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

Abstract

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.

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics

“…This enzyme assay connects any ADP formation that occurs upon phosphorylation of RMP to the oxidation of NADH, which can be followed by UV absorbance change at 340 nm ( Supplementary Fig. 8) [48][49][50] .…”

“…Non-specific small molecule ENT inhibitors have been identified 51 , although gene-specific inhibitors have not been identified. Homozygous germline SLC29A3 knockout is associated with histiocytosis (macrophage expansion) in mice and humans 50,51 , although it is unknown whether transient inhibition of SLC29A3 would have side effects. To gauge the relevance of SLC29A3 as a therapeutic target, animal virus restriction studies on SLC29A3 deficient models should be performed, and gene-specific inhibitors must be identified and tested for safety and efficacy in vivo.…”

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.…”