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.
KRAS-mutant colorectal cancers (CRC) are resistant to therapeutics, presenting a significant problem for ~40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant CRC. Using Kras-mutant mouse models and mouse-and patient-derived organoids we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small molecule targeting of this pathway, we acutely sensitize KRAS G12D models to rapamycin via suppression of c-MYC. We show that 45% of CRCs have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYCdependent co-targeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population who may benefit from its clinical application.
Enantiopure 6-alkylpipecolic acid hydrochlorides 1a-e were synthesized in five steps and 15-59% overall yields from alpha-tert-butyl beta-methyl N-(PhF)aspartate (3) via an approach featuring selective hydride reduction to the corresponding aspartate beta-aldehyde 2, aldol condensations with the enolates of various methyl alkyl ketones, and diastereoselective intramolecular reductive aminations. The influence of the 6-position substituent on the equilibrium and the energy barrier for isomerization of the amide N-terminal to pipecolate was then explored via the synthesis of N-acetyl N'-methylpipecolinamide (16) and its (2S,6R)-6-tert-butylpipecolinamide counterpart 17, and their conformational analysis by proton NMR spectroscopy and coalescence experiments. The presence of the tert-butyl substituent augmented the population of the amide cis-isomer and lowered the barrier for pipecolyl amide isomerization in water. Compared with the results from our previous examination of N-acetyl-5-tert-butylproline N'-methylamides (Beausoleil, E.; Lubell, W. D. J. Am. Chem. Soc. 1996, 118, 12902), the consequences of the bulky 6-alkyl substituent on the acetamide geometry and isomerization barrier were less pronounced in the pipecolate series relative to the respective proline amides.
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.
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