-phosphate of the bound nucleotide substrate (13,31). Modeling studies suggest that the K65R mutation in HIV-1 RT prevents optimal positioning of the NRTI-TP in the active site, which results in decreased catalytic efficiency of incorporation (27,29). In the ternary RT-T/P-dNTP complexes, the side chain of K70 does not interact with either the bound nucleotide or the nucleic acid substrate and exhibits minimal noncovalent interactions with other amino acid residue side chains.…”

Computational model of hepatitis B virus DNA polymerase: Molecular dynamics and docking to understand resistant mutations

“…16 The K65R mutation causes changes in the electrostatic interactions and destabilizes the positioning and alignment of the phosphate groups in the active site, slowing the catalytic step. 17 This mutation confers intermediate resistance against ddC, tenofovir, adefovir, and lamivudine, when associated with other mutations. Although no mutational data has been reported to date for the lysine corresponding to K65R in HDP, rtK32, it could be expected to play a similar role to that played by K65 in HIV-1 RT for binding of similar analogs and the development of resistance to inhibitors.…”