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.

FIGURE 1

Assay to screen for polymerase inhibitors. (A) Polymerase activity separates a short TAMRA-labeled oligonucleotide from its BHQ2-labeled complement. The TAMRA-labeled oligonucleotide was exposed to an excitation wavelength of λ_ex = 525 nm and fluorescence emission at λem = 598 nm was monitored over time. (B) Hpol η^1-437 activity was monitored and the mean (± standard deviation) from the resulting data sets was plotted as a function of time and the slope of the initial portion of the velocity curve (inset) was used to estimate the rate of polymerase-catalyzed strand-displacement: v₀ = 10.2 ± 0.4 nM min⁻¹.

FIGURE 2

Determination of IC₅₀ for ITBA-12 mediated inhibition of hpol η activity. (A) The chemical structure of ITBA-12, the first inhibitor identified in the screen, is shown. (B) Hpol η (10 nM) activity was monitored using the fluorescence-based assay in the presence of increasing amounts of ITBA-12: DMSO control (black), 1 μM (blue), 5 μM (cyan), 10 μM (green), 25 μM (orange), 50 μM (red), 100 μM (magenta) and 250 μM (purple). (C) Hpol η activity was plotted as a function of the log of inhibitor concentration and fit to equation 1 to determine the IC₅₀ value. The mean (± standard deviation) of three data sets is shown.

FIGURE 3

Determination of ITBA-12 specificity for inhibition of hpol η. The IC₅₀ values for ITBA-12 inhibition of different polymerases are shown. The mean (± standard deviation) of IC₅₀ values obtained for three data sets is shown.

FIGURE 4

Structure-activity relationships for ITBA derivatives and inhibition of hpol η. (A) The chemical structure of the ITBA scaffold with the position of the R and Ar substituents indicated. (B) The identity of the R and Ar substituents is listed for each of the twenty ITBA derivatives tested for activity against hpol η. (C) Hpol η activity was measured in the presence of either DMSO or 50 μM of the indicated ITBA derivative. The numbering scheme on the X-axis matches the numbering in panel B.

FIGURE 5

Determination of the IC₅₀ value for ITBA-16 mediated inhibition of hpol η. (A) The chemical structure of ITBA-16 is shown. (B) Hpol η activity was measured in the presence of increasing amounts of ITBA-16. The IC₅₀ was found to be roughly half that of ITBA-12.

FIGURE 6

Determination of ITBA-16 specificity for inhibition of hpol η. The IC₅₀ values for ITBA-16 inhibition of different polymerases are shown. The mean (± standard deviation) of IC₅₀ values obtained for three data sets is shown.