Drug Phosphorylation and Aging

药物磷酸化与老化

• 批准号: 10371082
• 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
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371082
• 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; 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; Work; adenylate kinase; age effect; age related; biophysical techniques; experience; genetic variant; insight; inter-individual variation; lens; mass spectrometric imaging; mutant; nucleotide analog; pre-exposure prophylaxis; sexual HIV transmission; young adult

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（AK 2）是通过腺嘌呤核苷酸的相互转换来调节细胞内稳态的关键调节剂 ATP、ADP和AMP。我们最近证明AK 2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）在细胞和组织中是HIV感染的假定位点。TFV是一种核苷酸反向 作为替诺福韦酯前体药物与其他药物联合处方的转录酶抑制剂， 治疗艾滋病毒。TFV需要两个连续的磷酸化步骤，以成为一个磷酸化酶。 活跃替诺福韦酯也是FDA唯一批准的HIV暴露前预防（PrEP）的组成部分 方案. AK 2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， 约1200个个体，并确定可能影响TFV激活的AK 2遗传变异。到目前为止，体外研究 已经表明，其中几种变体确实影响AK 2对TFV的活性。在前进的过程中， 将具体测试老化对AK 2表达和活性的影响。确定TFV的活性- 激活激酶，特别是AK 2，在老年人和年轻人中表现出不同的活性， 由于老年人（≥50岁）约占每年新感染艾滋病毒人数的17%，因此这一点非常重要。 该提议的目的是：1）测试AK 2是参与细胞凋亡的主要AK酶的假设。 TFV的磷酸化。我们将在培养的CD 4 + T细胞中沉默9种单独的AK酶中的每一种的表达。 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- 表达并纯化以测试其活性。生物物理方法将被应用，以获得一个 对亲和力的理解此外，我们将测试与年龄相关的修饰对AK 2表达的影响 2）验证激活TFV的激酶的模式和活性在老年人和老年人之间存在差异的假设。 成年人（65-80岁）和年轻人（18-30岁）的循环CD 4 + T细胞和CD 4 + T细胞， 结直肠组织此外，我们将测试老年人的TFV激活是否与年轻人不同。 成年人口服替诺福韦酯后，通过表征磷酸化TFV的水平， 循环和结直肠组织驻留的CD 4 + T细胞。MALDI-质谱成像将用于 显示老年人与年轻人结肠直肠组织CD 4 + T细胞中磷酸化TFV的分布。