HIV Integrase Modeling and Computer-Aided Inhibitor and Microbicide Development

HIV 整合酶建模以及计算机辅助抑制剂和杀菌剂开发

• 批准号: 10702372
• 负责人: MARC NICKLAUS
• 金额: $ 3.61万
• 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/10702372
• 关键词: 3-Dimensional; AIDS therapy; Acids; Active Sites; Anti-HIV Agents; Award; Binding; Biological; Biological Assay; Biological Testing; Catalytic Domain; Cell Nucleus; Cells; Characteristics; Chemicals; Clinical Trials; Collection; Complex; Computer Assisted; Computing Methodologies; Custom; DNA; DNA Repair Enzymes; DNA biosynthesis; Data; Databases; Development; Docking; Dose; Drug Design; Drug toxicity; Enzyme Inhibitor Drugs; Enzymes; Exhibits; FDA approved; Funding; Future; Genome; Goals; Grant; HIV; HIV Integrase; HIV Integrase Inhibitors; HIV resistance; HIV-1; HIV/AIDS; Homologous Gene; Homology Modeling; Integrase; Integrase Inhibitors; Ions; Joints; Knowledge; Lead; Leadership; Legal patent; Length; Libraries; Life Cycle Stages; Ligands; Methods; Modeling; Molecular; Molecular Target; Multi-Drug Resistance; Nucleotides; Pathogenesis; Patients; Peptide Hydrolases; Perception; Pharmaceutical Preparations; Phase; Phenotype; Preventive; Process; Protein Precursors; Proteins; Publishing; Quantitative Structure-Activity Relationship; RNA-Directed DNA Polymerase; Reaction; Replication-Associated Process; Resources; Retrieval; Reverse Transcriptase Inhibitors; Russia; Sampling; Series; Services; Side; Source; Structural Models; Structure; System; Techniques; Testing; Therapeutic; Topical application; Toxic effect; Vaginal Gel; Viral; Virion; Virus; Virus Replication; Work; X-Ray Crystallography; analog; anti-HIV microbicide; base; cell type; comorbidity; compound 30; design; enzyme model; gag-pol Fusion Proteins; human DNA; in silico; in vivo; inhibitor; metal chelator; microbicide; migration; molecular modeling; new therapeutic target; novel; pharmacophore; prevent; programs; protein complex; protein expression; response; screening; success; viral DNA

The principal objective of this project is to elucidate the structure of the HIV-1 integrase protein, complexed with DNA and/or inhibitors, to use the structural knowledge thus obtained to design better inhibitors of this enzyme with the goal of developing new anti-HIV drugs, and to apply any other computer-aided drug design method that may be helpful in identifying new, promising HIV-1 integrase inhibitors. HIV integrase (IN) is the virally encoded enzyme responsible for integration of the retroviral DNA into the host genome. This step in the life cycle of HIV is essential for viral replication. Inhibition of integration is seen as an attractive target in the development of anti-AIDS therapies because no cellular homologue to IN is known, thus raising the hope that effective anti-IN based drugs with low-toxicity can be developed. The emergence of multidrug-resistant virus phenotypes during administration of cocktails of protease and reverse transcriptase (RT) inhibitors has further highlighted the need for alternative therapeutic approaches. IN is a 32kDa protein that is a product of the gag-pol fusion protein precursor contained in the virus particle. Upon completion of proviral DNA synthesis by RT, IN cleaves two nucleotides from each viral DNA end ("3'-processing"). After subsequent migration to the host cell's nucleus, IN catalyzes the insertion of the recessed 3'-terminus, generated during the 3'-processing step, into one strand of the host DNA. This reaction is termed 3' end joining (also known as integration or strand transfer) and occurs for both ends of the viral DNA simultaneously. The subsequent gap-joining is presumed to be performed by cellular DNA repair enzymes to yield a fully integrated proviral DNA. Previous work, mainly based on 3D-pharmacophore searches in the NCI database, had yielded a number of inhibitors of IN. With the advent of more, and better, experimental structures (by X-ray crystallography and NMR) of HIV-1 IN as well as of closely related enzymes such as ASV integrase, it has become possible to model larger structures including multimeric models of the full-length protein, for which experimental structures are not available as of yet. We have generated such structures by means of molecular modeling techniques using all available experimental evidence. Special emphasis