Investigation of Structure‐Activity Relationships for Small‐Molecule Inhibitors of Human DNA Polymerase Eta

Efficient DNA replication by high‐fidelity polymerases (pols) can be perturbed by both endogenous and exogenous sources of DNA damage, resulting in accumulation of replication intermediates that activate DNA damage response pathways. DNA damage and replication stress are hallmarks of cancer and cells have intrinsic mechanisms that rely on replication stress response pols to assist in replication fork progression following DNA damage. Many chemo‐ and radio‐therapies rely on targeting DNA, thereby, blocking DNA replication to limit tumor growth. These genotoxic drugs can be rendered ineffective through Translesion DNA synthesis (TLS), involving Y‐family pols, namely human polymerases eta (η), kappa (κ), iota (ι) and Rev1. Most importantly, both in vitro and in vivo studies have highlighted the role of hpol η in bypassing lesions caused by doxorubicin and platinum based drugs. Therefore, targeted inhibition of these specialized pols will improve the ability to destroy cancer cells that rely on these enzymes to survive treatment with genotoxic anti‐cancer drugs. The main objective of our study was to identify small‐molecule inhibitors of hpol η and determine if these molecules show synergy with genotoxic anti‐cancer drugs in an effort to ameliorate existing cancer therapeutics to improve patient outcomes. Catalysis by hpol η was measured in the presence of eighty‐eight small molecules at a single dose of 20 μM from a novel library of more than 100 indole thio‐barbituric acids (ITBA) and indole barbituric acid (IBA) derivatives. From this initial study, we identified several potent inhibitors of hpol η activity. Michaelis‐Menten kinetic analysis and in‐silico docking approaches were employed to determine the mechanism of action for the most potent inhibitors. One of these compounds, PNR‐7‐02, an ITBA derivative with both N‐ napthoyl moiety and 5‐chloro substituent on the indole ring inhibits hpol η activity with an IC 50 value near 8 μM. In silico docking and Michaelis‐Menten kinetics revealed that PNR‐7‐02 likely inhibits hpol η activity through a partial competitive/allosteric mechanism of action. PNR‐7‐02 inhibited hRev1 and hpol lambda (λ) with low micromolar IC 50 values. Another ITBA derivative with a 4‐(2‐ethoxyethyl) morpholine substitution at position 5 on the indole ring (PNR‐9‐59) inhibited hpol η with an IC 50 of 13 μM. PNR‐9‐59 showed improved drug‐likeness as well as improved specificity for hpol η against A‐, B‐ and X‐family pols and also within Y‐family pols. In summary, our work has improved the potency and drug‐likeness of small‐molecule inhibitors of hpol η, and it stands as the first detailed analysis of structure‐activity relationships for inhibitors of a TLS pol involved in resistance to chemotherapeutics. Support or Funding Information This work was supported by NIH grants GM 084460 and CA 183895 (to R.L.E.).