HIV-1 Inhibition Using Tat peptide derivatives

使用 Tat 肽衍生物抑制 HIV-1

• 批准号: 8494511
• 负责人: Fatah Kashanchi
• 金额: $ 35.06万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8494511
• 关键词: Acquired Immunodeficiency Syndrome; Acute Disease; Affect; Age-Years; Animal Model; Animals; Anti-HIV Agents; Binding; Biological Assay; Biotin; Cell Culture Techniques; Cell Line; Cells; Chronic; Clinic; Complex; Cyclin-Dependent Kinases; Cyclins; DNA; Development; Diagnosis; Disease Progression; Drug resistance; Epidemic; Excretory function; G0 Phase; Generations; Genetic Transcription; HIV; HIV-1; Highly Active Antiretroviral Therapy; In Vitro; Infection; Inhibitory Concentration 50; Lead; Learning; Life; Life Cycle Stages; Medical; Metabolism; Mutate; Nature; Nucleosomes; Pathogenesis; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phosphorylation; Phosphotransferases; Play; Population; Process; Proteins; Quantitative Structure-Activity Relationship; SMARCA4 gene; Scheme; Site; Specificity; Staining method; Stains; Study models; Testing; Therapeutic; Time; Toxic effect; Vaccines; Viral; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; Work; absorption; chromatin remodeling; cytotoxicity; design; in vivo; inhibitor/antagonist; interfacial; mimetics; novel; resistant strain; small molecule; virus pathogenesis; young adult

DESCRIPTION (provided by applicant): UNAIDS and WHO estimate that of the 40 million people living with HIV/AIDS in the world, approximately 2.8 million are 50 years and older. In the US, an estimated 1 million people are infected with HIV, although one- third of this population is unaware of their diagnosis. While HIV infection is commonly thought to affect younger adults, there are an increasing number of patients over 50 years of age living with the condition. With the introduction of highly active antiretroviral therapy (HAART) in the mid-1990s, survival following HIV diagnosis has risen dramatically and HIV infection has evolved from an acute disease process to being managed as a chronic medical condition. Over the past two decades, significant progress has been made in understanding HIV pathogenesis and disease progression, which has allowed the identification of a multitude of drug and vaccine targets. Although currently available drug therapies have greatly increased the time from HIV infection to development of AIDS, drug resistance is an inevitable consequence that limits the duration of successful treatment. In this proposal we are targeting a step in viral life cycle that is essential for viral survival and to date there are no therapeutics against this particular pathway. HIV-1 uses Tat as its viral activator and viruses mutated in Tat are not viable. We are planning to continue our work on Tat peptide derivatives (mimetics) inhibitors that could potentially inhibit virus replication both in cell culture and our humanized animal models. From our original submission, we have successfully been able to trim the original Tat 15 aa peptide inhibitor to 5 aa and have found the new peptide to be able to bind to cdk2, and cdk9 tightly and inhibit their kinase activity both in vitro and in vivo. The new peptide and its mimetic (which was not planned in the original proposal) show promising results in inhibiting HIV-1 replication in cell lines and PBMCs through various mechanisms. In the current proposal we are completely focusing on the mimetics and not the peptides anymore. We will extend our initial observations in PBMCs to various pharmacological parameters including cytotoxicity, inhibitory concentrations, and its ability to inhibit replication of numerous viral strains including patient isolates and drug and resistant strains. The inhibition studies will be performed with the two 2 generation mimetics, F07#13 and F07#19, which will allow us to select the best possible inhibitors for a given HIV clade or stain. We will also ask about the functional consequence of SWI/SNF phosphorylation by cdk2/E in relation to HIV-1 chromatin remodeling of nucleosomes. Finally, we will examine the effect of cdk9 phosphorylation by cdk2/E in presence of chromatinized HIV-1 LTR DNA. We will use in vitro biotin DNA transcription assays that uses Tat for activated transcription with and without added cdk2/E and ask in cdk9 can get phosphorylated at any of the 175, 186, 262, 347 and 363 sites. Collectively, our new mimetic inhibitors and better definition of its targets will aid in bringing a new Tat inhibitor to the clinic.

描述（由申请人提供）：联合国艾滋病规划署和世界卫生组织估计，世界上有 4000 万艾滋病毒/艾滋病感染者，其中约 280 万年龄在 50 岁及以上。在美国，估计有 100 万人感染了艾滋病毒，尽管其中三分之一的人不知道自己的诊断结果。虽然人们普遍认为 HIV 感染会影响年轻人，但 50 岁以上的患者中患有这种疾病的人数正在不断增加。随着 20 世纪 90 年代中期高效抗逆转录病毒疗法 (HAART) 的引入，HIV 诊断后的生存率急剧上升，HIV 感染已从急性疾病过程演变为慢性疾病。在过去的二十年中，在了解艾滋病毒发病机制和疾病进展方面取得了重大进展，这使得能够确定多种药物和疫苗靶点。尽管目前可用的药物疗法大大延长了从HIV感染到发展为艾滋病的时间，但耐药性是不可避免的结果，限制了成功治疗的持续时间。在这项提案中，我们的目标是病毒生命周期中对于病毒生存至关重要的一个步骤，迄今为止，还没有针对这一特定途径的治疗方法。 HIV-1 使用 Tat 作为其病毒激活剂，而在 Tat 中突变的病毒无法存活。我们计划继续研究 Tat 肽衍生物（模拟物）抑制剂，该抑制剂可能会抑制细胞培养物和人源化动物模型中的病毒复制。从我们最初提交的材料来看，我们已经成功地将原来的 Tat 15 个氨基酸肽抑制剂修剪为 5 个氨基酸，并发现新的肽能够与 cdk2 和 cdk9 紧密结合，并在体外和体内抑制它们的激酶活性。新的肽及其模拟物（最初的提案中没有计划）在通过各种机制抑制细胞系和 PBMC 中的 HIV-1 复制方面显示出有希望的结果。在当前的提案中，我们完全专注于模拟物，而不是肽。我们将把对 PBMC 的初步观察扩展到各种药理学参数，包括细胞毒性、抑制浓度及其抑制多种病毒株（包括患者分离株、药物株和耐药株）复制的能力。抑制研究将使用两种第二代模拟物 F07#13 和 F07#19 进行，这将使我们能够为给定的 HIV 进化枝或染色剂选择最佳的抑制剂。我们还将询问 cdk2/E 磷酸化 SWI/SNF 与 HIV-1 染色质核小体重塑相关的功能后果。最后，我们将检查在染色质化 HIV-1 LTR DNA 存在的情况下 cdk2/E 对 cdk9 磷酸化的影响。我们将使用体外生物素 DNA 转录测定，使用 Tat 在添加和不添加 cdk2/E 的情况下进行激活转录，并询问 cdk9 可以在 175、186、262、347 和 363 位点中的任何一个位点磷酸化。总的来说，我们的新模拟抑制剂及其靶标的更好定义将有助于将新的 Tat 抑制剂推向临床。