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

HIV整合酶的设计与合成作为潜在的抗艾滋病药物

• 批准号: 7592570
• 负责人: TERRENCE BURKE
• 金额: $ 25.97万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592570
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Aldehydes; Amides; Amino Acid Sequence; Amino Acids; Anti-HIV Agents; Antiviral Agents; Area; Binding; Biochemical; Biological Assay; CCR; Catalytic Domain; Cell Nucleus; Cells; Chemistry; Class; Clinical; Clinical Trials; Collaborations; Complementary DNA; Complex; Cultured Cells; Cytidine Deaminase; Cytoplasmic Protein; DNA; Development; Drug Industry; Enzymes; Exhibits; Family; Fluorescence Anisotropy; Generations; Genome; Glycine; HIV; HIV Integrase; HIV-1; HIV-1 integrase; Human; Hydrazones; Infection; Integrase; Integrase Inhibitors; Investigation; Ions; Isoindol-1-one; Isothiocyanates; Laboratories; Lead; Libraries; Life Cycle Stages; Literature; Mediating; Metals; Molecular; Mutation; Numbers; Oximes; Parents; Peptide Library; Peptide Sequence Determination; Peptides; Peptoids; Phase; Phthalimides; Planning Techniques; Plant Resins; Process; Proteins; RNA; Reaction; Reporting; Research; Resort; Reverse Transcription; Roentgen Rays; Route; Series; Solid; Structure; System; TSG101 gene; Techniques; Therapeutic; Thinking; Tsg101 protein; United States National Institutes of Health; Variant; Viral; Work; analog; base; chelation; cofactor; design; human CEM15 protein; human TSG101 protein; inhibitor/antagonist; member; mimetics; multicatalytic endopeptidase complex; novel therapeutics; phthalimide; tool; vector; vif Gene Products

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

针对HIV-1生命周期中三个不同点的抑制剂正在准备中。这些抑制剂有望作为潜在的新疗法和药理学探针来研究病毒复制的生化机制。研究的三个领域是：(1)HIV-1整合酶（IN），其抑制剂可能会破坏病毒cDNA与宿主基因组的结合；(2) HIV p6Gag蛋白与人Tsg101蛋白结合，其中抑制剂可能破坏病毒组装和出芽；(3)人胞苷脱氨酶APOBEC3G （A3G）与HIV-1蛋白Vif结合，其中抑制剂可能导致前病毒DNA的超突变。(1) HIV-1 IN抑制剂：尽管文献中已经报道了大量的抑制剂，但双酮酸（DKA）衍生的药物最近显示出特别的前景，这类药物在抗艾滋病临床试验中显示出显著的前景。这些药物被认为是通过在IN催化位点螯合Mg2+离子起作用，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。我们之前报道过双水杨酸肼类的IN抑制剂，我们也假设它们通过金属螯合起作用。然而，该系列的成员只有在使用Mn2+作为辅助因子时才表现出有效的抑制作用，而当使用与生理相关的Mg2+时则没有。我们最近的研究结果表明，双芳酰肼可以通过包含二羟基苯甲酰取代基在Mg2+存在下获得抑制效力。使用2,3-二氢-6,7-二羟基- 1h -异吲哚-1- 1环体系作为构象约束的2,3-二羟基苯甲酰等价物，也可以实现in催化ST与3-P反应的良好选择性。在异吲哚-1- 1环体系中加入第二个氧基，得到基于邻苯二胺的异吲哚-1,3(2H)-二酮，其3- p和ST抑制能力普遍增强。利用基于HIV-1的载体在培养细胞中的抗病毒作用表明，某些双环构象约束类似物对HIV-1感染细胞具有亚微摩尔的抗病毒效力。这项工作表明，4,5-二羟基邻苯二胺核为进一步开发IN抑制剂提供了一个结构简单的起点。(2) Tsg101结合抑制剂：HIV p6Gag蛋白与人Tsg101蛋白的结合已被证明是病毒出芽所必需的，并涉及p6蛋白的一个关键的9-mer p - e - p - p - p - t - a - p - p - e - e序列。我们正在制备这种9-mer序列的肽和模拟肽变体，作为tsg101结合拮抗剂，这可能导致一类新的病毒出芽抑制剂。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，我们采用了一种新的肽类变体家族，其中包含腙酰胺作为NSG替代品。这些化合物可以很容易地以文库的方式制备，方法是在固相树脂裂解后，将一系列醛与单个hplc纯化的肼前体反应。将这些腙还原为n取代的肽类肼，为文库多样化提供了一条便捷的途径。我们扩展了这些研究，用其他非天然氨基酸类似物替换了母体p6衍生的9-mer序列中的几个关键残基。指导我们方法的统一原则是将氨基氧基功能结合到残基中，这些残基可以在单一的最后一步中被功能化，从而提供肟衍生物库。这种方法已经鉴定出几种低微摩尔亲和力的Tsg101结合拮抗剂。结合亲和力的持续优化正在通过各种手段继续进行，包括使用闭合环的复分解化学进行大环化。抑制剂与Tsg101蛋白络合的x射线晶体研究正在进行中，为基于结构的技术设计高亲和力的第二代类似物提供信息。(3) HIV-1蛋白Vif结合人胞苷脱氨酶APOBEC3G （A3G）蛋白体靶向抑制剂：在感染靶细胞后，在HIV-1 RNA逆转录成DNA的过程中，人A3G酶在负链上脱氨C残基，将其转化为U残基。这导致正链DNA中的A残基编码取代了原来的G残基，其净效应是原病毒DNA的超突变。HIV-1 23kda细胞质蛋白Vif通过与A3G结合并靶向其蛋白酶体降解来克服A3G的抗病毒作用。Vinay Pathak博士（CCR， NCI， NIH）的实验室已经表明，Vif蛋白序列S(144)LQYLA（149）对于将A3G靶向到蛋白质体至关重要。我们最初的工作是制备异硫氰酸荧光素（FITC）衍生的肽偶联物，用于Robert Fisher博士（SAIC, Inc., NCI-Frederick）的实验室，以开发Vif荧光各向异性结合测定。我们将与Pathaks博士实验室合作，对SLQYLA序列进行结构-活性研究，以开发高亲和力、细胞渗透性的类似物，有效阻断Vif靶向A3G的蛋白体。这项工作计划的技术之一是使用如上所述的tsg101结合抑制剂的腙和肟肽库