POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Discovery of Potent Inhibitors for the Large Neutral Amino Acid Transporter 1 (LAT1) by Structure-Based Methods.

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure-activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-µM range.