Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Zhang, Zhijun; Walker, Michelle; Xu, W.; Shim, Jae Hoon; Girardet, Jean‐Luc; Hamatake, Robert; Hong, Zhi

doi:10.1128/aac.00127-06

Cited by 47 publications

(63 citation statements)

References 31 publications

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“…Previous biochemical studies by our group have shown that INDOPY-1 binds preferentially following incorporation of pyrimidines, stabilizes the RT-DNA/DNA complex in its post-translocated state, and blocks DNA synthesis in a reversible manner (5). While the resistance profile for this inhibitor is unique, changes in susceptibility to INDOPY-1 have been associated with a number of mutations that likewise change susceptibility to NRTIs (5,6). Here we studied the biochemical phenotypes associated with these changes to provide novel insight into both mechanisms of resistance, and the binding properties of INDOPY-1.…”

Section: Discussionmentioning

confidence: 99%

“…Together, these findings suggest that the binding site for indolopyridones and nucleotide substrates can at least partially overlap. The resistance profile of INDOPY-1 provides further independent evidence for this notion (5,6). In vitro selection experiments and phenotypic susceptibility measurements with clinical isolates and constructs generated by site-directed mutagenesis suggest that most mutations associated with decreased susceptibility to INDOPY-1 are clustered around the dNTP binding site.…”

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confidence: 91%

“…The combination of M184V and Y115F is associated with decreased susceptibility to guanosine analogue abacavir (ABC) (9). Of note, K65R, which is associated with decreased susceptibility to tenofovir (TFV) (10), confers increased susceptibility to INDOPY-1 (5,6). The inhibitor is generally sensitive against a background of thymidine analogue-associated mutations (TAMs) or NNRTI-associated mutations, respectively, with the exception of the novel mutation L234F that is located in close proximity to the NNRTIbinding pocket (11).…”

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confidence: 99%

“…The prototype compound INDOPY-1 ( Fig. 1) has been shown to be active against NNRTI-resistant HIV strains (6). INDOPY-1, unlike NNRTIs, but like natural deoxyribonucleoside triphosphates (dNTPs), can bind to and stabilize RT-DNA/DNA complexes (5).…”

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confidence: 99%

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Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Ehteshami

Scarth

Tchesnokov

et al. 2008

Journal of Biological Chemistry

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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. NRTIassociated 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).The polymerase active site of the reverse transcriptase (RT) 3 enzyme of the human immunodeficiency virus type 1 (HIV-1) is a target for two classes of approved antiretroviral drugs referred to as nucleoside analogue RT inhibitors (NRTIs) and non-nucleoside analogue RT inhibitors (NNRTIs). Once phosphorylated, NRTIs act as chain-terminators that compete with natural nucleotide substrates while NNRTIs comprise a structurally diverse family of compounds that bind to a hydrophobic pocket near the active site of RT and appear to affect the chemical step of the reaction and not nucleotide binding (reviewed in Refs. 1-4).Indolopyridones represent a newly discovered class of inhibitors that interfere with RT function through a mechanism of action that is distinct from that described for NRTIs and NNRTIs (5). The prototype compound INDOPY-1 (Fig. 1) has been shown to be active against NNRTI-resistant HIV strains (6). INDOPY-1, unlike NNRTIs, but like natural deoxyribonucleoside triphosphates (dNTPs), can bind to and stabilize RT-DNA/DNA complexes (5). Footprinting experiments and binding studies revealed that the complex with INDOPY-1 is trapped in the post-translocational state that likewise allows dNTP binding. However, in contrast to NRTI or dNTP substrates, binding of INDOPY-1 depends on the chemical nature of the ultimate base pair at the 3Ј-end of the primer and no...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 91%

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confidence: 99%

mentioning

confidence: 99%

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Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Ehteshami

Scarth

Tchesnokov

et al. 2008

Journal of Biological Chemistry

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“…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).…”

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confidence: 99%

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

Auger

Beilhartz

Zhu

et al. 2011

Journal of Biological Chemistry

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The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre-and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA⅐DNA primer/template substrates versus DNA⅐DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA⅐DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.HIV RT is a multifunctional enzyme that catalyzes the conversion of the single-stranded RNA HIV genome to doublestranded DNA that is then incorporated into the host genome by the HIV integrase. In the process, RT must support RNAdirected and DNA-directed DNA synthesis along with ribonuclease H (RNase H) 2 activities to create an integration-competent product. A key step during reverse transcription is the incomplete hydrolysis of the (ϩ)-stranded RNA that leaves behind a purine-rich RNA sequence referred to as the polypurine tract (PPT). The remnant RNA PPT sequence serves as a primer to initiate synthesis of the (ϩ)-strand DNA (second strand) (1-3). The unique structure of the RNA PPT sequence has been shown to be a key determinant of the RNase H cleavage specificity at the PPT/U3 junction (4). A recent report described the mechanism by which RT discriminates between polymerase and RNase H activities thereby enabling more efficient initiation of polymerization from the RNA PPT primer versus other remnant RNA primers (5). Using single-molecule spectroscopy experiments, it was shown that RT binds nucleic acid substrates in two distinct orientations in a manner that is governed by the sugar backbone composition of the four or five nucleotides at each end of the primer. Depending on the binding orientation, RT either initiates polymerization at the 3Ј-end of the primer (polymerase binding mode on a DNA primer), or alternatively, RNA hydrolysis through the RNase H domain (RNase H bindi...

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Crystal Structure of HIV‐1 Reverse Transcriptase Bound to a Non‐Nucleoside Inhibitor with a Novel Mechanism of Action

et al. 2010

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Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Cited by 47 publications

References 31 publications

Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

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

Crystal Structure of HIV‐1 Reverse Transcriptase Bound to a Non‐Nucleoside Inhibitor with a Novel Mechanism of Action

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