Integrase

整合酶

• 批准号: 8938113
• 负责人: stephen h hughes
• 金额: $ 112.52万
• 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/8938113
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Affect; Anti-HIV Agents; Barrier Contraception; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Cell Culture Techniques; Cells; Chemistry; Chimeric Proteins; Chromatin; Clonal Expansion; Collaborations; Complex; Cultured Cells; DNA; DNA Integration; Data; Development; Drug Targeting; Drug resistance; Drug toxicity; Drug usage; Enzymes; Genes; Genome; Goals; HIV; HIV-1; In Vitro; Individual; Infection; Insertional Activations; Integrase; Integration Host Factors; Length; Life; Ligand Binding Domain; Link; Maps; Measures; Modeling; Mutation; Nuclear; Nucleosides; Nucleosomes; Oncogenes; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Play; Preventive; Problem Solving; Process; Prophylactic treatment; Protease Inhibitor; Protein Binding Domain; Proteins; Recombinants; Regimen; Reporting; Research; Resistance; Resistance profile; Resolution; Retroviridae; Role; Site; Specificity; Spumavirus; Structure; Subfamily lentivirinae; TAF3 gene; Technology; Testing; Therapeutic Index; Toxic effect; Toxicity Tests; Vaccines; Variant; Viral; Viral Load result; Virus; Work; base; combinatorial; design; drug resistant virus; effective therapy; gene therapy; genome-wide; inhibitor/antagonist; lens epithelium-derived growth factor; mutant; novel; preference; prevent; research study; resistance mutation; transmission process; viral DNA; virology

HIV-1 is the causative agent of AIDS. The three viral enzymes - RT, IN, and protease (PR) - have essential roles in the replication of HIV-1 and are the targets for all of the most potent anti-HIV drugs. Although considerable progress has been made in treating HIV-infected patients with three- and four-drug regimens, there is an immediate need for the development of effective ways to prevent new infections. A potent preventive vaccine would be ideal; however, despite a huge effort, the goal of developing an effective vaccine remains elusive. In the absence of an effective vaccine, reducing the transmission of HIV-1 must rely on barrier methods and/or drug treatments. There are two ways that anti-HIV drugs can be used to reduce viral transmission: (1) effective therapy in infected patients can reduce the viral load, making it less likely that an infected individual will transmit the virus to a partner; and (2) treating the uninfected partner with an anti-HIV drug can block transmission. Because most new infections are caused by a single virus, blocking transmission is an attractive option and there is now good evidence that giving an anti-HIV drug to the uninfected partner can significantly reduce viral transmission if the uninfected partner is compliant. Because of the problem of drug resistance, it would be better to use drugs with nonoverlapping resistance profiles for treatment and prophylaxis. Treatment would have to be long term and, for this reason, drug toxicity is an important consideration, which argues against the use of nucleoside RT inhibitors (NRTIs). It would also be better to block infection before the viral DNA is integrated, which argues against the use of PR inhibitors. Therefore, the two remaining options among the major classes of anti-HIV drugs are nonnucleoside RT inhibitors (NNRTIs) and IN strand-transfer inhibitors (INSTIs). We are using a combined approach that involves structural analysis, biochemistry, virology, modeling, toxicity testing, and chemistry to design, synthesize, and evaluate new NNRTIs and INSTIs. We have made good progress in developing new compounds that are effective against the wild-type (WT) and common drug-resistant viruses and that have good therapeutic indexes in tests done in cultured cells. Our progress with the IN research is reported below; progress with the