Genetic Variation of the Kinases That Phosphorylate Tenofovir and Emtricitabine in Peripheral Blood Mononuclear Cells

Abstract

Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation.

HIV Antiretroviral Pre‐Exposure Prophylaxis: Development Challenges and Pipeline Promise

“…Although there are competing methods for incorporation of the dNTP data into complex PK‐viral dynamics models of these interactions, the data help explain the need for at least 6–7 weekly doses of TDF/FTC in women (whose primary risk is receptive vaginal intercourse (RVI)), less frequent 4 per week dosing in MSM and TGW (whose primary risk is receptive anal intercourse (RAI)), and how only a few doses seems to provide rapid protection in MSM and TGW as in Ipergay . Another source of PK variability is that the specific intracellular kinases that phosphorylate TFV and FTC vary anatomically among the cells in blood, cervicovaginal tissue, and colorectal tissue; the kinases are also genetically polymorphic …”

“…Assuming similar adherence, oral TDF/FTC dosing prevents colorectal infection with less frequent dosing (4 per week) compared to preventing vaginal infection (6–7 doses per week), despite the higher risk of infection with RAI. Mucosal TDF and FTC PK and dNTP differences between colorectal and cervicovaginal tissue provide a plausible explanation these outcome difference . However, because systemic NRTI and dNTP PK is no different in MSM (primary RAI risk) compared with women (primary RVI risk), systemic concentration cannot contribute to differences in PrEP effectiveness in MSM and women. When modeling plasma TFV concentration‐seroconversion response relationships across all of the primary RCTs using daily dosing, most of the variation in the relationship is explained by adjusting for anatomic differences of TFV‐DP concentrations at mucosal sites of HIV acquisition and the higher RAI risk in MSM; both are mucosal differences, not systemic …”

“…15 Another source of PK variability is that the specific intracellular kinases that phosphorylate TFV and FTC vary anatomically among the cells in blood, cervicovaginal tissue, and colorectal tissue; the kinases are also genetically polymorphic. [29][30][31] Vaginal microbiome and genital inflammation An abnormal nonlactobacillus dominant vaginal microbiome was associated with reduced cervicovaginal lavage fluid (CVL) and plasma TFV concentrations in different studies, as well as a threefold reduction in HIV protection in the CAPRISA 004 study of TFV vaginal gel. 32,33 Some post hoc analysis concerns were allayed by finding nonlactobacillus dominant microbiome in similar proportions of high and low adherence groups and both TFV and placebo arms.…”

Pharmacology of Antiretrovirals in the Female Genital Tract for HIV Prevention

“…Although current research on the activation of TFV in the female genital tract is lacking, a study carried out by Lade et al suggested several kinases, notably adenylate kinase 2, pyruvate kinase muscle, and pyruvate kinase liver/red blood cell,were responsible for the transformation of TFV to the active metabolite of TFVdp in peripheral blood mononuclear cells (PBMCs) and female genital tract biopsies, differing from the kinase activity observed in the colorectal tissues which relied on adenylate kinase 2 and creatinine kinase muscle . Genetic diversity in these enzymes, as well as those responsible for FTC activation, may also impact local drug disposition, but due to low levels of polymorphisms in these specific kinases, the overall impact at the population level may be low . Further research must be conducted to gain a clearer picture of these mechanisms and their connection to the success of PrEP therapy.…”

“…Genetic diversity in these enzymes, as well as those responsible for FTC activation, may also impact local drug disposition, but due to low levels of polymorphisms in these specific kinases the overall impact at the population level may be low. 34 Further research must be conducted to gain a clearer picture of these mechanisms and their connection to the success of PrEP therapy.…”