Abstract
Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.