was placed on obtaining a model of the enzyme's active site with the viral DNA apposed to it as it might be after 3'-processing but before strand transfer, as described in Karki et al., 2004. This model is useful for structure-based inhibitor design of inhibitors which retain activity in vivo. We have made use of these structural models to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors for which no experimental complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevents the exposure of the 3'-processed end of the viral DNA to human DNA. These models were success fully used for inhibitor development, utilizing resources including those described in our database project, in particular through in silico screening of a database of more than 26 million purchasable screening samples. Current efforts have been focusing on ligand-based inhibitor design, making use of the structural information coming from those few molecules that have made it into late-phase clinical trials or been approved as anti-HIV drug. Based on these structural motifs, a series of novel compounds not covered by IN-related patents were designed and submitted for quotation for synthesis via the newly implemented Semi-Custom Online Synthesis Request System (SCSORS) mentioned in the Database project. From the more than 8,000 compounds quoted by more than 10 different suppliers world-wide, a set of nearly 100 was chosen and submitted for purchase. About 30 compounds were obtained from this set. Some of them exhibited moderate anti-IN activity. Based on these compounds and additional QSAR models as well as structure-based activity predictions, we designed a set of about 2000 possible novel analogs, a subset of which was synthesized by the original supplier identified through SCSORS. A recent extension of this project has been our work on HIV microbicides supported by Intramural-to-Russia Program award funds. The goal of this project is to develop novel HIV microbicides for preventive topical application such as in vaginal gels. While microbicidal activity need not be (solely) based on anti-IN activity, our current efforts are based on a combination of molecular targets including integrase. The other currently used target are reverse transcriptase (RT) and protease (PR). Both ligand-based (SAR/QSAR) inhibitor design approaches and structure-based approaches (docking) have been applied in this project. Several different types of cell-based and ex vivo assays of 48 compounds identified in our current CADD efforts have been conducted. Four compounds with interesting activities were identified. Elaboration of these hits by additional computer-aided drug design approaches and subsequent synthesis as well as additional purchases have been completed. The compilation of all these compounds, for a total of nearly 240 samples, have been assayed in a battery of tests, comprising two cell-based and three enzymatic assays. For 28 of the most-interesting hits, cell-based dose-response assays with six concentrations performed. The two most interesting ones among the nine active compounds found have been investigated as potential future lead compounds. The project "Exploration of Chemical-Biological Space via a Very Large Database of Synthesizable Compounds to Discover Novel Anti-HIV Agents" aims at developing a workflow for efficient retrieval from the SAVI database of compounds with complex predicted desirable ADMET characteristics to create a "SAVI-ADMET" subset; collection, analysis, curation, and usage of data (including protein expression, targets, and molecular mechanisms) for building of (Q)SAR models from publicly and commercially available databases about the main mechanisms of pathogenesis of HIV/AIDS and HIV-associated comorbidities (on the Russian side); identifying with these (Q)SAR models in SAVI-ADMET single- and multi-target ligands active against HIV-1 and against different types of HIV-related comorbidities; synthesis and biological testing of 50-100 selected compounds either by Russian or U.S. collaborators. After the source of the SAVI building blocks was changed to the building block set from Enamine, compounds were identified according to the criteria above and have been ordered for synthesis and for subsequent assaying. Continued work in this project is done by our Russian collaborators.

