Natural Product-Inspired Method for Enhancing HIV Protease Inhibitors

描述(申请人提供)：高效抗逆转录病毒疗法(HAART)在控制艾滋病毒感染/艾滋病方面非常有效。然而，由于治疗相关的并发症，患者对HAART的依从性往往是不同的。这是一个严重的问题，助长了耐药性的出现。导致HAART相关并发症的一个问题是HIV蛋白酶抑制剂代谢稳定性差，细胞渗透率低。我们小组一直在探索一种新的方法来解决这些限制。这一策略直接受到天然产物FK506的药理学特性的启发，尽管FK506在体外是P450酶的优秀底物，但它在人体内的半衰期长得惊人(T1/2~40小时)。我们最近想知道这种明显的矛盾是否可能是由于这种化合物对FK506结合蛋白(FKBP)的高亲和力造成的。血细胞，包括红细胞和白细胞，表达异常高水平的FKBP，但它们实际上缺乏P450酶。因此，我们假设，在这个受保护的细胞利基中，亲和力驱动的积累可能会限制对关键代谢酶的接触，从而延长药物的寿命。此外，FK506吸收迅速(约1小时)，对生物膜具有很高的渗透性，并通过其FKBP结合部分自然地靶向于白细胞。总而言之，这些特性似乎与艾滋病毒蛋白酶抑制剂面临的主要问题一致。基于这些观察，我们将FKBP结合基团与氨丙那韦类似物连接，创建了一种既能与FKBP结合又能与HIV蛋白酶结合的双功能分子。我们发现，所得化合物具有抗蛋白酶活性(IC50~20 nM)。此外，它现在被隔离在血细胞中(至少增加了8倍)，在小鼠身上的半衰期增加了约20倍。这种化合物的寿命优于利托那韦增强的阿普雷那韦，而且，它的代谢稳定性现在不依赖于利托那韦联合给药。基于这些有希望的初步发现，我们现在建议仔细探索控制这种行为的分子机制。具体地说，我们推断细胞的分配和寿命在一定程度上是由化合物对FKBP的亲和力决定的。为了探索这一中心假设，我们提出了以下具体目标：(1)合成一系列与FKBP结合的Amprenavir衍生物，并测量它们对白蛋白、FKBP和HIV蛋白酶的相对亲和力；(2)探索细胞分裂如何与结合亲和力和相对蛋白质表达水平相关。通过这些观察，我们希望了解三元复合体(即FKBP-药物-HIV蛋白酶)中的关键结构-活性关系。这项研究的直接目标是创造有效、安全和长寿的HIV蛋白酶抑制剂，选择性地针对表达FKBP的HIV感染细胞。这项研究意义重大，因为它解决了艾滋病治疗中的一个重要问题。这项拟议的工作具有创新性，因为它将探索一种全新的、“以天然产品为灵感”的战略。

DESCRIPTION (provided by applicant): Highly active anti-retroviral therapy (HAART) has been remarkably effective for managing HIV infection / AIDS. However, patient compliance with HAART is often variable due to treatment- related complications. This is a serious problem that facilitates the emergence of drug resistance. One issue that contributes to HAART-associated complications is the poor metabolic stability and low cellular penetration of the HIV protease inhibitors. Our group has been exploring a new method for addressing these limitations. This strategy is directly inspired by the pharmacological properties of the natural product, FK506, which has a surprisingly long halftime in humans (t1/2 ~ 40 hrs) despite being an excellent substrate for P450 enzymes in vitro. We recently wondered whether this apparent contradiction might arise from this compound's high affinity for the FK506-binding protein (FKBP). Blood cells, including both erythrocytes and leukocytes, express unusually high levels of FKBP but they are virtually devoid of P450 enzymes. Therefore, we hypothesized that affinity-driven accumulation within this protected cellular niche might limit exposure to key metabolic enzymes and, thereby, extend drug lifetime. Moreover, FK506 is rapidly absorbed (~1 hr), highly penetrant to biological membranes and naturally targeted to leukocytes via its FKBP-binding moiety. Together, these properties appear to align with the major problems facing HIV protease inhibitors. Based on these observations, we tethered an FKBP-binding group to an amprenavir analog, creating a bifunctional molecule that can bind both FKBP and HIV protease. We found that the resulting compound retained anti-protease activity (IC50 ~ 20 nM). Moreover, it was now sequestered into blood cells (by at least 8-fold) and its half-life was increased by ~ 20-fold in mice. The lifetime of this compound was superior to that of ritonavir-boosted amprenavir and, moreover, its metabolic stability was now independent of ritonavir co-administration. Based on these promising initial findings, we now propose to carefully explore the molecular mechanisms governing this behavior. Specifically, we reason that cellular partitioning and lifetime are dictated, in part, by the affinity of the compound for FKBP. To explore this central hypothesis, we propose the following specific aims: (1) synthesize a collection of FKBP-binding amprenavir derivatives and measure their relative affinities for albumin, FKBP and HIV protease and (2) explore how cellular partitioning correlates with binding affinities and relative protein expression levels. From these observations, we expect to understand the key structure-activity relationships in the context of the ternary complex (i.e. FKBP-drug-HIV protease). The immediate goal of this study is to create potent, safe and long-lived HIV protease inhibitors that selectively target FKBP-expressing, HIV-infected cells. This study is significant because it addresses an important problem in the treatment of AIDS. The proposed work is innovative because it will explore a fundamentally new, "natural product-inspired" strategy.