Drug Phosphorylation and Aging

药物磷酸化与老化

• 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
• 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

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 ATP、ADP 和 AMP。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）存在于假定的 HIV 感染部位的细胞和组织中。 TFV 是核苷酸反向 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 治疗艾滋病毒。 TFV 需要两个连续的磷酸化步骤才能发挥药理学作用 积极的。替诺福韦二吡呋酯也是 FDA 唯一批准的 HIV 暴露前预防 (PrEP) 的组成部分 养生法。 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 对约 1200 名个体进行了研究，并识别出可能影响 TFV 激活的 AK2 遗传变异。迄今为止，体外研究 研究表明，其中一些变体确实会影响 AK2 对 TFV 的活性。在前进的过程中， 将具体测试衰老对 AK2 表达和活性的影响。确定 TFV- 的活性是否 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 重要性，因为老年人（≥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 TFV 的磷酸化。我们将沉默培养的 CD4+ 中 9 种单独 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- 表达并纯化以测试其活性。将应用生物物理方法以获得 了解结合亲和力。此外，我们将测试年龄相关修饰对 AK2 表达的影响 和活动； 2) 测试以下假设：激活 TFV 的激酶的模式和活性在老年人之间有所不同 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 结直肠组织。此外，我们将测试老年人和年轻人的 TFV 激活是否不同 成人口服替诺福韦二吡呋酯后，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。 MALDI-质谱成像将用于 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。