A MULTIPRONGED, PROTEIN DEGRADATION-BASED APPROACH TO INHIBIT HIV TRANSMISSION

描述（由申请人提供）：阻断艾滋病毒传播是病毒学的一项关键挑战，也是一项紧迫的公共卫生需求。多管齐下的治疗方法为靶向这种快速进化的病原体提供了最有希望的方法。两种病毒蛋白靶点是整合酶和Vif，整合酶将逆转录的病毒基因组插入宿主细胞的DNA中，Vif将细胞抗病毒蛋白APOBEC 3G连接到E3泛素连接酶，导致其泛素化和蛋白酶体降解。我们之前开发了PROTAC（PROteolysis Targeting Chimeras），这是一种合成分子，其功能类似于Vif，通过将靶蛋白募集到E3连接酶，导致蛋白酶体降解。在这里，我们提出了一种新的，三管齐下的抗HIV方法，使用PROTAC降解整合酶，同时阻断整合酶和Vif功能。在概念验证实验中，我们将通过噬菌体展示鉴定整合酶的配体，该配体阻断与宿主因子透镜上皮衍生生长因子（LEDGF）的相互作用。同时，我们将优化与细胞蛋白Elongin B/C和Cullin 5相互作用的Vif片段，这些蛋白形成Vif途径的E3连接酶。然后，我们将创建一种结合这两个部分和一个（D-Arg）8细胞渗透性部分的异双功能肽，并测试其（a）在体外与E3连接酶和整合酶结合的能力;（B）在无细胞系统和培养细胞中使整合酶泛素化并降解，同时阻断整合酶-LEDGF和Vif-ABOPEC 3G相互作用;以及（c）减少细胞培养物中HIV传播的能力。一种重要病毒蛋白的降解加上阻断两种关键的宿主-病毒蛋白相互作用代表了抗HIV药物设计的新范式，并提供了作为治疗先导的巨大希望。虽然目前的抗HIV治疗策略在管理现有感染方面取得了成功，但需要新的创新方法来阻止HIV病毒在受感染细胞中的建立。在本申请中，我们提出了一种非常规的化疗方法来破坏HIV传播。也就是说，我们不是产生简单地抑制HIV编码蛋白质功能的小分子，而是提出诱导这种蛋白质的降解，从而将其从细胞中消除以阻断其功能。

DESCRIPTION (provided by applicant): Blockade of HIV transmission represents a key challenge in virology, and a pressing public health need. Multipronged therapeutic approaches offer the most promise for targeting this rapidly-evolving pathogen. Two viral protein targets are integrase, which inserts the reverse-transcribed viral genome into the host cell's DNA, and Vif, which links the cellular antiviral protein APOBEC3G to an E3 ubiquitin ligase, causing its ubiquitination and proteasomal degradation. We previously developed PROTACs (PROteolysis TArgeting Chimeras), which are synthetic molecules that function like Vif by recruiting a target protein to an E3 ligase, leading to proteasomal degradation. Here, we propose a novel, three-pronged anti-HIV approach using a PROTAC to degrade integrase while blocking integrase and Vif functions. In proof-of-concept experiments, we will identify by phage display a ligand for integrase that blocks interaction with the host factor lens epithelium-derived growth factor (LEDGF). Simultaneously, we will optimize fragments of Vif that interact with the cellular proteins Elongin B/C, and Cullin 5, which form the E3 ligase of the Vif pathway. We will then create a heterobifunctional peptide combining these two moieties plus a (D-Arg)8 cell permeability moiety and test it for the ability to (a) bind to both the E3 ligase and integrase in vitro; (b) cause integrase to be ubiquitinated and degraded in cell-free systems and in cultured cells, while blocking integrase-LEDGF and Vif-ABOBEC3G interactions; and (c) decrease HIV transmission in cell culture. The degradation of an essential viral protein coupled with blocking two key host-virus protein interactions represents a new paradigm in anti-HIV drug design, and offers great promise as a therapeutic lead. While current anti-HIV therapeutic strategies have been successful in managing existing infections, new and innovative approaches are needed to block the establishment of the HIV virus in infected cells. In this application, we propose an unconventional chemotherapeutic approach to disrupt HIV transmission. Namely, instead of generating small molecules that simply inhibit the function of a HIV-encoded protein, we propose to induce the degradation of such a protein, thus, eliminating it from cell in order to block its function.