Bisphosphonate inhibitors of NRTI excision

描述（由申请人提供）：fda批准的17种治疗HIV-1感染的抗逆转录病毒药物中有8种是NRTI，如AZT。NRTI是HIV-1 RT使用的dNTP底物的3‘-羟基缺失类似物，因此当NRTI被添加到新生DNA链的3’端时，病毒DNA合成被终止。HIV-1对AZT（以及包括阿巴卡韦在内的其他NRTI）的耐药性是一个严重的治疗问题，它是由rt催化的合并NRTI的磷酸化水解切除引起的。RT中被称为TAMs（胸腺嘧啶类似物突变）的特定突变增强了切除。切除合并的NRTI后，病毒DNA合成可以恢复完成病毒复制。NRTI切除的特异性抑制剂可以防止合并NRTI的去除，从而恢复NRTI的活性，如AZT和阿巴卡韦，以对抗含有HIV-1的TAM。理想情况下，这些抑制剂不应减少NRTI的掺入，因为这将明显地拮抗它们的抗逆转录病毒活性。我们假设可以识别选择性NRTI切除抑制剂，因为NRTI掺入和切除反应使用不同的底物，并且具有不同的限速步骤。我们的初步数据显示，某些双膦酸盐化合物可能在这种情况下有效用。因此，我们建议(1)筛选和表征现有的双膦酸盐库。这包括开发快速引物解锁筛选试验，以及筛选命中点的详细生化和病毒学分析；(2)优化双膦酸盐抑制剂对NRTI切除的效价。这包括预测最佳双膦酸盐类似物药效团的分子建模，以及选定的与RT -模板/ azt引物共价二元配合物结合的双膦酸盐类似物的晶体学研究；(3)合成并表征Aim 2预测的新的抑制剂类似物；(4)评价改善双膦酸盐细胞摄取的方法。这些方法包括使用药物载体和对双膦酸盐进行化学修饰以改善细胞摄取和抗病毒活性。

DESCRIPTION (provided by applicant): Eight of the 17 FDA-approved antiretroviral drugs for treating HIV-1 infection are NRTI such as AZT. NRTI are 3'-hydroxyl lacking analogs of the dNTP substrates used by HIV-1 RT, thus when an NRTI is added to the 3'-end of the nascent DNA chain, viral DNA synthesis is terminated. HIV-1 resistance to AZT (and other NRTI including abacavir) is a serious therapeutic problem and arises from RT-catalyzed phosphorolytic excision of the incorporated NRTI. Excision is enhanced by specific mutations in RT termed TAMs (thymidine analog mutations). After excision of the incorporated NRTI, viral DNA synthesis can resume to complete viral replication. Specific inhibitors of NRTI excision would be therapeutically useful by preventing removal of incorporated NRTI, thereby restoring activity of NRTI such as AZT and abacavir against TAM containing HIV-1. Optimally, such inhibitors should not reduce incorporation of NRTI as this would obviously antagonize their antiretroviral activity. We hypothesize that selective inhibitors of NRTI excision can be identified because NRTI incorporation and excision reactions use different substrates and have different rate limiting steps. Our preliminary data show certain bisphosphonate compounds may have utility in this context. We therefore propose (1) To screen and characterize an existing library of bisphosphonates. This includes development of a rapid primer unblocking screening assay, and detailed biochemical and virological analyses of screening hits; (2) To optimize the potency of bisphosphonate inhibitors of NRTI excision. This includes molecular modeling to predict the optimal bisphosphonate analog pharmacophore, and crystallographic studies of selected bisphosphonate analogs bound to covalent binary complexes of RT - template/AZT-primer; (3) To synthesize and characterize new inhibitor analogs predicted from Aim 2.; (4) To evaluate approaches to improve cell uptake of bisphosphonates. These include the use of drug carriers and chemical modification of bisphosphonates to improve cell uptake and antiviral activity.