Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: HIV-1 reverse transcriptase (RT) is an essential enzyme, targeting half of approved anti-AIDS drugs. While nucleoside RT inhibitors (NRTIs) are DNA chain terminators, the nucleotide-competing RT inhibitor (NcRTI) INDOPY-1 blocks dNTP binding to RT. Lack of structural information hindered INDOPY-1 improvement. Here we report the HIV-1 RT/DNA/INDOPY-1 crystal structure, revealing a unique mode of inhibitor binding at the polymerase active site without involving catalytic metal ions. The structure may enable new strategies for developing NcRTIs.

SUBMITTER: Ruiz FX

PROVIDER: S-EPMC7737671 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

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

Ruiz F Xavier FX Hoang Anthony A Das Kalyan K Arnold Eddy E

Journal of medicinal chemistry 20191025 21

HIV-1 reverse transcriptase (RT) is an essential enzyme, targeting half of approved anti-AIDS drugs. While nucleoside RT inhibitors (NRTIs) are DNA chain terminators, the nucleotide-competing RT inhibitor (NcRTI) INDOPY-1 blocks dNTP binding to RT. Lack of structural information hindered INDOPY-1 improvement. Here we report the HIV-1 RT/DNA/INDOPY-1 crystal structure, revealing a unique mode of inhibitor binding at the polymerase active site without involving catalytic metal ions. The structure ...[more]

PMID: 31603676

Similar Datasets

Identification and characterization of a novel HIV-1 nucleotide-competing reverse transcriptase inhibitor series.

Project description:Several groups have recently reported on the identification of nucleotide-competing reverse transcriptase inhibitors (NcRTIs), a new class of RT inhibitors. NcRTIs reversibly inhibit binding of the incoming nucleotide to the RT active site but do not act as chain terminators, unlike the nucleos(t)ide reverse transcriptase inhibitor (NRTI) class. We identified a novel benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI chemical series. Structure-activity relationship evaluation of this series with both RT and viral replication assays led to the identification of compound A, a new NcRTI. Compound A inhibited HIV-1 RT in a primer extension assay (50% inhibitory concentration, 2.6 nM) but had no measurable activity against human DNA polymerase γ at 10 μM. It potently inhibited HIV-1 replication in vitro (50% effective concentration, 1.5 nM). The antiviral potency of compound A was unaffected by the presence of nonnucleotide RT inhibitor (NNRTI) mutations tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V. In vitro selection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

| S-EPMC3716156 | biostudies-literature

Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors".

Project description:Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).

| S-EPMC2573056 | biostudies-literature

Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer.

Project description:Nucleotide-competing reverse transcriptase inhibitors were shown to bind reversibly to the nucleotide-binding site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). Here, we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R, and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analog phosphonoformic acid. However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on these data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.

| S-EPMC3682535 | biostudies-literature

Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain.

Project description:HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH dimer, in the presence and absence of Mg(2+) , were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (2011) Chem Biol Drug Des 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

| S-EPMC3465473 | biostudies-literature

Subtype-specific analysis of the K65R substitution in HIV-1 that confers hypersusceptibility to a novel nucleotide-competing reverse transcriptase inhibitor.

Project description: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.

| S-EPMC4432161 | biostudies-literature

Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.

Project description:AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

| S-EPMC313966 | biostudies-literature

Allosteric suppression of HIV-1 reverse transcriptase structural dynamics upon inhibitor binding.

Project description:Efavirenz is a second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) and a common component of clinically approved anti-AIDS regimens. NNRTIs are noncompetitive inhibitors that bind in a hydrophobic pocket in the p66 subunit of reverse transcriptase (RT) ?10 Å from the polymerase active site. Hydrogen exchange mass spectrometry (HXMS) shows that efavirenz binding reduces molecular flexibility in multiple regions of RT heterodimer in addition to the NNRTI binding site. Of the 47 peptic fragments monitored by HXMS, 15 showed significantly altered H/D exchange rates in the presence of efavirenz. The slow cooperative unfolding of a ?-sheet in the NNRTI binding pocket, which was previously observed in unliganded RT, is dramatically suppressed by efavirenz. HXMS also defines an extensive network of allosterically coupled sites, including four distinct regions of allosteric stabilization, and one region of allosteric destabilization. The effects of efavirenz binding extend > 60 Å from the NNRTI binding pocket. Allosteric changes to the structural dynamics propagate to the thumb and connection subdomains and RNase H domain of the p66 subunit as well as the thumb and palm subdomains of the p51 subunit. These allosteric regions may represent potential new drug targets.

| S-EPMC3010017 | biostudies-literature

Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase.

Project description:Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information.

| S-EPMC2443788 | biostudies-literature

Structural Aspects of Drug Resistance and Inhibition of HIV-1 Reverse Transcriptase.

Project description:HIV-1 Reverse Transcriptase (HIV-1 RT) has been the target of numerous approved anti-AIDS drugs that are key components of Highly Active Anti-Retroviral Therapies (HAART). It remains the target of extensive structural studies that continue unabated for almost twenty years. The crystal structures of wild-type or drug-resistant mutant HIV RTs in the unliganded form or in complex with substrates and/or drugs have offered valuable glimpses into the enzyme's folding and its interactions with DNA and dNTP substrates, as well as with nucleos(t)ide reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTIs) drugs. These studies have been used to interpret a large body of biochemical results and have paved the way for innovative biochemical experiments designed to elucidate the mechanisms of catalysis and drug inhibition of polymerase and RNase H functions of RT. In turn, the combined use of structural biology and biochemical approaches has led to the discovery of novel mechanisms of drug resistance and has contributed to the design of new drugs with improved potency and ability to suppress multi-drug resistant strains.

| S-EPMC2850067 | biostudies-literature

Structural basis of reverse nucleotide polymerization.

Project description:Nucleotide polymerization proceeds in the forward (5'-3') direction. This tenet of the central dogma of molecular biology is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand synthesis where reverse polymerization (3'-5') would present a "simpler" solution. Interestingly, reverse (3'-5') nucleotide addition is catalyzed by the tRNA maturation enzyme tRNA(His) guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNA(His) guanylyltransferase-tRNA(His) complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme's active site from the opposite direction (180° flip) compared with similar nucleotide substrates of canonical 5'-3' polymerases, and the finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that reverse polymerization appeared early in evolution and resembles a mirror image of the forward process.

| S-EPMC3876247 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data