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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

Distribution of nucleotide kinase genetic variants detected in 57 MTN-001 participants.Each colored oval is representative of a nucleotide kinase AK2 (blue), CKM (red), PKM (green), and PKLR (yellow). Non-overlapping regions demonstrate the number of participants observed to carry single nucleotide variants (SNVs) and deletions within only one gene (n = 40; AK2 n = 6, CKM n = 8, PKM n = 14, PKLR n = 12). Overlapping regions demonstrate the number of participants that were observed to carry SNVs and deletions in more than one gene (n = 17). For example, moving down the left-hand side of the diagram, six participants carried AK2 genetic variants alone, two participants carried variants for AK2 and PKM, one participant carried variants for AK2, PKM, and PKLR, and one participant carried variants for AK2 and PKLR.
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f0015: Distribution of nucleotide kinase genetic variants detected in 57 MTN-001 participants.Each colored oval is representative of a nucleotide kinase AK2 (blue), CKM (red), PKM (green), and PKLR (yellow). Non-overlapping regions demonstrate the number of participants observed to carry single nucleotide variants (SNVs) and deletions within only one gene (n = 40; AK2 n = 6, CKM n = 8, PKM n = 14, PKLR n = 12). Overlapping regions demonstrate the number of participants that were observed to carry SNVs and deletions in more than one gene (n = 17). For example, moving down the left-hand side of the diagram, six participants carried AK2 genetic variants alone, two participants carried variants for AK2 and PKM, one participant carried variants for AK2, PKM, and PKLR, and one participant carried variants for AK2 and PKLR.

Mentions: In order to test for the existence of genetic variants within the nucleotide kinases that activate TFV, we designed a targeted assay to sequence the exonic regions of AK2, CKM, PKM, and PKLR. GUK1 was not sequenced in the experiments described herein as we demonstrated siRNA knockdown of this enzyme had no significant impact on the formation of TFV-MP for PBMC, vaginal, or colorectal tissues. Of the 142 MTN-001 participants sequenced in this study, we observed 57 subjects (40%, 57/142) to carry single-base variations or deletions in their DNA that may result in a mutation at the amino acid level. Though this assay was targeted to exonic regions, we detected genetic variation within flanking intronic regions as well. The distribution of the variants and deletions detected for each kinase are depicted using a Venn diagram in Fig. 3. In 40 participants, we observed genetic variants within only one nucleotide kinase, while 17 individuals carried variants within two (n = 16) or three (n = 1) nucleotide kinases.


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)

Distribution of nucleotide kinase genetic variants detected in 57 MTN-001 participants.Each colored oval is representative of a nucleotide kinase AK2 (blue), CKM (red), PKM (green), and PKLR (yellow). Non-overlapping regions demonstrate the number of participants observed to carry single nucleotide variants (SNVs) and deletions within only one gene (n = 40; AK2 n = 6, CKM n = 8, PKM n = 14, PKLR n = 12). Overlapping regions demonstrate the number of participants that were observed to carry SNVs and deletions in more than one gene (n = 17). For example, moving down the left-hand side of the diagram, six participants carried AK2 genetic variants alone, two participants carried variants for AK2 and PKM, one participant carried variants for AK2, PKM, and PKLR, and one participant carried variants for AK2 and PKLR.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: Distribution of nucleotide kinase genetic variants detected in 57 MTN-001 participants.Each colored oval is representative of a nucleotide kinase AK2 (blue), CKM (red), PKM (green), and PKLR (yellow). Non-overlapping regions demonstrate the number of participants observed to carry single nucleotide variants (SNVs) and deletions within only one gene (n = 40; AK2 n = 6, CKM n = 8, PKM n = 14, PKLR n = 12). Overlapping regions demonstrate the number of participants that were observed to carry SNVs and deletions in more than one gene (n = 17). For example, moving down the left-hand side of the diagram, six participants carried AK2 genetic variants alone, two participants carried variants for AK2 and PKM, one participant carried variants for AK2, PKM, and PKLR, and one participant carried variants for AK2 and PKLR.
Mentions: In order to test for the existence of genetic variants within the nucleotide kinases that activate TFV, we designed a targeted assay to sequence the exonic regions of AK2, CKM, PKM, and PKLR. GUK1 was not sequenced in the experiments described herein as we demonstrated siRNA knockdown of this enzyme had no significant impact on the formation of TFV-MP for PBMC, vaginal, or colorectal tissues. Of the 142 MTN-001 participants sequenced in this study, we observed 57 subjects (40%, 57/142) to carry single-base variations or deletions in their DNA that may result in a mutation at the amino acid level. Though this assay was targeted to exonic regions, we detected genetic variation within flanking intronic regions as well. The distribution of the variants and deletions detected for each kinase are depicted using a Venn diagram in Fig. 3. In 40 participants, we observed genetic variants within only one nucleotide kinase, while 17 individuals carried variants within two (n = 16) or three (n = 1) nucleotide kinases.

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