title: Fluorescence Monitoring of the Oxidative Repair of DNA Alkylation Damage by ALKBH3, a Prostate Cancer Marker.
abstract: The 2-oxoglutarate-dependent iron enzyme ALKBH3 is an antitumor target and a potential diagnostic marker for several tumor types, including prostate cancer. However, there is at present no simple way to measure this enzyme's activity. Here we describe a fluorogenic probe design (MAQ) that is directly responsive to ALKBH3 repair activity. It makes use of the fluorescence-quenching properties of 1-methyladenine; removal of the alkyl group results in a >10-fold light-up signal. The probe is specific for ALKBH3 over its related homologue ALKBH2 and can be used to identify and measure the effectiveness of enzyme inhibitors. Measurements of the enzyme substrate parameters show that MAQ displays Km and kcat values essentially the same as those of the native substrate. Finally, we show that the probe functions efficiently in cells, allowing imaging and quantitation of ALKBH3 activity by microscopy and flow cytometry. We expect that MAQ probes will be broadly useful in the study of the basic biology of ALKBH3 and in clinical cancer applications as well.
authors.affiliation: Department of Chemistry, Stanford University , Stanford, California 94305, United States.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Beharry
authors.affiliation: Department of Cancer Biology, Beckman Research Institute , Duarte, California 91010, United States.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lacoste
authors.affiliation: Department of Cancer Biology, Beckman Research Institute , Duarte, California 91010, United States.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: O'Connor
authors.affiliation: Department of Chemistry, Stanford University , Stanford, California 94305, United States.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D010084:Oxidation-Reduction; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D018906:Antineoplastic Agents, Alkylating; D014408:Biomarkers, Tumor; D005456:Fluorescent Dyes; C008407:1-methyladenine; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D000225:Adenine