title: N-Aroyl indole thiobarbituric acids as inhibitors of DNA repair and replication stress response polymerases.
abstract: 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 η). In our initial screen, we identified the indole thiobarbituric acid (ITBA) derivative 5-((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 η, 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 η was the most potently inhibited enzyme (30 ± 3 μM), with hpol β, hpol γ, and hpol κ exhibiting comparable but higher IC50 values of 41 ± 24, 49 ± 6, and 59 ± 11 μM, respectively. The other polymerases tested had IC50 values closer to 80 μM. 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 η. Two compounds with N-naphthoyl Ar-substituents, ITBA-16 and ITBA-19, were both found to have improved potency against hpol η with IC50 values of 16 ± 3 μM and 17 ± 3 μM, respectively. Moreover, the specificity of ITBA-16 was improved relative to that of ITBA-12. The presence of a chloro substituent at position 5 on the indole ring appears to be crucial for effective inhibition of hpol η, with the indole N-1-naphthoyl and N-2-naphthoyl analogues being the most potent inhibitors of hpol η. 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, United States.
authors.forename: Grace E
authors.initials: GE
authors.lastname: Coggins
authors.forename: Leena
authors.initials: L
authors.lastname: Maddukuri
authors.forename: Narsima R
authors.initials: NR
authors.lastname: Penthala
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Hartman
authors.forename: Sarah
authors.initials: S
authors.lastname: Eddy
authors.forename: Amit
authors.initials: A
authors.lastname: Ketkar
authors.forename: Peter A
authors.initials: PA
authors.lastname: Crooks
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
mesh_terms: D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000737:chemistry* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013312:Stress, Physiological; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013858:Thiobarbiturates / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C587061:5-((1-(2-bromobenzoyl)-5-chloro-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione; D004791:Enzyme Inhibitors; C587062:ITBA-16; D007211:Indoles; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries; D013858:Thiobarbiturates; C029684:thiobarbituric acid