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J Am Chem Soc. 2016 Mar 23;138(11):3647-50. doi: 10.1021/jacs.6b00986. Epub 2016 Mar 15.

Fluorescence Monitoring of the Oxidative Repair of DNA Alkylation Damage by ALKBH3, a Prostate Cancer Marker.

Author information

1
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
2
Department of Cancer Biology, Beckman Research Institute , Duarte, California 91010, United States.

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.

PMID:
26967262
PMCID:
PMC5105888
DOI:
10.1021/jacs.6b00986
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Notes The authors declare no competing financial interest.

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