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Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

¹ Radiation Oncology Research Laboratory and Marlene and Stewart Greenebaum Cancer Center, ² Department of Biochemistry and Molecular Biology, School of Medicine, ³ Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland; and ⁴ Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri

Requests for reprints: Alan E. Tomkinson, Radiation Oncology Research Laboratory, Department of Radiation Oncology, School of Medicine, Bressler Research Building, 7-025, 655 West Baltimore Street, University of Maryland, Baltimore, MD 21201. Phone: 410-706-2365; Fax: 410-706-6666; E-mail: atomkinson{at}som.umaryland.edu and Alexander D. MacKerell, Jr., Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201. E-mail: amackere{at}rx.umaryland.edu.

Key Words: Computer-aided drug design • DNA replication • DNA repair • DNA ligation • DNA ligase inhibitors

Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]