Indolopyridones Inhibit Human Immunodeficiency Virus Reverse Transcriptase with a Novel Mechanism of Action

Abstract

We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed "hot spots" for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3 end.The reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) remains a major target in antiretroviral therapy, with the current standard of care being the use of two nucleoside or nucleotide analogue reverse transcriptase inhibitors (NRTIs), combined with one nonnucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (32).Upon entry into the cell, the virus is uncoated, and the viral RT enzyme converts its single-stranded RNA genome into double-stranded proviral DNA. Inhibition of this crucial event in the early viral life cycle ultimately precludes the virus from proliferating. Following the intracellular phosphorylation of NRTIs, NRTI-triphosphates compete with natural deoxyribonucleoside triphosphate (dNTP) pools and bind to the RT active site. They act as chain terminators due to the lack of a 3Ј-hydroxyl group (31). In contrast, NNRTIs represent a chemically diverse class of compounds that bind to a pocket in the vicinity of the catalytic site (25,29,30). Binding of these inhibitors is noncompetitive with respect to both dNTPs and template/primer (16).Despite the potency of combinations of NRTIs and NNRTIs, the emergence of mutations conferring resistance remains a major cause for treatment failure. The advent of novel RT inhibitors with a different mechani...

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

“…The conformational landscape of the binding sites may therefore be very similar. The ability of fragment 5 to inhibit MoMLV RT contrasts with INDOPY-1, which lacks activity against a murine retroviral RT (23), suggesting that this fragment binds to a site distinct from this NcRTI. Moreover, compounds 4, 5, and 8 are unlikely to target the NNIBP because NNRTIs are usually highly specific for the HIV-1 RT and do not inhibit MoMLV RT activity (24).…”

“…also more potent against RT K65R but is fivefold less potent for RT M184V (23). Hydroxyaniline 8 retained potency against both mutants (Table S4).…”

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

“…PPT primer ends with a purine (dGTP), which is not ideally suited for INDOPY-1 binding (15). For these reasons, we designed tailored chimeric PPT primers that mimic the initiation of the (ϩ)-strand DNA synthesis and favor PFA or INDOPY-1 binding.…”

“…For these reasons, we designed tailored chimeric PPT primers that mimic the initiation of the (ϩ)-strand DNA synthesis and favor PFA or INDOPY-1 binding. We thus synthesized a pre-translocated sequence (PPTϩ3D) to favor PFA binding (13) and a posttranslocated sequence (PPTϩ2D) that ends in a pyrimidine (dCTP) for INDOPY-1 (15).…”

“…The observed preference of PFA for the pre-translocational form of the polymerase⅐DNA complex was recently validated by the first crystal structure of PFA bound to a DNA polymerase, which showed PFA binding and stabilization of the closed enzyme conformation leading to the formation of an untranslocated form of the polymerase⅐DNA complex (14). In contrast, the more recently discovered scaffold of indolopyridones (INDOPY-1) (15,16) traps RT in the post-translocational state (15). Owing to its proposed binding mechanism, INDOPY-1 has been referred to as a nucleotide-competing RT inhibit (17).…”