Optimizing HIV reverse transcriptase inhibitors through structural, kinetic and cellular approaches

 描述（由申请人提供）：逆转录病毒HIV是全球艾滋病流行的病原体，自1981年发现以来一直是一个主要的健康问题。目前，最成功的治疗方案是高效抗逆转录病毒疗法（HAART），它结合了目前FDA批准的26种治疗艾滋病毒药物中的三种或四种。这些药物中有一半针对病毒复制所必需的逆转录酶（RT）。在RT抑制剂中，五种是非核苷类逆转录酶抑制剂（NNRTI），其结合到RT的变构口袋。NNRTI本身可以抑制聚合酶功能，或与结合到酶的活性位点的核苷/核苷酸逆转录酶抑制剂（NRTI）协同抑制聚合酶功能。目前，在HAART中，NNRTI总是与NRTI联合使用，以最大限度地发挥协同作用和疗效。虽然HAART已成功地延长了HIV感染者的预期寿命，但这种治疗方案存在一些限制，包括单个药物的溶解度，长期毒副作用和耐药性。因此，本研究的长期目标是开发更安全的下一代治疗药物，具有改善的药理学特性（PK/PD）和溶解度，可以克服耐药变体。使用计算方法，我们以前已经开发了新的NNRTI，对HIV-1以及具有常见RT突变的HIV-1耐药变体具有非常有效的抗病毒活性（最佳EC 50 = 530 pM）。此外，我们的化合物表现出比FDA批准的第二代NNRTI依曲韦林和利匹韦林低得多的细胞毒性和更高的溶解度。为了进一步将这些化合物开发成有用的治疗剂，将使用预稳态动力学测定结合亲和力和缔合/解离速率。此外，RT-抑制剂的相互作用将阐明与X-射线晶体学提供一个平台，合理的药物设计。此外，我们实验室开发的新型NNRTI的抗病毒活性将使用基于细胞的测定来测量，并且将检查与当前FDA批准的NRTI组合时的效力变化，以找到显示最高协同效应的组合。最后，NRTI-NNRTI协同作用的分子机制将通过解决四元RT：DNA：NRTI：NNRTI结构来研究。总的来说，这项研究将使我们能够开发出具有更好药理学特性的更有效的NNRTI，并确定NNRTI和NRTI的最佳组合用于联合治疗，降低治疗失败率并延迟耐药性的发展。

 DESCRIPTION (provided by applicant): Retrovirus HIV, the etiological agent of the global AIDS epidemic, has been a major health concern since its discovery in 1981. Currently, the most successful treatment regimen is the highly active anti-retroviral therapy (HAART), which combines three or four of the current 26 FDA-approved HIV drugs in treatment. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. Among the RT inhibitors, five are non-nucleoside reverse transcriptase inhibitors (NNRTIs) which bind to an allosteric pocket of RT. NNRTIs can inhibit the polymerase function by themselves, or synergistically with nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) that bind to the active site of the enzyme. Currently, NNRTIs are always combined with NRTIs in HAART to maximize synergy and efficacy. Although HAART has been successful in extending the life expectancy of HIV-infected individuals, several limitations to this treatment regimen exist, including solubility of individual drug, long-term toxic side effects and drug resistance. Therefore, the long term goal of this research is to develop safer, next generation therapeutics with improved pharmacological properties (PK/PD) and solubility that can overcome resistant variants. Using computational approaches, we have previously developed novel NNRTIs that have very potent antiviral activity (best EC50 = 530 pM) against HIV-1 as well as resistant variants of HIV-1 with common RT mutations. Moreover, our compounds exhibit much lower cytotoxicity and higher solubility than the FDA-approved second generation NNRTIs etravirine and rilpivirine. In order to further develop these compounds into useful therapeutics, binding affinity and association/dissociation rates will be determined using pre-steady state kinetics. Moreover, RT- inhibitor interactions will be elucidated with x-ray crystallography to provide a platform for rational drug design. Furthermore, antiviral activity of novel NNRTIs developed in our lab will be measured using cell-based assays, and changes in potency when combined with current FDA-approved NRTIs will be examined to find the combination that displays the highest synergistic effect. Finally, the molecular mechanism of NRTI-NNRTI synergy will be investigated by solving a quaternary RT:DNA:NRTI:NNRTI structure. Overall, this research will allow us to develop more potent NNRTIs with improved pharmacological properties, and identify the optimal combination of NNRTI and NRTI for combination therapy that should lower treatment failure rate and delay development of resistance.