Drug Phosphorylation and Aging

药物磷酸化与老化

基本信息

批准号：
10611341
负责人：
Benjamin Carl Orsburn
金额：
$ 55.56万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10611341
关键词：
3-nitrotyrosine Address Adenine Nucleotides Adherence Adult Affinity Age Age Years Aging Anti-Retroviral Agents Binding CD4 Positive T Lymphocytes CRISPR/Cas technology Cell Separation Cells Clinical Research Colorectal Complementary DNA Data Diphosphates Dose Drug Kinetics Elderly Enzymes Exhibits FDA approved Failure Genomic DNA Goals HIV HIV Infections Hepatitis B Therapy Homeostasis Human Immune In Vitro Individual Kinetics Mass Spectrum Analysis Measures Metabolic Biotransformation Modification Nucleotides Oral Pathway interactions Pattern Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Pharmacogenetics Pharmacology Pharmacotherapy Phosphorylation Phosphotransferases Play Population Prodrugs Proteomics RNA-Directed DNA Polymerase Regimen Reporting Reverse Transcriptase Inhibitors Role Site Spatial Distribution Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization System Tenofovir Testing Tissues Variant Visualization Work adenylate kinase age effect age related biophysical techniques experience genetic variant insight inter-individual variation lens mass spectrometric imaging mutant nucleotide analog pharmacologic pre-exposure prophylaxis sexual HIV transmission young adult

项目摘要

Adenylate kinase 2 (AK2) is a key regulator of cellular homeostasis via the interconversion of adenine nucleotides ATP, ADP, and AMP. We recently demonstrated that AK2 plays a crucial role in the activation of the antiretroviral drug tenofovir (TFV) in cells and tissues that are putative sites of HIV infection. TFV is a nucleotide reverse transcriptase inhibitor that is prescribed as a tenofovir disoproxil prodrug in combination with other drugs for the treatment of HIV. TFV requires two sequential phosphorylation steps in order to become pharmacologically active. Tenofovir disoproxil is also a component of the only FDA approved HIV pre-exposure prophylaxis (PrEP) regimen. The identification of AK2 as a TFV-activating kinase spurred us to sequence the human genomic DNA of ~1200 individuals and identify AK2 genetic variants that could impact TFV activation. Thus far, in vitro studies have revealed that several of these variants do indeed impact AK2 activity towards TFV. In moving forward, an effect of aging on AK2 expression and activity will be tested specifically. Determining whether the activity of TFV- activating kinases, particularly AK2, could exhibit differential activity in older versus younger adults is of importance since older adults (≥50 years of age) account for an approximate 17% of new HIV infections annually. The aims of this proposal are to: 1) test the hypothesis that AK2 is the primary AK enzyme involved in the phosphorylation of TFV. We will silence the expression of each of the 9 individual AK enzymes in cultured CD4+ cells using a CRISPR/Cas9 system and test for activity towards TFV. In addition, AK enzymes will be cDNA- expressed and purified to test for their activities. Biophysical approaches will be applied in order to gain an understanding of binding affinity. Further, we will test the impact of age-related modifications on AK2 expression and activity; 2) test the hypothesis that the patterns and activity of kinases that activate TFV differ between older adults (ages 65-80) and younger (ages 18-30) adults in circulating CD4+ T cells and CD4+ T cells that reside in colorectal tissue. In addition, we will test whether activation of TFV in older adults differs from that of younger adults following oral dosing with tenofovir disoproxil, via characterization of the levels of phosphorylated TFV in circulating and colorectal tissue resident CD4+ T cells. MALDI-mass spectrometry imaging will be employed to visualize the distribution of phosphorylated TFV in colorectal tissue CD4+ T cells of older versus younger adults.

腺苷酸激酶 2 (AK2) 是通过腺嘌呤核苷酸相互转化来调节细胞稳态的关键因子我们最近证明 AK2 在抗逆转录病毒的激活中起着至关重要的作用。药物替诺福韦 (TFV) 在 HIV 感染的细胞和组织中是一种核苷酸逆转。转录酶抑制剂，作为替诺福韦二吡呋酯前药与其他药物联合使用，用于治疗 HIV的治疗需要两个连续的磷酸化步骤才能发挥药理学作用。替诺福韦二吡呋酯也是 FDA 唯一批准的 HIV 暴露前预防 (PrEP) 的成分。 AK2 作为 TFV 激活激酶的鉴定促使我们对人类基因组 DNA 进行测序。迄今为止，体外研究对约 1200 名个体进行了研究，并鉴定出可能影响 TFV 激活的 AK2 基因变异。研究表明，其中一些变体确实会影响 AK2 对 TFV 的活性。将具体测试衰老对AK2表达和活性的影响，以确定TFV-的活性。激活激酶，特别是 AK2，在老年人和年轻人中可能表现出不同的活性老年人（≥50 岁）每年约占新发 HIV 感染的 17%。该提案的目的是：1) 检验 AK2 是参与 AK 酶的主要酶的假设我们将沉默培养的 CD4+ 中 9 种单独的 AK 酶的表达。使用 CRISPR/Cas9 系统检测细胞并测试对 TFV 的活性。此外，AK 酶将是 cDNA-。将应用生物物理方法进行表达和纯化以测试其活性。此外，我们将测试年龄相关修饰对 AK2 表达的影响。和活性；2) 检验老年人之间激活 TFV 的激酶的模式和活性不同的假设成人（65-80 岁）和年轻人（18-30 岁）循环 CD4+ T 细胞和驻留在体内的 CD4+ T 细胞此外，我们将测试老年人的 TFV 激活是否与年轻人不同。口服替诺福韦二吡呋酯后的成人，通过表征磷酸化 TFV 的水平循环和结直肠组织驻留 CD4+ T 细胞将用于检测。可视化老年人与年轻人结直肠组织 CD4+ T 细胞中磷酸化 TFV 的分布。