Despite the potential significance of targeting pol κ in cancer therapeutics, limited investigations have been reported concerning the discovery of pol κ inhibitors. Although compounds with inhibitory activity against pol κ have been identified, most of these inhibitors were discovered as part of screens of natural products against any DNA polymerase and their therapeutic potential may be limited primarily due to either low potency or poor selectivity. For example, a derivative of a natural product kohamaic acid, (1S*,4aS*,8aS*)-17-(1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethylnaphthalen-1-yl)heptadecanoic acid and a derivative of vitamin K2
and vitamin K3
, MK-2, have been shown to inhibit pol κ activity with IC50
s of 7.2 µM and 35.3 µM, respectively 
. Pol κ activity is also inhibited by C12:0-Acyl juglone and C18:1-Acyl juglone with IC50
s of 6.8 µM and 8.1 µM, respectively 
. Glycyrrhetinic acid is another compound that inhibits pol κ activity (IC50
of 15.8 µM) 
. Penicilliols A and B are other natural products that inhibit pol κ; however, the activities of these compounds are higher against mouse pol ι than pol κ with an IC50
against pol ι of 19.8 µM and 32.5 µM for penicilliols A and B, respectively 
. Collectively, these compounds have low potency as well as poor selectivity against pol κ, since they inhibit many other DNA polymerases with similar potency as they inhibit pol κ. Additionally, although penta-1,2,3,4,6-O
-galloyl-beta-D-glucose exhibits nanomolar potency against pol κ (IC50
of 30 nM), it is more potent against pol α (IC50
of 13 nM) 
. Thus, the value of this compound as pol κ inhibitors is lowered by poor selectivity. In contrast to the aforementioned compounds, 3-O
-methylfunicone has been determined to exhibit high selectivity against pol κ. However, its low potency (IC50
of 12.5 µM) limits its utilization as a pharmaceutical or as a tool compound to probe pol κ biology 
. Collectively, these studies emphasize the importance of the identification of pol κ inhibitors with improved potency.
Here we reported the first study to utilize the combination of qHTS and a series of secondary validation assays, including primer extension assays on non-damaged templates, replication bypass assays using site-specifically modified oligodeoxynucleotides, cell survival assays, and DNA intercalation assays for the discovery of small molecule inhibitors of pol κ. A total of 60 compounds identified through qHTS were selected as proof-of-principal chemicals and validated in radioactive primer extension assays with electrophoretic separation. The majority of these hits inhibited pol κ activity at the top concentration tested (80 µM), demonstrating the sensitivity of the qHTS assay to accurately identify positive hits and the reliability of the primer extension assays using non-damaged DNAs to confirm the hits.
After elimination of weak inhibitors, compounds with potentially reactive functionalities or other undesirable chemical features, as well as compounds for which there were no convenient commercial sources, further validation of the remaining three compounds, candesartan cilexetil, manoalide, and MK-886 were conducted by radioactive gel-based primer extension assays. The results revealed that these compounds were capable of inhibiting the ability of pol κ to catalyze synthesis on either a control non-damaged DNA template or a template adducted with the acrolein-derived ring-opened reduced form of γ-HOPdG in a dose-dependent manner with similar potency. Since the predominant role of pol κ is in DNA lesion bypass, these results demonstrated that the primer extension assays using damage-containing DNAs can effectively measure the ability of the compounds to inhibit a biologically relevant activity of pol κ.
In order to assess the ability of these compounds to target intracellular pol κ, cell survival assays were carried out by exposing cells to the combination of pol κ inhibitors and UV. The results showed that candesartan cilexetil could potentiate cellular toxicity induced by UV in XP-V cells. It cannot be ruled out that the cellular effect of candesartan cilexetil may be partly due to its effect on other proteins in addition to pol κ, including pol η and pol ι, since the compound also inhibited the activities of these polymerases in vitro
(); however, our in vitro
results clearly show that pol κ is inhibited by this compound. Additionally, it has been shown that the depletion of either pol η or pol ι in XP-V cells did not enhance UV cytotoxicity 
. Collectively, these observations suggest that pol κ is inhibited by this compound in the cells, and thus validate the usefulness of this cell-based assay in identifying compounds with potential to inhibit intracellular pol κ.
Although manoalide and MK-886 could inhibit pol κ activity in vitro
, these compounds were unable to enhance UV-induced toxicity in XP-V cells under the conditions tested. Both manoalide and MK-886 have anti-inflammatory activity; manoalide is well-known as a non-specific phospholipase A2
, and MK-886 inhibits leukotriene synthesis by blocking 5-lipoxygenase-activating protein 
. The reason for the inability of these compounds to potentiate UV cytotoxicity could be due to their significantly lower binding affinity to intracellular pol κ relative to other cellular targets. Alternatively, these compounds may take a long time to enter the cells and bind to pol κ. Moreover, it is possible that only a small fraction of intracellular pol κ is inhibited by these compounds and the remaining pol κ may be sufficient to process UV-induced DNA lesions, resulting in unaltered cellular sensitivity to UV. Given the presence of multiple back-up TLS polymerases, nearly-complete inhibition of the activity of all intracellular pol κ may be essential for cells to present an apparent phenotype. Further understanding of the inability of these compounds to target intracellular pol κ could involve structure-activity relationship analyses. In fact, several structural analogues of these compounds exist such as secomanoalide and luffariellolide for manoalide 
and L538,916 for MK-886 
, thus enabling the initiation of such studies.
In summary, we presented herein the development of new strategies for the discovery of small molecules that could inhibit pol κ activity both in vitro and in vivo. The identification of chemotypes with established drug properties targeting pol κ validates this qHTS platform, as well as the secondary assays and sets the stage for exploration of significantly larger diverse collections to discover compounds with high potency and specificity towards pol κ and thus could potentially be used as pharmaceuticals. Therefore, these studies would move the research effort one step closer to the development of pol κ-targeted novel combination cancer therapeutics.