N-Aroyl Indole Thiobarbituric Acids as Inhibitors of DNA Repair and Replication Stress Response Polymerases

Using a robust and quantitative assay, we have identified a novel class of DNA polymerase inhibitors that exhibits some specificity against an enzyme involved in resistance to anti-cancer drugs, namely, human DNA polymerase eta (hpol eta). In our initial screen, we identified the indole thiobarbituric acid (ITBA) derivative 54(1-(2-bromobenzoyl)-5-chloro-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (ITBA-12) as an inhibitor of the Y-family DNA member hpol eta, an enzyme that has been associated with increased resistance to cisplatin and doxorubicin treatments. An additional seven DNA polymerases from different subfamilies were tested for inhibition by ITBA-12. Hpol eta was the most potently inhibited enzyme (30 +/- 3 mu M), with hpol beta, hpol gamma, and hpol kappa exhibiting comparable but higher IC50 values of 41 +/- 24, 49 +/- 6, and 59 +/- 11 mu M, respectively. The other polymerases tested had IC50 values closer to 80 mu M. Steady-state kinetic analysis was used to investigate the mechanism of polymerase inhibition by ITBA-12. Based on changes in the Michaelis constant, it was determined that ITBA-12 acts as an allosteric (or partial) competitive inhibitor of dNTP binding. The parent ITBA scaffold was modified to produce 20 derivatives and establish structure-activity relationships by testing for inhibition of hpol eta. Two compounds with N-naphthoyl Ar-substituents, ITBA-16 and ITBA-19, were both found to have improved potency against hpol eta with IC50 values of 16 +/- 3 mu M and 17 +/- 3 mu M, respectively. Moreover, the specificity of ITBA-16 was improved relative to that of ITBA-12. The presence of a chloro substituent at position S on the indole ring appears to be crucial for effective inhibition of hpol eta with the indole N-1-naphthoyl and N-2-naphthoyl analogues being the most potent inhibitors of hpol eta. These results provide a framework from which second-generation ITBA derivatives may be developed against specialized polymerases that are involved in mechanisms of radio- and chemo-resistance.