Capsid-Targeting Small Molecules Blocking HIV through Novel Mechanism of Action

产品说明：高效抗逆转录病毒疗法（HAART）已成功控制HIV感染患者的病毒载量，并显著降低了与HIV-1感染相关的发病率和死亡率。然而，抗药性HIV-1菌株的出现和在个体之间传播，降低了目前可用的抗病毒药物的疗效。因此，需要鉴定通过新机制起作用并且与现有疗法没有交叉耐药性的新型抗逆转录病毒药物（ART）。 HIV-1衣壳（CA）是复制周期早期和晚期事件所必需的病毒蛋白，迄今为止一直是一个未开发的目标，因此为发现通过新的作用机制发挥作用的新型ART提供了极好的机会。我们筛选了一个内部的化合物化学库，并确定了18 E8，一种干扰HIV-1 CA多聚化的小分子。我们证明，在细胞为基础的测定与完全感染的艾滋病毒，18 E8显示广泛的抗逆转录病毒活性（EC 50低至1？M）针对多种实验室菌株和多种多重耐药临床分离株。在另外的初步实验中，我们表明18 E8通过结合HIV-1 CA发挥其抗逆转录病毒活性。该筛选中的另外6个命中尚未表征。 为了深入了解18 E8的作用模式并确定其影响的病毒复制周期的具体步骤，我们进行了药物添加时间实验， 18 E8在细胞培养中失去抗病毒活性之前可以推迟多长时间加入。令人惊讶的是，我们的初步数据表明，18 E8靶向逆转录后HIV复制周期的早期步骤。18 E8似乎不影响小瓶生命周期的后期阶段。基于这些数据，我们假设18 E8的独特作用机制是由于其结合方式不同于影响病毒生命周期其他步骤的其他CA靶向化合物。我们将探索这一假设，并使用相同的方法来评估和表征以下两个目标中的额外复合命中：具体目标1。18 E8和其他化合物的功能和机理表征CA与18 E8和其他化合物相互作用的晶体学表征。拟议研究的可交付成果包括至少一种具有新作用机制的新型CA靶向抗病毒药物，其生物学和结构作用机制可指导新型抗病毒药物的设计。

DESCRIPTION: Highly Active Antiretroviral Therapies (HAART) have been successful at controlling viral load in HIV-infected patients and have significantly decreased the morbidity and mortality associated with HIV-1 infection. However, drug resistant HIV-1 strains arise and are transmitted between individuals, reducing the efficacy of currently available antivirals. Thus, identification of novel antiretrovirals (ARTs) that act by new mechanisms and have no cross-resistance with existing therapeutics is required. HIV-1 capsid (CA) is a viral protein essential for early and late events of the replication cycle and so far has been an untapped target, thus providing excellent opportunities for the discovery of novel ARTs that act by novel mechanisms of action. We screened an in-house chemical library of compounds and identified18E8, a small molecule that interferes with multimerization of HIV-1 CA. We demonstrated in cell-based assays with fully-infectious HIV that 18E8 showed broad antiretroviral activity (EC50 as low as ~1?M) against multiple laboratory strains and several multi-drug resistant clinical isolates. In additional preliminary experiments we showed that 18E8 exerts its antiretroviral activity by binding to HIV-1 CA. Six additional hits from this screening have not yet been characterized. In order to gain insight into the mode of action of 18E8 and to determine the specific step of the viral replication cycle that it affects, we performed time-of-drug-addition experiments that define how long the addition of 18E8 could be postponed before losing its antiviral activity in cell culture. Surprisingly, our preliminary data suggest that 18E8 targets an early step in the HIV replication cycle, after reverse transcription. 18E8 did not appear to affect late stages of the vial life cycle. Based on these data, we hypothesize that the unique mechanism of action of 18E8 is due to its binding in a manner different than other CA-targeting compounds that affect other steps of the virus life cycle. We will explore this hypothesis and use the same approaches to evaluate and characterize additional compound hits in the following two aims: Specific Aim 1. Functional and mechanistic characterization of 18E8 and other compounds Specific Aim 2. Crystallographic characterization of CA interactions with 18E8 and other compounds. The deliverables of the proposed studies include at least one novel CA-targeting antiviral with a new mechanism of action, its biological and structural mechanism of action that could guide the design of novel antivirals.