Design and Synthesis of HIV Integrase as Potential Anti-AIDS Drugs

针对HIV-1生命周期中三个不同点的抑制剂正在研制中。这些抑制剂旨在作为潜在的新疗法和药理探针来研究病毒复制的生化机制。这三个领域的研究是：(1)HIV-1整合酶(IN)，其中抑制剂可能会破坏病毒的cDNA进入宿主基因组；(2)HIV p6Gag蛋白与人Tsg101蛋白的结合，其中抑制剂可能会扰乱病毒的组装和萌发；(3)人胞苷脱氨酶APOBEC3G(A3G)与HIV-1蛋白Vif的结合，其中抑制剂可能会导致前病毒DNA的高度突变。(1)HIV-1 IN抑制剂：尽管文献中报道了大量的抑制剂，但二酮酸(DKA)衍生的药物最近显示出特别的前景，这一类型的成员在抗艾滋病临床试验中显示出巨大的前景。这些试剂被认为是通过在IN催化部位螯合镁离子发挥作用，在那里它们选择性地抑制链转移(ST)反应而不是3-加工(3-P)反应。我们以前曾报道过双水杨酰肼类IN抑制剂，我们也假设它是通过金属螯合发挥作用的。然而，这一系列的成员只有当使用Mn2+作为辅因子时才显示出有效的抑制作用，而当使用与生理相关的Mg2+时则不显示出有效的抑制作用。我们最近的发现表明，双芳酰肼在有镁离子存在的情况下，可以通过引入二羟基苯甲酰取代基来获得抑制活性。使用2，3-二氢-6，7-二羟基-1H-异吲哚-1-酮环体系作为构象受限的2，3-二羟基苯甲酰基当量，也可以实现IN催化的ST与3-P反应的良好选择性。在异吲哚-1-单环体系中加入第二个氧基，得到了邻苯二甲酰亚胺基异吲哚-1，3(2H)-二酮类化合物，其3-P和ST抑制活性普遍增强。利用HIV-1载体在培养细胞中的抗病毒作用表明，某些双环构象受限类似物对HIV-1感染的细胞显示出亚微摩尔的抗病毒活性。这项工作表明，4，5-二羟基邻苯二甲酰亚胺核为进一步开发IN抑制剂提供了一个结构简单的起点。(2)Tsg101结合抑制物：HIV p6Gag蛋白与人Tsg101蛋白的结合是病毒萌发所必需的，并涉及p6蛋白的关键9-聚P-E-P-T-A-P-P-E-E序列。我们正在准备作为Tsg101结合拮抗剂的9-聚体序列的多肽和多肽模拟变体，这可能会导致一类新的病毒萌发抑制物。一种方法是用N-取代甘氨酸(NSG)残基(称为类肽)取代Pro4残基。然而，这是一个综合问题。因此，我们求助于一类新的肽类变异体，这些变异体将肼类酰胺作为NSG的替代物。在固相树脂裂解后，通过将一系列的醛与单一的高效液相纯化的酰肼前体反应，可以很容易地在库中制备这些化合物。将这些肼还原为N-取代的肽酰肼，为文库多样化提供了一条简便的途径。我们扩展了这些研究，用其他非天然氨基酸类似物取代了亲本p6衍生的9-聚体序列中的几个关键残基。指导我们方法的一个统一的原则是将氨氧基官能团结合到残基中，这些残基可以在单一的最后一步中官能化，从而提供一个肟衍生物文库。这种方法已经鉴定了几种低微摩尔亲和力的Tsg101结合拮抗剂。通过各种方法继续进行结合亲和力的优化，包括使用闭环复分解化学进行大环化。与Tsg101蛋白络合的抑制剂的X射线晶体研究正在进行中，以期为利用基于结构的技术设计更高亲和力的第二代类似物提供信息。(3)HIV-1蛋白与人胞苷脱氨酶APOBEC3G(A3G)结合的蛋白酶体靶向性抑制Vif：感染靶细胞后，在将HIV-1RNA逆转录入DNA的过程中，人A3G酶将负链中的C残基脱氨，将其转化为U残基。这导致编码正链DNA中的A残基而不是原来的G残基，净影响是前病毒DNA的超突变。HIV-1 23 kDa细胞质蛋白Vif通过与A3G结合并靶向蛋白酶体降解来克服A3G的抗病毒作用。维奈·帕萨克博士(CCR，NCI，NIH)的实验室已经证明，Vif蛋白序列S(144)LQYLA(149)对于将A3G靶向蛋白质体起关键作用。我们最初的工作是制备异硫氰酸荧光素(FITC)衍生的多肽结合物，供Robert Fisher博士(SAIC，Inc.，NCI-Frederick)用于开发VIF荧光各向异性结合分析。我们将与巴塔克斯博士的实验室合作，对SLQYLA序列进行结构活性研究，以开发能够有效阻断Vif靶向A3G的高亲和力、细胞渗透性类似物。在这项工作中计划的技术之一是使用上文所述的用于Tsg101结合抑制剂的肼和肟多肽库

Inhibitors directed against three distinct points in the HIV-1 life cycle are being prepared. These inhibitors are intended to serve as potential new therapeutics and as pharmacological probes to investigate biochemical mechanisms of viral replication. The three areas of investigation are: (1) HIV-1 integrase (IN), where inhibitors may disrupt incorporation of viral cDNA into the host genome; (2) Binding of HIV p6Gag protein to human Tsg101 protein, where inhibitors may disrupt viral assembly and budding and (3) Binding of human cytidine deaminase APOBEC3G (A3G) to the HIV-1 protein Vif, where inhibitors may result in hyper-mutation of proviral DNA. (1) HIV-1 IN inhibitors: Although a large number of inhibitors have been reported in the literature, diketoacid (DKA)-derived agents have recently shown particular promise, with members of this genre showing significant promise in anti-AIDS clinical trials. These agents are thought to function by chelating Mg2+ ions at the IN catalytic site, where they selectively inhibit strand transfer (ST) reactions over 3-processing (3-P) reactions. We had previously reported the bis-salicylhydrazides class of IN inhibitors that we also hypothesized function by metal chelation. However, members of this series exhibit potent inhibition only when Mn2+ is used as cofactor and not when the physiologically-relevant Mg2+ is used. Our recent findings have shown that bis-aroylhydrazides could acquire inhibitory potency in the presence of Mg2+ through the inclusion of dihdroxybenzoyl substituents. Good selectivity for IN-catalyzed ST versus 3-P reactions could also be achieved using a 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one ring system as a conformationally-constrained 2,3-dihydroxybenzoyl equivalent. Adding a second oxo-group