Natural Product-Inspired Method for Enhancing HIV Protease Inhibitors

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）对管理HIV感染/ AIDS非常有效。然而，由于治疗相关的并发症，患者对HAART的依从性通常是可变的。这是一个严重的问题，促进了耐药性的出现。导致HAART相关并发症的一个问题是HIV蛋白酶抑制剂的代谢稳定性差和细胞渗透性低。我们的团队一直在探索解决这些限制的新方法。该策略直接受到天然产物FK 506的药理学性质的启发，FK 506在人体中具有令人惊讶的长半衰期（t1/2 ~ 40小时），尽管是体外P450酶的优良底物。我们最近想知道这种明显的矛盾是否可能源于这种化合物对FK 506结合蛋白（FKBP）的高亲和力。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，在这种受保护的细胞生态位内的亲和力驱动的积累可能会限制关键代谢酶的暴露，从而延长药物寿命。此外，FK 506吸收迅速（约1小时），对生物膜具有高度渗透性，并通过其FKBP结合部分天然靶向白细胞。总之，这些特性似乎与HIV蛋白酶抑制剂面临的主要问题一致。基于这些观察结果，我们将FKBP结合基团与安普那韦类似物连接，产生了一种可以结合FKBP和HIV蛋白酶的双功能分子。我们发现所得化合物保留了抗蛋白酶活性（IC 50 ~ 20 nM）。此外，它现在被隔离到血细胞中（至少8倍），其半衰期在小鼠中增加了约20倍。这种化合物的寿命是上级的利托那韦加强安普那韦，而且，它的代谢稳定性现在是独立的利托那韦共同管理。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，我们的理由是，细胞的分区和寿命，在一定程度上，由FKBP的化合物的亲和力。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FKBP结合安普那韦衍生物，并测量它们对白蛋白、FKBP和HIV蛋白酶的相对亲和力;（2）探索细胞分配如何与结合亲和力和相对蛋白质表达水平相关。从这些观察结果中，我们期望了解三元复合物（即FKBP-药物-HIV蛋白酶）背景下的关键结构-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，选择性靶向表达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。拟议的工作是创新的，因为它将探索一个全新的，“自然产品启发”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.