ART558

Zatreanu et al. report a first-in-class chemical probe, ART558, that inhibits the DNA polymerase POLQ (theta) involved in MMEJ DNA repair. ART558 meets all relevant criteria for an in vitro chemical probe, including potent biochemical inhibition (IC50 = 7.9 nM), potent on-mechanism cellular activity (IC50 = 150 nM in MMEJ reporter assay), and excellent selectivity against the most concerning potential off-targets (inactive against human polymerases alpha, gamma, eta, and nu, PARP1 and PARP2, and 78 oncology-relevant kinases). ART558 is reported alongside a matched negative control enantiomer, ART615, which provides further confidence in the results of all reported cellular experiments. The profound POLQ selectivity of ART558 relative to other polymerases is likely due to its unique allosteric mechanism of action--enzyme kinetics suggest that ART558 is non-competitive with respect to dNTP's and uncompetitive with respect to dsDNA. Further structural work should focus on elucidating the precise binding site for ART558, as it could both provide insight into developing selective inhibitors of other DNA polymerases and also facilitate the development of next-generation POLQ inhibitors with superior potency/DMPK and therapeutic potential. Zatreanu et al. report clear evidence of intracellular ART558 target engagement, including an elegant system that demonstrates a profound ART558-dependent (but not ART615-dependent) increase in YFP-POLQ residence time at sites of laser-induced DNA damage. Zatreanu et al. present a battery of cellular data showing that ART558 only has profound effects on bulk cellular phenotypes (2D cell proliferation, DNA damage, organoid growth, apoptosis, ect.) in cell lines harboring synthetic lethal sensitizing mutations (BRACA1-/- and/or 53BP1-/-). Indeed, these results recapitulate previously reported genetic synthetic lethality screens which demonstrated that POLQ loss is a dependency in BRACA1-/- cell lines. Therefore, ART558 is an unusual example of a targeted small-molecule recapitulating a genetic synthetic lethality, proving beyond a reasonable doubt that ART558 directly inhibits POLQ function in cells. That being said, experimentalists planning to use ART558 in sensitized mutant cell lines should be aware of these on-target cytotoxic effects, which can often muddy the interpretation of other assays (especially transcriptomics) if a sensible [ART558] and treatment time are not used. Finally, it is important to emphasize that ART558 is fundamentally unsuitable for in vivo experiments because of incredibly high intrinsic clearance. All in vivo experiments presented by Zatreanu et al. were conducted with a related analog, ART812, which has stabilizing substitutions both in the central five-membered ring and in the phenyl moiety (ART812 structure in Supplemental Figure 3 of Zatreanu et al.). Future disclosures are likely to highlight additional ART558 analogs that are suitable for in vivo experiments. Taken together, ART558 is an excellent cellular probe for the previously orphan target POLQ.