Phosphorylation of Drugs in Colonic Tissue

结肠组织中药物的磷酸化

• 批准号: 10653625
• 负责人: Craig Walter Hendrix
• 金额: $ 72.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10653625
• 关键词: 3-nitrotyrosine; AIDS prevention; Active Sites; Adherence; Age; Aging; Anal Sex; Biochemistry; Biopsy; Buffers; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cells; Clinical; Clinical Research; Clinical Trials; Colon; Creatine; Creatine Kinase; Data; Deuterium; Development; Diagnosis; Diphosphates; Elderly; Energy Metabolism; Environment; Enzymes; Exhibits; FDA approved; Family; Family member; Genes; Genetic; Genetic Variation; Genomics; Goals; HIV; HIV Infections; Homeostasis; Human; Hydrogen; Infection; Kinetics; Longevity; MM form creatine kinase; Maps; Mass Spectrum Analysis; Measures; Modification; Muscle; Mutation; Nucleotides; Participant; Pattern; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphocreatine; Phosphorylation; Phosphotransferases; Play; Point Mutation; Post-Translational Protein Processing; Prevention strategy; Process; Prodrugs; Production; Proteins; Proteomics; Regimen; Reporting; Resolution; Reverse Transcriptase Inhibitors; Risk; Role; Route; Sampling; Site; Spatial Distribution; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; T-Cell Activation; Tenofovir; Testing; Tissues; United States; Variant; age effect; base; clinically relevant; dimer; energy balance; enzyme activity; imaging approach; in silico; inorganic phosphate; insight; inter-individual variation; male; mass spectrometric imaging; men; muscle pharmacology; muscular structure; mutant; novel; pre-exposure prophylaxis; preservation; recruit; response; transmission process

Creatine kinases (CK) are abundant enzymes that play a central role in regulating cellular energy homeostasis. They are particularly important in environments of high energy flux. CK enzymes catalyze the interconversion of phosphocreatine and ADP with creatine and ATP, thereby buffering ATP concentrations during periods of rapid cellular energy expenditure. There are four main CK family members. We have reported that creatine kinase muscle (CKM) plays an important role in the phosphorylation of tenofovir (TFV) in colonic tissues, a putative site of HIV infection. TFV is a nucleotide reverse transcriptase inhibitor that is prescribed as a tenofovir disoproxil or tenofovir alafenamide prodrug in combination with other drugs for the treatment and prevention of HIV infection. TFV must be phosphorylated twice, resulting in the formation of TFV-diphosphate (TFV-DP) in order to becomes pharmacologically active. We have found that CKM carries out the final phosphorylation step in colonic tissue that is required for TFV activation. Of note, genomic analysis that we have performed has revealed at least 15 novel variants predicted to result in a loss or decrease of CKM activity. Studies proposed here will investigate these variants mechanistically by introducing point mutations into CKM in colonic CD4+ T cells and testing the impact on TFV-DP production. Hydrogen-deuterium exchange mass spectrometry will be used to probe the impact of naturally occurring mutations on CKM structure. In addition, we will test whether other CK family members are able to phosphorylate TFV, which could be particularly important when seeking to understand TFV pharmacology in the range of tissues in which TFV is active. Further, we will use a mass spectrometry-based imaging approach to elucidate the distribution of CKM and pharmacologically active TFV-DP in colonic tissue and colonic CD4+ T cells. We will examine this across the life span through the recruitment of healthy participants across a range of ages. In addition, we will probe whether CD4+ T cell activation impacts CKM expression and activity, and/or TFV activation in colonic CD4+ T cells. These studies will provide mechanistic insight into the variables that regulate TFV-DP levels in colonic tissue that can be leveraged in optimizing TFV-based drug regimens, while lending unique insight into CK biochemistry and colonic pharmacology more broadly.

肌酸激酶（CK）是一种在调节细胞能量平衡中发挥核心作用的酶。 它们在高能量通量的环境中特别重要。CK酶催化 磷酸肌酸和ADP与肌酸和ATP，从而缓冲ATP浓度期间的快速 细胞能量消耗有四个主要的CK家族成员。我们曾报道肌酸激酶 肌钙蛋白（CKM）在结肠组织中替诺福韦（TFV）的磷酸化中起重要作用， 艾滋病毒感染。TFV是一种核苷酸逆转录酶抑制剂，作为替诺福韦酯或替诺福韦酯处方。 替诺福韦艾拉酚胺前药与其它药物组合用于治疗和预防HIV感染。 TFV必须被磷酸化两次，导致形成TFV-二磷酸（TFV-DP），以便成为 活跃的。我们发现CKM在结肠组织中进行最后的磷酸化步骤 这是激活TFV所必需的。值得注意的是，我们进行的基因组分析显示，至少有15个 预测导致CKM活性丧失或降低的新变体。这里提出的研究将调查 通过将点突变引入结肠CD 4 + T细胞中的CKM中并测试这些突变， 影响TFV-DP生产。氢-氘交换质谱法将用于探测 天然突变对CKM结构的影响。此外，我们将测试其他CK家族是否 成员能够磷酸化TFV，这在试图理解TFV时可能特别重要 在TFV具有活性的组织范围内的药理学。此外，我们将使用基于质谱的 结肠组织中CKM和TFV-DP分布的影像学研究 和结肠CD 4 + T细胞。我们将通过招募健康的参与者来检查整个生命周期的这一点 在不同的年龄段。此外，我们将探讨CD 4 + T细胞活化是否影响CKM表达， 活性和/或结肠CD 4 + T细胞中的TFV活化。这些研究将提供对 调节结肠组织中TFV-DP水平的变量，可用于优化基于TFV的药物 方案，同时更广泛地提供对CK生物化学和结肠药理学的独特见解。