X-ray crystal structures at 2.9 Å resolution are reported for complexes of catechol diethers 1 and 2 with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.
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.
Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPolmediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol ␥ that may manifest in toxicity.N ucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antivirals that target the human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT). All FDA-approved NRTIs are nucleoside analogs that lack a 3=-hydroxyl moiety to terminate DNA chain extension upon incorporation by RT into the growing proviral DNA. While significant health advances have been achieved with the use of NRTIs, the necessity for lifelong treatment to control HIV infection is limited by NRTI-associated toxicities that arise from virus-versus-host polymerase selectivity wherein NRTIs also serve as substrates for host polymerases (1, 2).The most severe NRTI-associated toxicities predominantly manifest in mitochondrial dysfunction (1-6) and are attributed primarily to incorporation by the human mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) (7-9). However, there are observed discrepancies between toxicity and the potential to inhibit mtDNA Pol ␥, suggesting alternative mechanisms and evaluation of additional host cell polymerases as potential perpetrators of antiviral toxicity (10, 11). Understanding the propensity for host cell polymerases to incorporate nucleoside analogs is critical for assessing safety in the design and development of antiviral, -parasitic, -bacterial, and -cancer nucleoside analog therapies. For instance, development of the antiviral ribonucleoside triphosphate analog BMS-986094 for the treatment of hepatitis C virus was halted in phase II after nine patients were hospitalized and one died (12). Investigational in vitro studies had identified that BMS-986094 was incorporated by the human mitochondrial RNA polymerase 30-fold more ...
Catechol diether compounds have nanomolar antiviral and enzymatic activity against HIV with reverse transcriptase (RT) variants containing K101P, a mutation that confers high-level resistance to FDA-approved non-nucleoside inhibitors efavirenz and rilpivirine. Kinetic data suggests that RT (K101P) variants are as catalytically fit as wild-type and thus can potentially increase in the viral population as more antiviral regimens include efavirenz or rilpivirine. Comparison of wild-type structures and a new crystal structure of RT (K101P) in complex with a leading compound confirms that the K101P mutation is not a liability for the catechol diethers while suggesting that key interactions are lost with efavirenz and rilpivirine.
The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed.
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