ABSTRACT
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.
Subject(s)
DNA End-Joining Repair , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Ligands , DNA/metabolism , DNA Polymerase thetaABSTRACT
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Repair/drug effects , DNA-Directed DNA Polymerase/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Synthetic Lethal Mutations/drug effects , Allosteric Regulation , Animals , Apoptosis/drug effects , Apoptosis/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/radiation effects , DNA Damage/drug effects , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleases/antagonists & inhibitors , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Homologous Recombination/drug effects , Humans , Inhibitory Concentration 50 , Mice , Organoids/drug effects , Ovarian Neoplasms/genetics , Rats , Synthetic Lethal Mutations/genetics , Tumor Suppressor p53-Binding Protein 1/deficiency , Tumor Suppressor p53-Binding Protein 1/metabolism , DNA Polymerase thetaABSTRACT
Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.
Subject(s)
Antineoplastic Agents/pharmacology , Glutaminase/antagonists & inhibitors , Neoplasms/drug therapy , Pyridazines/pharmacology , Thiadiazoles/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Biological Availability , Cell Line, Tumor , Drug Discovery , Glutaminase/metabolism , Humans , Male , Mice, SCID , Molecular Docking Simulation , Neoplasms/metabolism , Neoplasms/pathology , Pyridazines/chemistry , Pyridazines/pharmacokinetics , Pyridazines/therapeutic use , Thiadiazoles/chemistry , Thiadiazoles/pharmacokinetics , Thiadiazoles/therapeutic useABSTRACT
As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.