Crystal Structure of<i>tert</i>-Butyldimethylsilyl-spiroaminooxathioledioxide-thymine (TSAO-T) in Complex with HIV-1 Reverse Transcriptase (RT) Redefines the Elastic Limits of the Non-nucleoside Inhibitor-Binding Pocket

Das, Kalyan; Bauman, J.D.; Rim, Angela S.; Dharia, Chhaya; Clark, Arnold M.; Camarasa, Marı́a-José; Balzarini, Jan; Arnold, Eddy

doi:10.1021/jm101536x

Cited by 67 publications

(41 citation statements)

References 56 publications

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“…It has been proposed this could be a mechanism for the (-)-3TC resistance of M184I/V mutant enzymes (34). Additionally, even bound nonnucleoside reverse transcriptase inhibitors (NNRTIs) can alter the conformations of the active site or nucleic acid binding channel (35)(36)(37). On the contrary, the substrate-binding site of Pol γ undergoes more precise structural changes, evidenced by an unchanged O-helix configuration in normal substrate and inhibitor structures.…”

Section: Different Mechanisms For Pol γ and Rt Substrate Specificity Andmentioning

confidence: 99%

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Sohl

Szymanski

Mislak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

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Section: Different Mechanisms For Pol γ and Rt Substrate Specificity Andmentioning

confidence: 99%

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Sohl

Szymanski

Mislak

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Both are potent inhibitors of HIV-1 RTs that are resistant to first generation NNRTIs (nevirapine, delavirdine, and efavirenz). RPV and ETV demonstrate "strategic flexibility" that allows efficient binding to a high plasticity NNIBP (20,21,26,72,73). These two drugs were discovered by a multidisciplinary approach that involved x-ray crystallography, structure-based drug design, and in vivo testing (21, 74 -80).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Mechanism of HIV-1 Resistance to Rilpivirine

Singh

Marchand

Devendra

et al. 2012

Journal of Biological Chemistry

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Background: Reverse transcriptase mutations E138K and M184I emerged most frequently in HIV-1 patients who failed rilpivirine/emtricitabine/tenofovir combination therapy. Results: M184I reduces polymerase efficiency, and E138K restores it. E138K also reduces rilpivirine binding affinity mainly by increasing its dissociation rate. Conclusion: E138K abrogates the polymerase defect of M184I and increases rilpivirine dissociation. Significance: Our results provide a biochemical explanation for the selection of E138K/M184I in patients who failed combination therapy.

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“…This hydrophobic pocket was originally identified as the binding site for non-nucleoside reverse transcriptase inhibitors (NNRTIs) including Nevirapine and Efavirenz. [21] NNRTIs do not resemble nucleoside analogues and thus function as uncompetitive inhibitors by binding to the RT-DNA-dNTP complex as opposed to free RT or RT-DNA complex. [22] Furthermore, NNRTIs bind to a pocket that lies~10 from the polymerase's active site and that is lined with large and hydrophobic amino acids including Y181, Y188, F227, and L234.…”

Section: Discussionmentioning

confidence: 99%

Insights into the Roles of Desolvation and π‐Electron Interactions during DNA Polymerization

2013

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This report describes the use of several isosteric non-natural nucleotides as probes to evaluate the roles of nucleobase shape, size, solvation energies, and π-electron interactions as forces influencing key kinetic steps of the DNA polymerization cycle. Results are provided using representative high- and low-fidelity DNA polymerases. Results generated with the E. coli Klenow fragment reveal that this high-fidelity polymerase utilizes hydrophobic nucleotide analogues with higher catalytic efficiencies compared to hydrophilic analogues. These data support a major role for nucleobase desolvation during nucleotide selection and insertion. In contrast, the low-fidelity HIV-1 reverse transcriptase discriminates against hydrophobic analogues and only tolerates non-natural nucleotides that are capable of hydrogen-bonding or π-stacking interactions. Surprisingly, hydrophobic analogues that function as efficient substrates for the E. coli Klenow fragment behave as noncompetitive or uncompetitive inhibitors against HIV-1 reverse transcriptase. In these cases, the mode of inhibition depends upon the absence or presence of a templating nucleobase. Molecular modeling studies suggest that these analogues bind to the active site of reverse transcriptase as well as to a nearby hydrophobic binding pocket. Collectively, the studies using these non-natural nucleotides reveal important mechanistic differences between representative high- and low-fidelity DNA polymerases during nucleotide selection and incorporation.

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Crystal Structure oftert-Butyldimethylsilyl-spiroaminooxathioledioxide-thymine (TSAO-T) in Complex with HIV-1 Reverse Transcriptase (RT) Redefines the Elastic Limits of the Non-nucleoside Inhibitor-Binding Pocket

Cited by 67 publications

References 56 publications

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Biochemical Mechanism of HIV-1 Resistance to Rilpivirine

Insights into the Roles of Desolvation and π‐Electron Interactions during DNA Polymerization

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