HIV Integrase Modeling and Computer-Aided Inhibitor Development

该项目的主要目标是阐明HIV-1整合酶蛋白与DNA和/或抑制剂复合物的结构，利用由此获得的结构知识来设计更好的该酶抑制剂，以开发新的抗hiv药物，并应用任何其他计算机辅助药物设计方法，可能有助于识别新的，有前途的HIV-1整合酶抑制剂。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对病毒复制至关重要。抑制整合被认为是开发抗艾滋病治疗的一个有吸引力的靶点，因为没有已知的in的细胞同源物，从而提高了开发有效的低毒性抗in药物的希望。在使用蛋白酶和逆转录酶（RT）抑制剂的混合物期间出现的多重耐药病毒表型进一步突出了替代治疗方法的必要性。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”）。在随后迁移到宿主细胞核后，IN催化在3‘加工步骤中产生的嵌入的3’末端插入到宿主DNA的一条链中。这种反应被称为3'端连接（也称为整合或链转移），并同时发生在病毒DNA的两端。随后的间隙连接被认为是由细胞DNA修复酶进行的，以产生完全整合的原病毒DNA。先前的工作主要基于NCI数据库中的3d药效团搜索，已经获得了许多in抑制剂。随着更多、更好的实验结构（通过x射线晶体学和核磁共振）的出现，HIV-1 IN以及密切相关的酶，如ASV整合酶，已经有可能模拟更大的结构，包括全长蛋白的多聚体模型，而实验结构目前还不可用。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。如Karki et al.， 2004所述，特别强调的是获得酶活性位点的模型，与之相对的病毒DNA可能是在3'加工之后，但在链转移之前。该模型可用于基于结构的抑制剂设计，使抑制剂在体内保持活性。我们已经利用这些结构模型来研究各种没有实验复杂结构的双酮酸HIV-1 IN抑制剂的潜在结合模式。结果表明，二酮酸类IN抑制剂可能在催化位点螯合金属离子，并阻止病毒DNA的3'加工端暴露于人类DNA。这些模型成功地用于抑制剂的开发，利用了包括数据库项目中描述的资源，特别是通过对数据库中超过2600万个可购买筛选样本的计算机筛选。目前的努力主要集中在基于配体的抑制剂设计上，利用那些已经进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。基于这些结构基序，设计了一系列未被in相关专利覆盖的新化合物，并通过数据库项目中提到的新实现的半定制在线合成请求系统（SCSORS）提交了合成报价。从全球10多家不同供应商提供的8000多种化合物中，选出了近100种供购买。目前已获得25个化合物，其中少数化合物具有一定的抗in活性。预计不久将有数十种其他化合物，并将在收到后立即进行检测。

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 has been chosen and submitted for purchase. About 25 compounds have been obtained so far from this set, with some anti-IN activity exhibited by a few. Several tens of additional compounds are expected soon and will undergo assaying as soon as they are received.