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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 28; pp. 8596 - 8601
• Main Authors: Sohl, Christal D., Szymanski, Michal R., Mislak, Andrea C., Shumate, Christie K., Amiralaei, Sheida, Schinazi, Raymond F., Anderson, Karen S., Yin, Y. Whitney
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 14.07.2015; National Acad Sciences
• Subjects: Biological Sciences; Crystallography, X-Ray; DNA Polymerase gamma; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - metabolism; Human immunodeficiency virus; Humans; Kinetics; Mitochondria - drug effects; Mitochondria - enzymology; Molecular Probes; Nucleic Acid Synthesis Inhibitors - pharmacology; Protein Conformation; Reverse Transcriptase Inhibitors - pharmacology; Substrate Specificity; nucleoside reverse transcriptase inhibitors; mitochondrial toxicity; human mitochondrial DNA polymerase; drug efficacy and toxicity; HIV reverse transcriptase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1421733112

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 (Polγ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Polγand 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 Polγdiscriminates 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 Polγfour 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.