to the isoindole-1-one ring system gave the phthalimide-based isoindole-1,3(2H)-diones, which generally exhibited enhanced of 3-P and ST inhibitory potencies. Antiviral effects in cultured cells using HIV-1 based vectors showed that the certain bicyclic conformationally constrained analogues exhibited sub-micromolar antiviral potencies against HIV-1 infected cells. This work indicates that the 4,5-dihydroxyphthalimide nucleus offers a structurally simple starting point for the further development of IN inhibitors. (2) Tsg101-binding inhibitors: Binding of the HIV p6Gag protein to human Tsg101 protein has been shown to be necessary for viral budding and to involve a critical 9-mer P-E-P-T-A-P-P-E-E sequence of the p6 protein. We are preparing peptide and peptide mimetic variants of this 9-mer sequence as Tsg101-binding antagonists that may lead to a new class of viral budding inhibitors. One approach was to replace the Pro4 residue with N-substitued glycine (NSG) residues (termed peptoids). However, this is synthetically problematic. Therefore, we resorted to a new family of peptoid variants that incorporate hydrazone amides as NSG surrogates. These can be prepared readily in library fashion by reacting a series of aldehydes with a single HPLC-purified hydrazide precursor following cleavage from the solid-phase resin. Reduction of these hydrazones to N-substitued peptoid hydrazides affords a facile route to library diversification. We have extended these studies by replacing several key residues in the parent p6-derived 9-mer sequence with other non-natural amino acid analogues. A unifying principal guiding our approach has been the incorporation of amino-oxy functionality into the residues that can be functionalized in a single final step to provide a library of oxime derivatives. This approach has resulted in the identification of several low micromolar affinity Tsg101 binding antagonists. Ongoing optimization of binding affinity is being continued by a variety of means, including macrocyclization using ring-closing metathesis chemistries. X-ray crystal studies of inhibitors complexed with Tsg101 protein are in progress to provide information for the design of higher affinity second generation analogues by structure-based techniques. (3) Inhibitors of proteosomal targeting of human cytidine deaminase APOBEC3G (A3G) binding by the HIV-1 protein Vif: After infection of a target cell and during reverse transcription of HIV-1 RNA into DNA, the human A3G enzyme deaminates C residues in the minus strand, converting them to U residues. This results in encoding of A residues in the plus strand DNA in place of the original G residues, with the net effect being hypermutation of proviral DNA. The HIV-1 23 kDa cytoplasmic protein Vif overcomes the antiviral effects of A3G by binding to it and targeting it for proteasomal degradation. The laboratory of Dr. Vinay Pathak (CCR, NCI, NIH) has shown that the Vif protein sequence S(144)LQYLA(149) is critical for targeting A3G to the proteosome. Our initial work has been to prepare fluoresceine isothiocyanate (FITC)-derived peptide conjugates for use in the laboratory of Dr. Robert Fisher (SAIC, Inc., NCI-Frederick) to develop Vif fluorescence anisotropy binding assays. In collaboration with Dr. Pathaks laboratory, we will conduct structure-activity studies on the SLQYLA sequence to develop high affinity, cell-permeable analogues that can effectively block proteosomal targeting of A3G by Vif. Among the techniques planned for this work is the use of hydrazone and oxime peptide libraries as described above for Tsg101-binding inhibitors