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

基本信息

批准号：
10926035
负责人：
MARC NICKLAUS
金额：
$ 2.77万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926035
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 drug 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 Metals Methods Modeling Molecular Molecular Target Multi-Drug Resistance Nucleotides Pathogenesis Patients Peptide Hydrolases Perception Pharmaceutical Preparations Phase Phenotype Preventive Probability 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 Replication Work X-Ray Crystallography analog anti-HIV microbicide cell type chelation comorbidity compound 30 design drug resistant virus enzyme model gag-pol Fusion Proteins human DNA in silico in vivo inhibitor microbicide migration molecular modeling new therapeutic target novel pharmacophore prevent programs protein expression response screening success viral DNA

项目摘要

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，以便使用因此获得的结构知识来设计这种酶的更好的抑制剂，以开发新的抗HIV药物，并开发新的抗HIV药物，并可以将任何其他计算机辅助药物设计方法应用于任何其他计算机辅助的药物设计方法，以识别任何新的promise promise new promise new new promise new new new new new new new new new new new new new new new new new new hiv-1。 HIV整合酶（IN）是导致逆转录病毒DNA整合到宿主基因组中的病毒编码酶。艾滋病毒生命周期的这一步骤对于病毒复制至关重要。抑制整合被视为抗AIDS疗法发展的有吸引力的靶标，因为尚无细胞同源物的知名度，因此提出了希望可以开发出有效的具有低毒性的抗内部药物。在给药蛋白酶和逆转录酶（RT）抑制剂时，多药耐药性病毒表型的出现进一步强调了替代治疗方法的需求。 IN是一种32KDA蛋白，是病毒颗粒中包含的GAG-POL融合蛋白前体的产物。在通过RT完成前病毒DNA合成后，在每个病毒DNA端的裂解中，裂解两个核苷酸（“ 3''PROCESSING”）。随后迁移到宿主细胞的核之后，在催化3'处理步骤中产生的嵌入式3'-末端的插入中，进入了宿主DNA的一条链。该反应称为3'端连接（也称为积分或链传递），并同时发生病毒DNA的两端。假定随后的间隙加入是通过细胞DNA修复酶进行的，以产生完全整合的前病毒DNA。以前的工作主要基于NCI数据库中的3D-Pharmacophore搜索，它产生了许多IN抑制剂。随着HIV-1和紧密相关的酶（例如ASV积分酶）的更多，更好，更好的实验结构（通过X射线晶体学和NMR）的出现，已经可以模拟包括全长蛋白的多主模型（包括全长蛋白质的多主模型）对哪些实验结构尚无可用的模型。我们已经使用所有可用的实验证据通过分子建模技术生成了此类结构。正如Karki等人2004年所述，该模型对基于体内活性的抑制剂抑制剂设计有用，特别强调具有与该酶的活性位点的模型一样，其病毒DNA与该酶的DNA相同，但在链传递之前可能是在链传递之前。我们利用这些结构模型来研究在没有实验性复合结构的抑制剂中，各种二酸HIV-1的潜在结合模式。结果表明，抑制剂中的二基酸酸可能会螯合催化位点中的金属离子，并防止病毒DNA的3'加工端暴露于人DNA。这些模型成功用于抑制剂开发，利用资源，包括我们数据库项目中描述的资源，尤其是通过对超过2600万可购买筛选样本的数据库进行的硅化筛选。当前的努力一直集中在基于配体的抑制剂设计上，利用来自将其纳入后期临床试验或被批准为抗HIV药物的少数分子的结构信息。基于这些结构图案，设计并提交了一系列未覆盖的内部专利的新颖化合物，并通过数据库项目中提到的新实施的半定量在线合成请求系统（SCSOR）提交了用于合成的报价。从全球10个以上不同的供应商引用的8,000多种化合物中，选择了近100套并提交购买。从这组获得了约30种化合物。其中一些表现出适度的抗内部活性。基于这些化合物和其他QSAR模型以及基于结构的活动预测，我们设计了一组约2000个可能的新型类似物，其中一个子集由通过SCSORS确定的原始供应商合成。该项目的最新扩展是我们在校园内到俄罗斯计划奖励基金支持的HIV杀菌剂的工作。该项目的目的是开发用于预防性局部应用（例如阴道凝胶）的新型HIV菌心。尽管微生物活性不一定是基于抗IN活性的，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。当前使用的另一个目标是逆转录酶（RT）和蛋白酶（PR）。该项目都应用了基于配体的（SAR/QSAR）抑制剂设计方法和基于结构的方法（对接）。已经进行了几种不同类型的基于细胞的基于细胞的和体内测定，这些测定已在我们当前的CADD工作中鉴定出48种化合物。确定了四种具有有趣活动的化合物。通过其他计算机辅助的药物设计方法和随后的合成以及其他购买的详细说明。所有这些化合物的汇编，总共有近240个样品，在一系列测试中进行了测定，其中包括两个基于细胞的和三个酶试验。对于28个最具兴趣的命中，基于细胞的剂量反应测定具有六个浓度。在发现的九种活性化合物中，最有趣的两个最有趣的化合物已被研究为潜在的未来铅化合物。该项目“通过非常大的合成化合物数据库探索化学生物学空间，以发现新型的抗HIV药物”旨在开发工作流程，以从具有复杂预测的预测的ADMET特性的化合物的SAVI数据库中有效检索，以创建“ Savi-Admet”子集；收集，分析，策划和使用数据（包括蛋白质表达，靶标和分子机制），从公共和市售数据库中构建（Q）SAR模型，涉及HIV/AIDS和HIV相关的合并症的主要机制（在俄罗斯方面）；用这些（Q）SAR模型在SAVI ADMET单一和多目标配体中与HIV-1相对的，以及与不同类型的HIV相关合并症相关；俄罗斯或美国合作者对50-100种选择化合物的合成和生物测试。在将SAVI构建块的来源更改为来自Enamine的构建块之后，根据上述标准确定化合物，并已订购进行合成和随后的测定。该项目的持续工作是由我们的俄罗斯合作者完成的。