Nucleoside Triphosphate Diphosphohydrolase 1 Exhibits Enzymatic Activity toward Tenofovir Diphosphate

Abstract

Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFVdiphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultra-highperformance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was utilized to measure the relative abundance of NTPDases in human colorectal tissue among adult healthy individuals (n=4). These analyses confirmed the high abundance of NTPDase 1 in human colorectal tissue. Taken together, results suggest that NTPDase 1 may contribute to the regulation of TFV-DP levels. The above data provide important insights into the dephosphorylation of TFV-DP.

Tenofovir Activation Is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice

“…Further, Entpd1, which is able to dephosphorylate TFV-DP to TFV, was increased in WT female and Ckb tm1Nnb male brains compared to WT male brains. 47 Together, the differential abundances of these enzymes likely compound the loss of Ckb, resulting in the significant reduction in the level of TFV-DP formation we observed in Ckb tm1Nnb brain lysates.…”

Special Section on Drug Metabolism and Precision Medicine—Editorial

“…The enzymes that can catalyze dephosphorylation of tenofovir-diphosphate, the pharmacologically active metabolite of tenofovir, have not been previously reported. This study highlights the fact that interindividual differences in dephosphorylation activity could impact tenofovir disposition and pharmacology in precision medicine (Seneviratne, 2023).…”

Cytosolic 5′-Nucleotidase III and Nucleoside Triphosphate Diphosphohydrolase 1 Dephosphorylate the Pharmacologically Active Metabolites of Gemcitabine and Emtricitabine

“…We did not test the nucleotidases that were not available commercially. In vitro incubations were conducted according to previously described methods, with slight modifications (Seneviratne, 2023). Specifically, in a reaction volume of 100 μL, 10 nM of human recombinant protein, 1 mM monophosphate substrate (dCMP, dFdCMP, and FTC-MP) or 0.1 mM triphosphate substrate (dCTP, dFdCTP, and FTC-TP), and 5 mM MgCl 2 were incubated in 50 mM TRIS-HCl (pH 7.5) buffer at 37°C for 30 minutes.…”

“…Enzyme kinetics of drug metabolite dephosphorylation mediated by NT5C3 for dCMP, dFdCMP, and FTC-MP, and NTPDase 1 for dCTP, dFdCTP, and FTC-TP was conducted as previously described with some modifications (Seneviratne, 2023). Initial experiments were conducted to ascertain the linearity of enzyme-mediated dephosphorylation of the aforementioned substrates with respect to time and enzyme concentrations.…”

“…Immunoblotting procedures were performed as previously described (Seneviratne, 2023). Mouse pancreatic (n = 3, male and n = 3, female) and colorectal tissues (n = 3, male and n = 3, female) were lysed in 1X cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM Phenylmethanesulfonyl Fluoride (Cell Signaling Technology, Beverly, MA, USA) and centrifuged at 14,000 x g for 10 min at 4°C, and the supernatant was collected.…”