Subtype-Specific Analysis of the K65R Substitution in HIV-1 That Confers Hypersusceptibility to a Novel Nucleotide-Competing Reverse Transcriptase Inhibitor

Abstract

Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype. C urrently, over 35 million people are living with HIV, according to the World Health Organization (http://www.who.int /hiv/en/). Genetic diversity is characteristic of HIV-1 due to the error-prone nature of its reverse transcriptase (RT) enzyme and its high viral replication rate. The HIV-1 epidemic has rapidly evolved to include 9 major known circulating subtypes (A to D, F to H, J, and K) and over 60 known circulating recombinant forms (CRFs) that show 25 to 35% overall genetic variation; this includes 10 to 15% diversity in RT (1-4). HIV-1 subtype C accounts for 50 to 55% of all HIV infections worldwide, while HIV-1 subtype B is the most prevalent subtype in developed countries and accounts for ϳ12% of global HIV infections. CRF01_AE and CRF02_AG are two globally predominant CRFs that are found in Southeast Asia and West/Central Africa, respectively.Current standard anti-HIV-1 therapy, termed highly active antiretroviral therapy (HAART), consists of three or more antiretroviral compounds from six distinct classes, including nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, entry inhibitors, and integrase inhibitors (INIs) (for reviews, see references 5 and 6). Most currently available antiretrovirals (ARVs) were developed based on the ability to block replication of subtype B viruses, but the development of resistance to all ARVs is a major obstacle in the face of long-term treatment success (7,8). The high genetic diversity of HIV-1 subtypes may lead to distinct pathways to drug resistance (9-12), necessitating the development of novel effective ARVs that possess distinct mechanisms and superior resistance profiles for all HIV-1 subtypes.Inhibitors that target RT constitute the largest class of ARVs and are ke...

Structural Basis of HIV-1 Inhibition by Nucleotide-Competing Reverse Transcriptase Inhibitor INDOPY-1

“…Additionally, compound A also has a hot spot for inhibition following a dT-terminated primer. 24 To understand this distinct activity spectrum of compound A and probe the present structure, we performed a rigid molecular docking (see Supporting Information results) at the INDOPY-1 binding site of RT/DNA/INDOPY-1 structure. Notably, the best pose obtained (Figure 4A) displays INDOPY-1-like stacking of the benzofuranopyrimidone core with the nucleotide base pair at the primer 3′-end and intercalation between the template bases d-0 and dA-1.…”

“… Its main features are (i) potency against Y115F and M184V RT, (ii) hypersusceptibility to K65R RT, and (iii) decrease in potency against W153L (see Supporting Information results). Additionally, compound A also has a hot spot for inhibition following a dT-terminated primer . To understand this distinct activity spectrum of compound A and probe the present structure, we performed a rigid molecular docking (see Supporting Information results) at the INDOPY-1 binding site of RT/DNA/INDOPY-1 structure.…”

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

“…Subtype B HIV-1 wild-type reverse transcriptase (RT) and RT containing the M184V substitution were generated as described previously (43,44). A test of the inhibitory effect of DMB220 on HIV-1 RT activity was performed as described previously (45). The reaction mixture (50 l) contained 50 mM Tris-HCl (pH 7.8), 5 mM MgC1 2 , 2 mM ATP, 60 mM KCl, 5 mM dithiothreitol (DTT), 1 M [ 3 H]dTTP, 5 g/ml of template/primer poly(rA)/oligo(dT) [12][13][14][15][16][17][18] (Midland Certified Reagent Company, Midland, TX), recombinant RTs of the same activity, and variable amounts of DMB220.…”

Avoiding Drug Resistance in HIV Reverse Transcriptase

“…307 The TFV resistance mutation K65R confers hypersusceptibility to INDOPY-1. 308 Hence, compounds that block RT by leading to dead-end reaction intermediates while engaging in interactions with conserved residues and main chain atoms may offer another strategy to minimize drug resistance mutations.…”

“…Notably, interactions of INDOPY-1 at the active site involve some conserved residues, including K65, R72, D110, D185, as well as the main chain N of G152. ,, Resistance to INDOPY-1 has been reported through the FTC/3TC resistance mutation M184V and through Y115F that is known to cause resistance to multiple NRTIs . The TFV resistance mutation K65R confers hypersusceptibility to INDOPY-1 . Hence, compounds that block RT by leading to dead-end reaction intermediates while engaging in interactions with conserved residues and main chain atoms may offer another strategy to minimize drug resistance mutations.…”