Human Mitochondrial Dna Polymerase With Anti-hiv Nucleotides

具有抗 HIV 核苷酸的人线粒体 DNA 聚合酶

• 批准号: 8553737
• 负责人: William Copeland
• 金额: $ 43.1万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553737
• 关键词: 2' -deoxyadenosine; Acquired Immunodeficiency Syndrome; Adult; Adverse effects; Anti-HIV Agents; Anti-Retroviral Agents; Antiviral Agents; Antiviral Therapy; Cells; Characteristics; Clinical; Clinical Trials; DNA; DNA biosynthesis; DNA polymerase gamma; DNA-Directed DNA Polymerase; Development; Didanosine; Discrimination; Disease; Enzymes; Equilibrium; Excision; Exhibits; Exonuclease; Fiber; Goals; HIV; HIV Infections; HIV-1; Human; In Vitro; Individual; Kinetics; Lamivudine; Life; Light; Mediating; Mitochondria; Mitochondrial DNA; Molecular; Muscle; Mutation; Myopathy; Neuropathy; Nuclear; Nucleosides; Nucleotides; Patients; Pattern; Phase; Phase II Clinical Trials; Phosphodiesterase I; Polymerase; Property; Proteins; RNA-Directed DNA Polymerase; Recombinant DNA; Reporting; Reverse Transcriptase Inhibitors; Role; Stavudine; Tenofovir; Thymidine; Toxic effect; Viral; Zalcitabine; Zidovudine; abacavir; aged; alovudine; analog; antiviral nucleoside analog; deoxyguanosine triphosphate; dideoxynucleotide; dideoxythymidine; experience; in vivo; inhibitor/antagonist; novel; nucleoside analog; nucleotide analog; pinacolyl methylphosphonic acid; tripolyphosphate; zidovudine triphosphate

We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2-3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2-3-dideoxy-CTP (ddCTP), 2-3didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-dGTP (CBV-TP). Human pol gamma readily incorporated all five analogs into DNA but with varying efficiencies. Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) 8805; ddI(didanosine) 8805; D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. With the exception of terminally incorporated 3TC, the pol gamma 3-5 exonuclease was inefficient at removing these five analogs from DNA and removal required enzyme levels exceeding substrate concentrations. Even though discrimination against inserting AZT and CBV makes them only moderate inhibitors in vitro, their inefficient excision suggest AZT and CBV may persist in vivo once incorporated into mtDNA by pol gamma. Finally, we found that the exonuclease activity is inhibited by AZT-monophosphate at concentrations known to occur in cells. Thus, although these analogs exert their greatest effect by insertion and chain termination of DNA synthesis, the persistence in DNA and inhibition of proofreading activity may also contribute to mitochondrial toxicity. To further investigate the potential mitochondrial toxicity from nucleoside reverse transcriptase inhibitors, we have performed studies on several new antiviral drugs. The potent antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase gamma to assess the potential for toxicity. Pol gamma incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol gamma substrate, suggesting minimal Pol gamma-mediated toxicity, although this should be examined under clinical settings. Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase gamma, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol gamma, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol gamma could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2',3'-didehydro-2',3'-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4'-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2',3'-didehydro-2',3'-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol gamma-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.

我们先前以前比较了纯化的人类重组DNA聚合酶伽玛上目前批准的五个抗病毒核苷酸类似物的抑制作用，插入和外核解解去除。确定了明显的KM和KCAT值，以掺入TTP，DCTP，DGTP，2-3-二维氧基-TTP（DDTTP），3-氮杂-TTP（AZT-TP），2-3-DIDEOXY-CTP（DDCTP（DDCTP），2-3DDIDEDRO-TTP（2-3DDIDEHDRO-TTP（D4TRO）） （ - ） - 2,3-二维氧基-3- thiacytidine（3TC-TP）和2,3-Didehydro-DGTP（CBV-TP）。人类Pol Gamma很容易将所有五个类似物纳入DNA，但效率不同。动力学研究表明，对批准的NRTI的线粒体毒性的明显体外层次结构是：DDC（Zalcitabine）8805； DDI（Didanosine）8805; D4T（Stavudine）>> 3TC（Lamivudine）> PMPA（Tenofovir）> AZT（Zidovudine）> CBV（Abacavir）。人类POL GAMMA在体外使用二乙氧基核苷酸和D4T-TP的体外效率和天然脱氧核苷三磷酸盐，而AZT-TP，3TC-TP和CBV-TP是中等链延长的抑制剂。除终端掺入3TC外，Pol Gamma 3-5核酸酶在从DNA中去除这五个类似物的效率低下，并且去除所需的酶水平超过底物浓度。即使对插入AZT和CBV的歧视使它们仅在体外中度抑制剂，但它们效率低下的切除表明AZT和CBV可能会在Vivo中持续存在，曾经由Pol Gamma掺入mtDNA中。最后，我们发现核酸酶活性被AZT-单磷酸盐以已知在细胞中发生的浓度抑制。因此，尽管这些类似物通过插入DNA合成的插入和链终止产生了最大的作用，但是DNA和抑制校对活性的持久性也可能有助于线粒体毒性。 为了进一步研究核苷逆转录酶抑制剂的潜在线粒体毒性，我们对几种新的抗病毒药药进行了研究。 有效的抗逆转录病毒4'-乙基-2-氟-2'-脱氧腺苷（EFDA）是治疗HIV感染的有前途的实验剂。稳态前动力学用于表征EFDA-三磷酸酯（EFDA-TP）与人线粒体DNA聚合酶伽玛的相互作用，以评估毒性的潜在。 Pol Gamma掺入EFDA-TP的效率比DATP少4,300倍，切除率类似于DDATP。这强烈表明EFDA是一个较差的POL GAMMA底物，表明POL GAMMA介导的毒性最少，尽管应在临床环境下检查。 两种新型胸苷类似物，3'-氟-3'-脱氧胸苷（FLT）和2'，3'-二氢-3'-二氧基-4'-4'-4'-乙基甲酰胺（ED4T），已被研究为用于治疗HIV的核苷逆转录酶抑制剂（NRTIS）。在II期临床试验中，ED4T似乎非常有前途，而在此阶段，毒性停止了FLT的发展。为了理解这些不同的毒性分子机制，使用稳态的态动力学研究来检查FLT和ED4T与野生型（WT）人线粒体DNA聚合酶γ的相互作用，以及病毒靶标蛋白，WT HIV-1逆转录酶（RT）。我们报告说，ED4T-三磷酸盐（TP）是第一个类似物，而不是天然核苷酸优于天然核苷酸，但经历了WT Pol Gamma的可忽略的掺入，在高抗逆转录病毒功效和最小宿主毒性之间具有理想的平衡。 WT Pol Gamma可以比RT更好地区分ED4T-TP 12,000倍，而FLT-TP的歧视差异仅为8.3倍。与本机核苷酸相比，与天然核苷酸相比，与结构相关的NRTI，2'，3'-二羟基-2'，3'-二氧甲基甲烷是唯一的另一个类似物，但在两种酶之间的区分中，它仅表现出13倍差（与ED4T相比12,000倍）。我们建议ED4T的4'-乙基尼尔组充当酶选择性部分，对于RT和WTpolγ之间的识别至关重要。我们还表明，在接受2'，3'-Didehydro-2'，3'-二维噻天基蛋白治疗HIV时，polγ突变R964C倾向于患者会导致线粒体毒性，从而导致FLT-TP和ED4T-TP的歧视丧失。这些模拟掺入的分子机制对于理解与Pol Gamma相关的毒性至关重要，它阐明了在临床试验期间观察到的独特毒性曲线。