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）通过腺嘌呤核苷酸的相互转化是细胞稳态的关键调节因子