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Discovery of Genetic Variants of the Kinases That Activate Tenofovir in a Compartment-specific Manner.

Lade JM, To EE, Hendrix CW, Bumpus NN - EBioMedicine (2015)

Bottom Line: Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention.Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues.Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Biophysics 307, Baltimore, MD 21205, USA ; Department of Medicine (Division of Clinical Pharmacology), Johns Hopkins University School of Medicine, 725 North Wolfe Street, Biophysics 307, Baltimore, MD 21205, USA.

ABSTRACT
Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry-liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.

No MeSH data available.


Related in: MedlinePlus

Schematic summarizing the intracellular activation of the antiretroviral drug TFV in cells and tissues susceptible to HIV infection.AK2 can transform TFV to TFV-MP in PBMC, colorectal, and vaginal tissues. PKM and PKLR can transform TFV-MP to the active anabolite TFV-DP in PBMC and vaginal tissue, while CKM can transform TFV-MP to TFV-DP in colorectal tissue.
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f0010: Schematic summarizing the intracellular activation of the antiretroviral drug TFV in cells and tissues susceptible to HIV infection.AK2 can transform TFV to TFV-MP in PBMC, colorectal, and vaginal tissues. PKM and PKLR can transform TFV-MP to the active anabolite TFV-DP in PBMC and vaginal tissue, while CKM can transform TFV-MP to TFV-DP in colorectal tissue.

Mentions: Guided by our preliminary findings, siRNA was used to knockdown the following nucleotide kinases AK2, GUK1, PKM, and PKLR in PBMC (n = 3), AK2, GUK1, and CKM in colorectal tissue (n = 3), and AK2, GUK1, PKM, and PKLR in vaginal tissue (n = 3). Decreased expression of each targeted kinase was evaluated relative to the non-targeting siRNA control using immunoblot analysis (Fig. 1). The intracellular formation of TFV metabolites was detected using uHPLC–MS/MS and the impact of enzyme loss-of-function on TFV-MP and TFV-DP formation following incubation with TFV are shown as bar graphs in Fig. 1. When AK2 was knocked down, TFV-MP decreased to 17 ± 1.4% (17 / 100 ± 1.4 / 100), 9.5 ± 1.1% (9.5 / 100 ± 1.1 / 100), and 12 ± 1.5% (12 / 100 ± 1.5 / 100; p-value = 3.4E − 6, 3.6E − 5, and 7.2E − 5) of the non-targeting siRNA control in PBMC, colorectal tissue, and vaginal tissue, respectively. Knockdown of AK2 protein expression also resulted in a decrease of TFV-DP to 13 ± 1.7% (13 / 100 ± 1.7/100), 15 ± 2.3% (15 / 100 ± 2.3 / 100), and 9 ± 2.2% (9 / 100 ± 2.2 / 100; p-value = 3.9E − 6, 4.5E − 6, and 2.7E − 5) of non-targeting control in PBMC, colorectal tissue, and vaginal tissue, respectively. Knockdown of PKM decreased TFV-DP to 33 ± 5.8% (33 / 100 ± 5.8 / 100) and 27 ± 4.3% (27 / 100 ± 4.3 / 100; p-value = 2.7E − 5 and 8.2E − 5) of control in PBMC and vaginal tissue, respectively. Additionally, knockdown of PKLR decreased TFV-DP to 78 ± 6.6% (78 / 100 ± 6.6 / 100) and 81 ± 7.4% (81 / 100 ± 7.4 / 100; p-value = 0.008 and 0.017) of control in PBMC and vaginal tissue, respectively. When CKM was knocked down in colorectal tissue, TFV-DP was decreased to 8 ± 2.9% (8 / 100 ± 2.9 / 100; p-value = 2.2E − 5) of control. The observed tissue-specific activation of TFV has been summarized in Fig. 2.


Discovery of Genetic Variants of the Kinases That Activate Tenofovir in a Compartment-specific Manner.

Lade JM, To EE, Hendrix CW, Bumpus NN - EBioMedicine (2015)

Schematic summarizing the intracellular activation of the antiretroviral drug TFV in cells and tissues susceptible to HIV infection.AK2 can transform TFV to TFV-MP in PBMC, colorectal, and vaginal tissues. PKM and PKLR can transform TFV-MP to the active anabolite TFV-DP in PBMC and vaginal tissue, while CKM can transform TFV-MP to TFV-DP in colorectal tissue.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588390&req=5

f0010: Schematic summarizing the intracellular activation of the antiretroviral drug TFV in cells and tissues susceptible to HIV infection.AK2 can transform TFV to TFV-MP in PBMC, colorectal, and vaginal tissues. PKM and PKLR can transform TFV-MP to the active anabolite TFV-DP in PBMC and vaginal tissue, while CKM can transform TFV-MP to TFV-DP in colorectal tissue.
Mentions: Guided by our preliminary findings, siRNA was used to knockdown the following nucleotide kinases AK2, GUK1, PKM, and PKLR in PBMC (n = 3), AK2, GUK1, and CKM in colorectal tissue (n = 3), and AK2, GUK1, PKM, and PKLR in vaginal tissue (n = 3). Decreased expression of each targeted kinase was evaluated relative to the non-targeting siRNA control using immunoblot analysis (Fig. 1). The intracellular formation of TFV metabolites was detected using uHPLC–MS/MS and the impact of enzyme loss-of-function on TFV-MP and TFV-DP formation following incubation with TFV are shown as bar graphs in Fig. 1. When AK2 was knocked down, TFV-MP decreased to 17 ± 1.4% (17 / 100 ± 1.4 / 100), 9.5 ± 1.1% (9.5 / 100 ± 1.1 / 100), and 12 ± 1.5% (12 / 100 ± 1.5 / 100; p-value = 3.4E − 6, 3.6E − 5, and 7.2E − 5) of the non-targeting siRNA control in PBMC, colorectal tissue, and vaginal tissue, respectively. Knockdown of AK2 protein expression also resulted in a decrease of TFV-DP to 13 ± 1.7% (13 / 100 ± 1.7/100), 15 ± 2.3% (15 / 100 ± 2.3 / 100), and 9 ± 2.2% (9 / 100 ± 2.2 / 100; p-value = 3.9E − 6, 4.5E − 6, and 2.7E − 5) of non-targeting control in PBMC, colorectal tissue, and vaginal tissue, respectively. Knockdown of PKM decreased TFV-DP to 33 ± 5.8% (33 / 100 ± 5.8 / 100) and 27 ± 4.3% (27 / 100 ± 4.3 / 100; p-value = 2.7E − 5 and 8.2E − 5) of control in PBMC and vaginal tissue, respectively. Additionally, knockdown of PKLR decreased TFV-DP to 78 ± 6.6% (78 / 100 ± 6.6 / 100) and 81 ± 7.4% (81 / 100 ± 7.4 / 100; p-value = 0.008 and 0.017) of control in PBMC and vaginal tissue, respectively. When CKM was knocked down in colorectal tissue, TFV-DP was decreased to 8 ± 2.9% (8 / 100 ± 2.9 / 100; p-value = 2.2E − 5) of control. The observed tissue-specific activation of TFV has been summarized in Fig. 2.

Bottom Line: Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention.Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues.Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Biophysics 307, Baltimore, MD 21205, USA ; Department of Medicine (Division of Clinical Pharmacology), Johns Hopkins University School of Medicine, 725 North Wolfe Street, Biophysics 307, Baltimore, MD 21205, USA.

ABSTRACT
Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry-liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.

No MeSH data available.


Related in: MedlinePlus