Drug Phosphorylation and Aging

药物磷酸化与老化

• 批准号: 10217514
• 负责人: Namandje N Bumpus
• 金额: $ 12.5万
• 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/10217514
• 关键词: Adenine Nucleotides; Adult; Affinity; Age; Age-Years; Aging; Anti-Retroviral Agents; Binding; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cells; Colorectal; Complementary DNA; Dose; Elderly; Enzymes; Exhibits; FDA approved; Genomic DNA; Goals; HIV; HIV Infections; Homeostasis; Human; Image; In Vitro; Individual; Mass Spectrum Analysis; Modification; Nucleotides; Oral; Pattern; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphorylation; Phosphotransferases; Play; Prodrugs; Regimen; Reverse Transcriptase Inhibitors; Role; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Tenofovir; Testing; Tissues; Variant; Work; adenylate kinase; age effect; age related; biophysical techniques; genetic variant; pre-exposure prophylaxis; 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内环境平衡的关键因子 ATP、ADP和AMP。我们最近证明了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)在细胞和组织中，这些细胞和组织可能是艾滋病毒感染的部位。TFV是一种核苷酸反转 转录酶抑制剂，作为替诺福韦异丙酚前体药物与其他药物联合治疗 艾滋病毒的治疗。TFV需要两个连续的磷酸化步骤才能成为药理上的 激活。替诺福韦也是FDA批准的唯一HIV暴露前预防(PrEP)的一种成分 养生法。AK2是一种TFV激活蛋白，这促使我们对人类基因组DNA进行了测序 并确定可能影响TFV激活的AK2基因变异。到目前为止，体外研究 已经揭示，这些变体中的几个确实影响了AK2对TFV的活性。在向前迈进的过程中，一个 将专门测试衰老对AK2表达和活性的影响。确定TFV的活性是否- 激活激酶，尤其是AK2，在老年人和年轻人中可能表现出不同的活性。 由于老年人(≥，50岁)约占每年新增艾滋病毒感染者的17%，这一点非常重要。 这一提议的目的是：1)检验AK2是参与 TFV的磷酸化。我们将沉默培养的CD4+中9个单独的AK酶的表达 使用CRISPR/CAS9系统的细胞，并测试对TFV的活性。此外，AK酶将是cDNA3- 对其进行表达和纯化，以检测其活性。将应用生物物理方法，以获得 对结合亲和力的理解。此外，我们将测试与年龄相关的修饰对AK2表达的影响 和活性；2)检验这样的假设，即激活TFV的激酶的模式和活性在老年人之间不同 成人(65-80岁)和年轻(18-30岁)成人循环中的CD4+T细胞和驻留在 结直肠组织。此外，我们将测试TFV在老年人和年轻人中的激活情况是否不同 成人口服替诺福韦异丙酚后，体内磷酸化TFV水平的变化 循环和结直肠组织中驻留的CD4+T细胞。将采用MALDI-MS成像技术 观察大肠组织中磷酸化TFV在老年人和年轻人中的分布情况。