title: Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response.
abstract: Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA, 94305, United States of America.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Beharry
authors.affiliation: Departments of Biological Engineering and Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States of America.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Nagel
authors.affiliation: Departments of Biological Engineering and Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States of America.
authors.forename: Leona D
authors.initials: LD
authors.lastname: Samson
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA, 94305, United States of America.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D004260:DNA Repair; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D019853:O(6)-Methylguanine-DNA Methyltransferase