HIV-1 RT research is reported separately for Project ZIA BC 010481. HIV-1 IN. Like RT, HIV-1 IN is an important drug target; however, as is the case for all anti-HIV drugs, treatment with INSTIs leads to resistance. We are making good progress on two fronts: understanding how mutations in IN confer resistance to the currently available compounds, and developing new INSTIs that are effective against the common drug-resistance mutations. Dr. Terry Burke is synthesizing new anti-IN compounds; Dr. Yves Pommier is using biochemical assays to test Dr. Burke's anti-IN compounds in vitro (using purified recombinant IN); and we are testing how the new inhibitors affect viral replication and measuring their toxicity in cultured cells. Until quite recently, we had no structural information to guide the development of IN inhibitors. However, Dr. Peter Cherepanov has obtained high-resolution structures of full-length foamy virus (FV) IN in complexes with both DNA substrates and anti-IN drugs. Dr. Cherepanov has joined our collaborative effort and has solved the structures of FV IN in complex with some of the more promising compounds developed by Dr. Burke. The active site of FV IN is similar, but not identical, to the active site of HIV-1 IN, and we are using Dr. Cherepanov's data to develop models of HIV-1 IN (both WT and mutant). These models have helped us understand how resistance arises and have been useful in the design of more effective compounds. Dr. Burke has recently synthesized several novel compounds that have IC50s in the low nanomolar range in a one-round replication assay, and that effectively inhibit both WT IN and most of the common drug-resistant variants. Based on tests done in cultured cells, these compounds have excellent therapeutic indexes (the CC50s are more than 3 logs higher than the IC50s). We also have three projects that involve studies of HIV-1 integration. In the first project, we are working with Drs. Alan Engelman and Vineet KewalRamani to define the host factors that are involved in transporting the preintegration complex (PIC) and to determine the exact roles they play in this process. In the second project, we are taking advantage of the fact that it is possible to redirect where HIV-1 DNA preferentially integrates. Redirecting HIV-1 DNA integration has the potential to make gene therapy safer because it may help solve the problems associated with the insertional activation of oncogenes; in addition, the technology can be used to determine where in the genome proteins/domains bind to chromatin. Different retroviruses have different integration-site preferences. There are good reasons to believe that such preferences are based on which host factor(s) the PIC interacts with; however, in most cases, it is unclear what the host factor(s) might be. Lentiviruses (including HIV-1) are the exception: HIV-1 IN is known to bind to lens epithelium-derived growth factor (LEDGF); the distribution of LEDGF on chromatin is the major factor that determines the local sites where HIV-1 DNA integrates. However, working with Dr. KewalRamani, we recently found that the host factor CPSF6, which binds to the viral CA protein, helps direct the PIC to nuclear speckles, which are associated with regions of the genome that are enriched in highly expressed genes. Thus, the interaction of CA and CPSF6 directs the PIC to broad regions of the genome, and the interaction of LEDGF and IN determines the exact sites where the HIV DNA is integrated. We, and others, showed that replacing the N-terminus of LEDGF with chromatin-binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. The initial experiments were done either with single CBDs or, in one case, with two linked domains taken from a larger protein. These analyses showed that the binding sites for CBD can be accurately determined by mapping redirected HIV-1 integration sites and