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

摘要 高效抗逆转录病毒疗法（HAART）已成功控制HIV感染者的病毒载量。 患者，并显着降低了与HIV-1感染相关的发病率和死亡率。然而，在这方面， 抗药性HIV-1菌株出现并在个体之间传播，降低了目前的治疗效果。 可用的抗病毒药物因此，鉴定通过新的机制起作用并且具有以下特征的新型抗逆转录病毒药物（ART）， 不需要与现有治疗剂的交叉抗性。 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， 一直是一个未开发的目标，从而为发现新的ART提供了极好的机会， 新的作用机制。我们筛选了一个内部的化合物化学库，并确定了18 E8，一个 干扰HIV-1 CA多聚化的小分子。我们在基于细胞的试验中证明， 18 E8显示出广泛的抗逆转录病毒活性（EC 50低至~1 μ M）， 实验室菌株和几种多重耐药临床分离株。在附加的初步实验中， 表明18 E8通过与HIV-1 CA结合发挥其抗逆转录病毒活性。这次筛选中又有六个匹配结果 尚未被定性。 为了深入了解18 E8的作用模式，并确定病毒的具体步骤， 复制周期，它的影响，我们进行了时间的药物添加实验，定义多久， 18 E8的加入可以推迟到在细胞培养中失去其抗病毒活性之前。令人惊讶的是，我们 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似乎不影响病毒生命周期的晚期阶段。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其他CA靶向 影响病毒生命周期其他步骤的化合物。我们将探讨这一假设，并使用相同的 在以下两个目标中评价和描述其他复合命中的方法： 具体目标1。18 E8和其他化合物的功能和机理表征 具体目标2。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新型CA靶向抗病毒药物， 作用机制，其生物和结构的作用机制，可以指导设计的新的 抗病毒药

ABSTRACT 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 viral 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.