Phosphorylation of Drugs in Colonic Tissue

结肠组织中药物的磷酸化

基本信息

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
项目类别：
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

项目摘要

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) 是一种丰富的酶，在调节细胞能量稳态中发挥着核心作用。它们在高能量通量的环境中尤其重要。 CK酶催化以下物质的相互转化磷酸肌酸和 ADP 与肌酸和 ATP 一起作用，从而在快速反应期间缓冲 ATP 浓度细胞能量消耗。 CK家族有四位主要成员。我们报道过肌酸激酶肌肉（CKM）在替诺福韦（TFV）在结肠组织中的磷酸化中发挥重要作用，结肠组织是一个假定的位点艾滋病毒感染。 TFV 是一种核苷酸逆转录酶抑制剂，处方为替诺福韦二吡呋酯或替诺福韦艾拉酚胺前药与其他药物联合用于治疗和预防 HIV 感染。 TFV 必须被磷酸化两次，从而形成 TFV-二磷酸 (TFV-DP)，才能成为药理活性。我们发现 CKM 在结肠组织中执行最终的磷酸化步骤这是 TFV 激活所需的。值得注意的是，我们进行的基因组分析揭示了至少 15 预计会导致 CKM 活性丧失或降低的新变异。这里提出的研究将调查通过将点突变引入结肠 CD4+ T 细胞中的 CKM 并测试对 TFV-DP 生产的影响。氢-氘交换质谱法将用于探测自然发生的突变对 CKM 结构的影响。另外，我们会测试其他CK家族是否成员能够磷酸化 TFV，这在寻求了解 TFV 时尤其重要 TFV 活跃的组织范围内的药理学。此外，我们将使用基于质谱的成像方法阐明结肠组织中 CKM 和药理活性 TFV-DP 的分布和结肠 CD4+ T 细胞。我们将通过招募健康的参与者来在整个生命周期中检查这一点跨越各个年龄段。此外，我们将探讨 CD4+ T 细胞激活是否影响 CKM 表达和结肠 CD4+ T 细胞中的活性和/或 TFV 激活。这些研究将为我们提供机制上的见解调节结肠组织中 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。