title: A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.
abstract: 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. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.
authors.affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, London, United Kingdom.
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Arnold
authors.affiliation: Division of Physical Biochemistry, MRC National Institute for Medical Research, London, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Kunzelmann
authors.affiliation: Division of Physical Biochemistry, MRC National Institute for Medical Research, London, United Kingdom.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
authors.affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, London, United Kingdom itaylor@nimr.mrc.ac.uk.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Taylor
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000212:Acyclovir / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000737:chemistry / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001087:Arabinonucleosides / Q000737:chemistry / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D002384:Catalysis / Q000187:drug effects; D000077866:Clofarabine; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015774:Ganciclovir / Q000737:chemistry / Q000494:pharmacology; D015497:HIV-1; D006868:Hydrolysis; D020559:Monomeric GTP-Binding Proteins / Q000032:analysis* / Q000378:metabolism*; D000076106:SAM Domain and HD Domain-Containing Protein 1
chemical_list: D000227:Adenine Nucleotides; D000998:Antiviral Agents; D001087:Arabinonucleosides; D003854:Deoxyribonucleotides; D000077866:Clofarabine; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D017766:Acid Anhydride Hydrolases; C020137:exopolyphosphatase; D020559:Monomeric GTP-Binding Proteins; D015774:Ganciclovir; D000212:Acyclovir