该项目的主要目标是阐明与 DNA 和/或抑制剂复合的 HIV-1 整合酶蛋白的结构，利用由此获得的结构知识设计更好的该酶抑制剂，以开发新的抗 HIV 药物，并应用任何其他可能有助于识别新的、有前途的 HIV-1 整合酶抑制剂的计算机辅助药物设计方法。 HIV 整合酶 (IN) 是病毒编码的酶，负责将逆转录病毒 DNA 整合到宿主基因组中。 HIV生命周期中的这一步骤对于病毒复制至关重要。整合的抑制被视为抗艾滋病疗法开发中有吸引力的目标，因为尚无与IN同源的细胞，因此提高了开发有效的低毒性抗IN药物的希望。在施用蛋白酶和逆转录酶（RT）抑制剂混合物期间出现的多重耐药病毒表型进一步凸显了对替代治疗方法的需求。 IN是一种32kDa的蛋白质，是病毒颗粒中含有的gag-pol融合蛋白前体的产物。通过 RT 完成原病毒 DNA 合成后，IN 从每个病毒 DNA 末端切割两个核苷酸（“3'-加工”）。随后迁移至宿主细胞核后，IN 催化将 3' 加工步骤中产生的凹进 3' 末端插入宿主 DNA 的一条链中。该反应称为 3' 末端连接（也称为整合或链转移），并且病毒 DNA 的两端同时发生。据推测，随后的间隙连接是由细胞 DNA 修复酶进行的，以产生完全整合的原病毒 DNA。之前的工作主要基于 NCI 数据库中的 3D 药效团搜索，已经产生了许多 IN 抑制剂。随着更多、更好的 HIV-1 IN 实验结构（通过 X 射线晶体学和 NMR）以及密切相关的酶（如 ASV 整合酶）的出现，建模更大的结构（包括全长蛋白质的多聚体模型）已成为可能，但目前尚无可用的实验结构。我们利用所有可用的实验证据，通过分子建模技术生成了这样的结构。特别强调获得酶活性位点的模型，其中病毒 DNA 与其相连，因为它可能是在 3' 加工之后但在链转移之前，如 Karki 等人，2004 年所述。该模型可用于保留体内活性的基于结构的抑制剂设计。我们利用这些结构模型来研究各种二酮酸 HIV-1 IN 抑制剂的潜在结合模式，但目前还没有可用的实验复合结构。 结果表明，二酮酸IN抑制剂可能会螯合催化位点的金属离子，并且还可以防止病毒DNA的3'加工末端暴露于人类DNA。这些模型成功地完全用于抑制剂开发，利用了包括我们数据库项目中描述的资源在内的资源，特别是通过对超过 2600 万个可购买筛选样品的数据库进行计算机筛选。目前的工作重点是基于配体的抑制剂设计，利用来自少数分子的结构信息，这些分子已进入后期临床试验或被批准作为抗艾滋病毒药物。基于这些结构主题，设计了一系列IN相关专利未涵盖的新型化合物，并通过数据库项目中提到的新实施的半定制在线合成请求系统（SCSORS）提交合成报价。从全球 10 多家不同供应商报价的 8,000 多种化合物中，选择了一组近 100 种化合物并提交购买。从该组中获得了大约 30 种化合物。其中一些表现出中等的抗IN活性。基于这些化合物和其他 QSAR 模型以及基于结构的活性预测，我们设计了一组约 2000 种可能的新型类似物，其中一部分是由通过 SCSORS 确定的原始供应商合成的。该项目最近的一个扩展是我们在艾滋病毒杀微生物剂方面的工作，并得到了俄罗斯校内计划奖励资金的支持。该项目的目标是开发新型艾滋病毒杀微生物剂，用于预防性局部应用，例如阴道凝胶。虽然杀菌活性不需要（仅仅）基于抗 IN 活性，但我们目前的努力是基于包括整合酶在内的分子靶标的组合。目前使用的其他靶标是逆转录酶（RT）和蛋白酶（PR）。基于配体的（SAR/QSAR）抑制剂设计方法和基于结构的方法（对接）均已在该项目中得到应用。已经对我们当前 CADD 工作中鉴定的 48 种化合物进行了几种不同类型的基于细胞和离体的测定。鉴定出四种具有有趣活性的化合物。通过额外的计算机辅助药物设计方法和随后的合成以及额外的购买对这些成功产品的阐述已经完成。所有这些化合物的汇编（总共近 240 个样品）已通过一系列测试进行了分析，其中包括两项基于细胞的分析和三项酶分析。对于 28 个最有趣的命中，进行了六种浓度的基于细胞的剂量反应测定。已发现的九种活性化合物中最有趣的两种已被研究作为未来潜在的先导化合物。 “通过非常大的可合成化合物数据库探索化学生物空间以发现新型抗 HIV 药物”项目旨在开发一个工作流程，用于从 SAVI 数据库中有效检索具有复杂的预测所需 ADMET 特性的化合物，以创建“SAVI-ADMET”子集；收集、分析、整理和使用数据（包括蛋白质表达、靶标和分子机制），以便从公共和商业数据库中构建有关艾滋病毒/艾滋病和艾滋病毒相关合并症主要发病机制的（Q）SAR模型（​​俄罗斯方面）；利用 SAVI-ADMET 中的这些 (Q)SAR 模型识别对 HIV-1 和不同类型的 HIV 相关合并症具有活性的单靶点和多靶点配体；由俄罗斯或美国合作者对 50-100 种选定的化合物进行合成和生物测试。在 SAVI 构建模块的来源更改为 Enamine 的构建模块集后，根据上述标准鉴定了化合物，并已订购进行合成和后续分析。该项目的后续工作由我们的俄罗斯合作者完成。