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

 描述(申请人提供)：逆转录病毒艾滋病毒，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种艾滋病毒药物中的3到4种进行治疗。这些药物中有一半针对的是对病毒复制至关重要的逆转录酶(RT)酶。在RT抑制剂中，有五种是非核苷类逆转录酶抑制剂(NNRTI)，它们与RT的变构口袋结合。NNRTI可以单独或与核苷/核苷酸逆转录酶抑制剂(NRTI)协同抑制聚合酶功能，NRTI与酶的活性部位结合。目前，在HAART中，NNRTIs总是与NRTIs相结合，以最大限度地发挥协同作用和疗效。尽管HAART已经成功地延长了艾滋病毒感染者的预期寿命，但这种治疗方案存在一些限制，包括单个药物的溶解、长期毒副作用和耐药性。因此，这项研究的长期目标是开发更安全的下一代疗法，具有更好的药理特性(PK/PD)和溶解性，可以克服耐药变体。使用计算方法，我们以前已经开发出对HIV-1以及具有常见RT突变的HIV-1的耐药变体具有非常强大的抗病毒活性(最佳EC50=530 PM)的新型NNRTI。此外，我们的化合物表现出比FDA批准的第二代NNRTIs依特拉韦林和利培韦林更低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药物，将使用预稳态动力学来确定结合亲和力和结合/解离速率。此外，RT-抑制剂的相互作用将用X射线结晶学来阐明，为合理的药物设计提供平台。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分析进行测量，并将检查与FDA批准的当前NRTI结合时效力的变化，以找到显示出最高协同效应的组合。最后，通过求解一个四元RT：DNA：NRTI：NNRTI结构来研究NRTI-NNRTI协同作用的分子机制。总体而言，这项研究将使我们能够开发更有效的NNRTI，具有更好的药理特性，并确定NNRTI和NRTI的最佳组合用于联合治疗，应降低治疗失败率并延缓耐药性的发展。

 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.