Anti-HIV and anti-reactivation activities of pyrrolopyridine-based ALLINIs

项目摘要 为全球3790万艾滋病毒感染者获得功能性治愈的首要障碍是 消除HIV-1病毒库。病毒整合酶（IN）在转录不活跃但 通过IN将前病毒DNA整合到宿主染色体中而使复制感受态细胞中。国产化 整合由宿主因子LEDGF/p75引导，其结合IN束缚到活性转录基因密集区 的DNA。HIV-1 IN是主要的抗病毒靶点之一，目前已开发出多种IN抑制剂 作为当前抗逆转录病毒疗法（ART）的关键组成部分，但受到病毒耐药性、交叉耐药性 和毒性。快速开发具有高遗传屏障、新作用模式（MOA）、安全性 DMPK配置文件是迫切需要的。变构IN抑制剂（ALLINI）是一类新的IN抑制剂， HIV-1 IN的非催化位点。STP 0404是一种基于吡咯并吡啶的ALLINI，具有出色的疗效、PK和 在临床前体外和动物研究中观察到的安全性特征。STP 0404成为第一个在人体中 (FIH)ALLINI化合物证明其出色的安全性和人体PK特征，每日一次口服给药 该制剂进一步支持其在HIV-1患者中抗病毒疗效的高级临床评估。到 这种效果，我建议探索一种新的第二代ALLINI化合物，我们的实验室设计的潜在 改善了遗传屏障，并可能用作HIV-1治疗剂。我发现了A128 T IN突变体 病毒对STP 0404的耐药性，我们的X射线结构研究证实了A128 T冲突的较长侧链 与STP 0404吡咯部分的3-甲基结合，导致与STP 0404的结合亲和力降低 和对STP 0404的病毒抗性。为了避免A128 T突变体与STP 0404的甲基接触， 我们的团队最近合成了一种新的STP 0404衍生物EKC 110，缺少甲基接触 A128 T侧链。在这里，我建议测试EKC 110显示改善遗传屏障的假设 与亲本STP 0404相比，具有改变的抗性突变谱。我们的初步数据显示 支持这一假设，另外新出现的证据报告ALLINI阻断LEDGF/p75结合 到IN，称为LEDGIN，可以驱动HIV-1的整合位点特异性朝向非活性转录区域 减少HIV-1从潜伏感染的T细胞库中的再活化。在目标1中，我将 利用分子、病毒学和细胞生物学技术研究新型STP 0404衍生物EKC 110 遗传屏障、病毒耐药性、抗病毒MOA，以确定结构修饰对HIV-1的影响 抗病毒活性。目的2：研究STP 0404和EKC 110对HIV-1整合位点选择的影响 使用生物化学、分子生物学和细胞生物学方法， 技术.总的来说，这些目标将证明我们的基于吡咯并吡啶的ALLINI如 EKC 110可能是一种新的抗逆转录病毒药物，不仅具有改善遗传屏障的作用，而且具有抑制HIV再活化的作用 作为一种“封锁和锁定”剂的能力，可用于病毒感染和持久性。

Project Summary The overarching obstacle in obtaining a functional cure for the 37.9 millions of people living with HIV globally is eliminating the HIV-1 reservoirs. Viral integrase (IN) establishes lifelong infection in transcriptionally inactive but replication competent cells by IN integrating proviral DNA into the host chromosome. The localization of integration is directed by host factor LEDGF/p75 binding IN tethering to active transcriptional gene dense regions of DNA. HIV-1 IN is one of the major antiviral targets, and multiple IN inhibitors (INSTs) have been developed as a key component of current antiretroviral therapy (ART) but are subjected to viral resistance, cross-resistance and toxicity. Rapid development of IN antivirals with high genetic barrier, new modes of action (MOA), with safe DMPK profiles are in immediate need. Allosteric IN inhibitors (ALLINIs) are a new class of IN inhibitors targeting non-catalytic sites of HIV-1 IN. STP0404 is a pyrrolopyridine-based ALLINI with outstanding efficacy, PK and safety profiles observed in both preclinical in vitro and animal investigations. STP0404 became the first-in-human (FIH) ALLINI compound demonstrating its outstanding safety and human PK profile for once-a-day oral formulations, which further supports advanced clinical evaluations for its antiviral efficacy in HIV-1 patients. To this effect, I propose to explore a new 2nd-generation ALLINI compound our laboratory designed for potentially improved genetic barrier and possible utility as an HIV-1 cure agent. I have identified A128T IN mutant rendering viral resistance to STP0404, and our X-ray structural study confirmed the longer side chain of A128T clashes against 3-methyl group at the pyrrole moiety of STP0404, contributing to the reduced binding affinity to STP0404 and viral resistance to STP0404. To avoid contact between the A128T mutant and the methyl group of STP0404, our group recently synthesized a new STP0404 derivative, EKC110, with missing the methyl group contacting the A128T side chain. Here, I propose to test the hypothesis that EKC110 shows improved genetic barrier with altered resistance mutation profiles, compared to the parental STP0404. Our preliminary data has supported this hypothesis, additionally emerging evidence has reported ALLINIs blocking LEDGF/p75 binding to IN, called LEDGINs, can drive the integration site specificity of HIV-1 toward non-actively transcribed regions of the chromosomes, reducing HIV-1 reactivation from latently infected T cell reservoirs. In Aim 1, I will investigate a new STP0404 derivative, EKC110 using molecular, virological and cell biological techniques for its genetic barrier, viral resistance, antiviral MOA to determine the impact of structural modifications on HIV-1 antiviral activity. Aim 2, I will investigate the effect of STP0404 and EKC110 on HIV-1 integration site selection and reactivation suppression from HIV-1 latently infected T cells using biochemical, molecular, and cell biological techniques. Collectively these aims will demonstrate the utility that our pyrrolopyridine based ALLINIs such as EKC110 can be a new ART agent with not only improved genetic barrier but also HIV reactivation inhibition capability as a “Block and Lock” agent, which can be used for both viral infection and persistence.