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A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

Arnold LH, Kunzelmann S, Webb MR, Taylor IA - Antimicrob. Agents Chemother. (2014)

Bottom Line: The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes.SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells.The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, London, United Kingdom.

No MeSH data available.


Endpoint assays of SAMHD1 catalysis, activation, and inhibition; results from the malachite green endpoint assays. The bar charts show the percentages of hydrolysis or activity with respect to the control compounds. The error bars represent the standard error of the mean (SEM) of three independent measurements. (A) Assay for compounds that are activators and substrates. The data are expressed relative to that of a dGTP control. (B) Assay for compounds that are hydrolyzed upon inclusion of a SAMHD1 activator (200 μM GTP). The data are expressed relative to that of a control TTP substrate. (C) Assay for SAMHD1 activation. The data are expressed as the percentage of TTP hydrolysis when a test compound was employed as an activator (200 μM) relative to that of the control GTP. (D) Assay for SAMHD1 inhibition. The data are expressed as the percentage of TTP hydrolysis after inclusion of a test compound in addition to the GTP activator at an equimolar concentration (200 μM).
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Figure 4: Endpoint assays of SAMHD1 catalysis, activation, and inhibition; results from the malachite green endpoint assays. The bar charts show the percentages of hydrolysis or activity with respect to the control compounds. The error bars represent the standard error of the mean (SEM) of three independent measurements. (A) Assay for compounds that are activators and substrates. The data are expressed relative to that of a dGTP control. (B) Assay for compounds that are hydrolyzed upon inclusion of a SAMHD1 activator (200 μM GTP). The data are expressed relative to that of a control TTP substrate. (C) Assay for SAMHD1 activation. The data are expressed as the percentage of TTP hydrolysis when a test compound was employed as an activator (200 μM) relative to that of the control GTP. (D) Assay for SAMHD1 inhibition. The data are expressed as the percentage of TTP hydrolysis after inclusion of a test compound in addition to the GTP activator at an equimolar concentration (200 μM).

Mentions: Endpoint assays were used to determine if individual compounds can allosterically activate SAMHD1 and also be hydrolyzed (Fig. 4A). Under these conditions, only dGTP, previously shown to be both a SAMHD1 activator and substrate (1), was hydrolyzed, reaffirming the requirement for a guanine base and the lack of a 2′-hydroxyl for a nucleoside triphosphate to both activate and be hydrolyzed. Although not a substrate, GTP activates SAMHD1 as effectively as dGTP (17); given the abundance of GTP with respect to the cellular dNTP pool, the panel of compounds was also tested to see if they were hydrolyzed by GTP-activated SAMHD1 (Fig. 4B). These data revealed that acyclovir triphosphate (acyclovir-TP), ganciclovir triphosphate (ganciclovir-TP), and dApNHpp were still refractory to hydrolysis but that clofarabine triphosphate (clofarabine-TP) was hydrolyzed at a rate comparable to that of the control TTP.


A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

Arnold LH, Kunzelmann S, Webb MR, Taylor IA - Antimicrob. Agents Chemother. (2014)

Endpoint assays of SAMHD1 catalysis, activation, and inhibition; results from the malachite green endpoint assays. The bar charts show the percentages of hydrolysis or activity with respect to the control compounds. The error bars represent the standard error of the mean (SEM) of three independent measurements. (A) Assay for compounds that are activators and substrates. The data are expressed relative to that of a dGTP control. (B) Assay for compounds that are hydrolyzed upon inclusion of a SAMHD1 activator (200 μM GTP). The data are expressed relative to that of a control TTP substrate. (C) Assay for SAMHD1 activation. The data are expressed as the percentage of TTP hydrolysis when a test compound was employed as an activator (200 μM) relative to that of the control GTP. (D) Assay for SAMHD1 inhibition. The data are expressed as the percentage of TTP hydrolysis after inclusion of a test compound in addition to the GTP activator at an equimolar concentration (200 μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291348&req=5

Figure 4: Endpoint assays of SAMHD1 catalysis, activation, and inhibition; results from the malachite green endpoint assays. The bar charts show the percentages of hydrolysis or activity with respect to the control compounds. The error bars represent the standard error of the mean (SEM) of three independent measurements. (A) Assay for compounds that are activators and substrates. The data are expressed relative to that of a dGTP control. (B) Assay for compounds that are hydrolyzed upon inclusion of a SAMHD1 activator (200 μM GTP). The data are expressed relative to that of a control TTP substrate. (C) Assay for SAMHD1 activation. The data are expressed as the percentage of TTP hydrolysis when a test compound was employed as an activator (200 μM) relative to that of the control GTP. (D) Assay for SAMHD1 inhibition. The data are expressed as the percentage of TTP hydrolysis after inclusion of a test compound in addition to the GTP activator at an equimolar concentration (200 μM).
Mentions: Endpoint assays were used to determine if individual compounds can allosterically activate SAMHD1 and also be hydrolyzed (Fig. 4A). Under these conditions, only dGTP, previously shown to be both a SAMHD1 activator and substrate (1), was hydrolyzed, reaffirming the requirement for a guanine base and the lack of a 2′-hydroxyl for a nucleoside triphosphate to both activate and be hydrolyzed. Although not a substrate, GTP activates SAMHD1 as effectively as dGTP (17); given the abundance of GTP with respect to the cellular dNTP pool, the panel of compounds was also tested to see if they were hydrolyzed by GTP-activated SAMHD1 (Fig. 4B). These data revealed that acyclovir triphosphate (acyclovir-TP), ganciclovir triphosphate (ganciclovir-TP), and dApNHpp were still refractory to hydrolysis but that clofarabine triphosphate (clofarabine-TP) was hydrolyzed at a rate comparable to that of the control TTP.

Bottom Line: The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes.SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells.The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, London, United Kingdom.

No MeSH data available.