Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067

• Published in: PloS one 2018-04, Vol.13 (4), p.e0195764-e0195764
• Main Authors: Figueroa, Dominique B, Tillotson, Joseph, Li, Maoji, Piwowar-Manning, Estelle, Hendrix, Craig W, Holtz, Timothy H, Bokoch, Kevin, Bekker, Linda-Gail, van Griensven, Frits, Mannheimer, Sharon, Hughes, James P, Grant, Robert M, Bumpus, Namandjé N
• Format: Article
• Language: English
• Subjects: Acquired immune deficiency syndrome; Adenylate kinase; Adenylate Kinase - genetics; AIDS; Analysis; Anti-HIV Agents - pharmacology; Antiretroviral agents; Antiretroviral drugs; Bioinformatics; Biology and Life Sciences; Blood; Care and treatment; Complications and side effects; Creatine; Creatine kinase; Creatine Kinase, MM Form - genetics; Deoxyribonucleic acid; Disease control; Disease prevention; DNA; DNA polymerases; Dosage and administration; E coli; Enzymatic activity; Epidemiology; Erythrocytes; Genetic diversity; Genetic variance; Genetic Variation; Genomes; Hepatocytes; High-Throughput Nucleotide Sequencing; HIV; HIV Infections - drug therapy; HIV Infections - epidemiology; HIV Infections - genetics; HIV Infections - virology; HIV-1 - drug effects; HIV-1 - enzymology; HIV-1 - genetics; Homeostasis; Human immunodeficiency virus; Humans; Infections; Inhibitors; Kinases; Laboratories; Liver; Medical research; Medicine; Medicine and Health Sciences; Metabolism; Muscles; Mutation; People and places; Pharmacology; Phosphorylation; Physical Sciences; Predictions; Public health; Pyruvate kinase; Pyruvate Kinase - genetics; Pyruvic acid; Research and analysis methods; RNA-directed DNA polymerase; South Africa - epidemiology; Tenofovir; Tenofovir - pharmacology; Thailand - epidemiology; United States - epidemiology; Womens health
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0195764

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.