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）是细胞稳态的关键调节剂，这是通过腺嘌呤核苷酸的相互作用 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 在艾滋病毒感染的推定部位的细胞和组织中的药物替诺福韦（TFV）。 TFV是核苷酸反向 转录酶抑制剂被处方为替诺福韦毒素前药与其他药物结合使用 艾滋病毒的治疗。 TFV需要两个顺序磷酸化步骤才能成为药物 积极的。 Tenofovir disoproxil也是唯一获得FDA批准的HIV预防前预防的组成部分（PREP） 方案。 AK2作为TFV激活激酶的识别刺激了我们对人基因组DNA进行测序 〜1200个个体并确定可能影响TFV激活的AK2遗传变异。远处的体外研究 已经透露，其中一些变体确实确实影响了AK2活动对TFV。在前进时， 衰老对AK2表达和活性的影响将进行特定测试。确定TFV-的活性是否 激活激酶，尤其是AK2，可能在老年人和年轻人中表现出差异性活动 由于老年人（≥50岁）以来的重要性占每年新艾滋病毒感染的17％。 该提议的目的是：1）检验以下假设：AK2是参与该的主要AK酶 TFV的磷酸化。我们将在培养的CD4+中沉默9种单独的AK酶的表达 使用CRISPR/CAS9系统的细胞并测试对TFV的活性。另外，AK酶将是cDNA- 表达和净化以测试其活动。将采用生物物理方法，以获得 了解绑定亲和力。此外，我们将测试与年龄相关的修改对AK2表达的影响 和活动； 2）检验以下假设：激活较老的TFV的激酶的模式和活性 成年人（65-80岁）和年轻（18-30岁）的成年人，循环CD4+ T细胞和CD4+ T细胞中的成年人（18-30岁） 结直肠组织。此外，我们将测试老年人中TFV的激活是否不同于年轻人 通过表征磷酸化TFV的水平，以替诺福韦的口服给药后的成年人 循环和结直肠组织居民CD4+ T细胞。 MALDI-MAS光谱成像将被聘为 可视化老年人与年轻人的结直肠组织CD4+ T细胞中磷酸化的TFV的分布。