that the distance between the CBD binding site and the integration site(s) is relatively small. We also showed that the binding sites for multiple-domain modules reflect the combinatorial interactions of the individual domains and chromatin and that the structural relationship of the domains helps define binding specificity. We have recently moved from an analysis of isolated CBDs to intact proteins. Although not every protein we have tried has worked, we have been able to get excellent data with a number of relatively large proteins, including TAF3, and we have been able explore how TAF3 interacts with its binding sites on chromatin using mutants with altered binding specificities (the TAF3 experiments are part of a collaboration with Dr. Robert Roeder). More recently, we have mapped the genome-wide distribution of the binding sites for the proteins in the integrator complex (with Drs. J. Skaar and M. Pagano) and WT and mutant DNMTs (with Drs. M. Noh and D. Allis). In the third project, we are working with Drs. Xiaolin Wu, Frank Maldarelli, John Coffin, John Mellors, and Mary Kearney to determine the distribution of integration sites in HIV-infected patients. We recently showed that there is extensive clonal expansion of HIV-infected cells in patients, and that, in some cases, integration of HIV DNA in specific oncogenes (MKL2 and BACH2) can contribute to this clonal expansion.

HIV-1是艾滋病的病因。三种病毒酶 - RT，IN和蛋白酶（PR） - 在HIV -1的复制中具有重要作用，并且是所有最有效的抗HIV药物的靶标。尽管在治疗三毒和四药治疗方案的HIV感染患者方面取得了长足进展，但仍需要开发有效的方法来预防新感染。有效的预防性疫苗将是理想的；但是，尽管付出了巨大的努力，但开发有效疫苗的目标仍然难以捉摸。在没有有效的疫苗的情况下，减少HIV-1的传播必须依靠屏障方法和/或药物治疗。可以使用抗HIV药物来减少病毒传播的方式有两种：（1）感染患者的有效治疗可以减少病毒载量，从而使感染者将病毒传播给伴侣的可能性较小； （2）用抗HIV药物治疗未感染的伴侣可以阻止传播。由于大多数新感染是由单个病毒引起的，因此阻止传播是一个有吸引力的选择，现在有充分的证据表明，如果未感染的伴侣合规，则将抗HIV药物给未感染的伴侣可以大大减少病毒传播。由于耐药性问题，最好使用具有非重叠抗性特征的药物进行治疗和预防。治疗必须是长期的，因此，药物毒性是一个重要的考虑因素，它反对使用核苷RT抑制剂（NRTIS）。在整合病毒DNA之前，最好阻止感染，这反对使用PR抑制剂。因此，主要类别的抗HIV药物中的剩余两种选择是非核苷RT抑制剂（NNRTIS）和链转移抑制剂（INSTIS）。我们正在使用涉及结构分析，生物化学，病毒学，建模，毒性测试以及化学的组合方法来设计，合成和评估新的NNRTIS和Instis。我们在开发针对野生型（WT）和常见药物抗药性病毒的新化合物方面取得了良好的进展，并且在培养细胞中进行的测试中具有良好的治疗指数。下面报告了我们在研究中的进展； HIV-1 RT研究的进展分别报道了Zia BC010481。HIV-1 IN。像RT一样，HIV-1是一个重要的药物靶标。但是，与所有抗HIV药物一样，使用研究所的治疗会导致抗药性。我们在两个方面取得了良好的进步：了解如何赋予对当前可用化合物的抗性突变，以及开发有效反对常见药物耐药突变的新研究。特里·伯克（Terry Burke）博士正在合成新的抗内化合物。 Yves Pommier博士正在使用生化测定法测试Burke博士在体外的抗内化合物（使用纯化的重组剂）；我们正在测试新抑制剂如何影响病毒复制并测量其在培养细胞中的毒性。直到最近，我们还没有结构信息来指导抑制剂的发展。然而，彼得·切尔帕诺夫（Peter Cherepanov）博士在与DNA底物和抗IN药物的复合物中获得了全长泡沫病毒（FV）的高分辨率结构。 Cherepanov博士加入了我们的合作努力，并与Burke博士开发的一些更有前途的化合物解决了FV的结构。 FV的活跃位点与HIV-1 IN的活性位点相似，但并不相同，我们正在使用Cherepanov博士的数据来开发HIV-1的模型（WT和突变体）。这些模型帮助我们了解了抗性的产生方式，并且在设计更有效的化合物的设计中很有用。 Burke博士最近合成了几种在单轮复制测定中具有IC50在低纳摩尔范围内的新型化合物，它们有效地抑制了WT中的WT和大多数常见的耐药变体。根据在培养细胞中进行的测试，这些化合物具有出色的治疗指数（CC50s比IC50高3个对数）。我们还有三个涉及HIV-1整合研究的项目。在第一个项目中，我们正在与Drs合作。 Alan Engelman和Vineet Kewalramani定义了运输前整合综合体（PIC）所涉及的宿主因素，并确定它们在此过程中所起的确切作用。在第二个项目中，我们利用了可以重定向HIV-1 DNA优先集成的事实。重定向HIV-1 DNA的整合具有使基因治疗更安全的潜力，因为它可能有助于解决与肠插入激活相关的问题。另外，该技术可用于确定基因组蛋白/结构域中与染色质结合的位置。不同的逆转录病毒具有不同的整合点偏好。有充分的理由相信，这种偏好是基于PIC与哪个宿主因素相互作用的；但是，在大多数情况下，尚不清楚宿主因素可能是什么。慢病毒（包括HIV-1）是例外：已知HIV-1与晶状体上皮衍生的生长因子（LEDGF）结合； LEDGF在染色质上的分布是确定HIV-1 DNA积分的局部位点的主要因素。但是，与Kewalramani博士合作，我们最近发现，与病毒CA蛋白结合的宿主因子CPSF6有助于将PIC引导到核斑点上，这些核斑点与高度表达基因富集的基因组区域有关。因此，CA和CPSF6的相互作用将PIC引导到基因组的宽区域，而LEDGF的相互作用并确定了HIV DNA积分的确切位点。我们和其他人表明，从其他蛋白质中代替LEDGF的N端（CBD）会改变HIV-1 DNA整合的特异性。最初的实验是用单个CBD进行的，或者在一种情况下是从较大蛋白质中取出两个链接结构域进行的。这些分析表明，可以通过绘制重定向的HIV-1积分位点来准确确定CBD的结合位点，并且CBD结合位点与积分位点之间的距离相对较小。我们还表明，多域模块的结合位点反映了各个域和染色质的组合相互作用，并且域的结构关系有助于定义结合特异性。我们最近从对孤立的CBD的分析转变为完整的蛋白质。尽管并非我们尝试过的每种蛋白质都起作用，但我们能够获得具有许多相对较大的蛋白质（包括TAF3）的出色数据，我们已经能够探索使用具有改变结合特异性的突变体在染色体上与其结合位点相互作用（TAF3实验是与Robert Roeder博士的合作的一部分）。最近，我们绘制了整合体复合物（J. Skaar和M. Pagano博士）以及WT和WT和突变的DNMT（带有M. Noh和D. Allis博士）的结合位点的结合位点的全基因组分布。在第三个项目中，我们正在与Drs合作。小小的Wu，Frank Maldarelli，John Coffin，John Mellors和Mary Kearney，以确定HIV感染的患者的整合位点的分布。我们最近表明，患者HIV感染细胞的克隆膨胀广泛，在某些情况下，特定癌基因中HIV DNA的整合（MKL2和BACH2）可以有助于这种克隆扩张。