Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta

Human DNA polymerase theta (Polθ), which is essential
for
microhomology-mediated DNA double strand break repair, has been proposed
as an attractive target for the treatment of BRCA deficient and other
DNA repair pathway defective cancers. As previously reported, we recently
identified the first selective small molecule Polθ in vitro
probe, 22 (ART558), which recapitulates the phenotype
of Polθ loss, and in vivo probe, 43 (ART812), which
is efficacious in a model of PARP inhibitor resistant TNBC in vivo.
Here we describe the discovery, biochemical and biophysical characterization
of these probes including small molecule ligand co-crystal structures
with Polθ. The crystallographic data provides a basis for understanding
the unique mechanism of inhibition of these compounds which is dependent
on stabilization of a “closed” enzyme conformation.
Additionally, the structural biology platform provided a basis for
rational optimization based primarily on reduced ligand conformational
flexibility.