The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

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.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

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.

Abstract Estrogen receptor alpha (ERα) is expressed in ∼75% of breast cancers where it binds and regulates specific target genes which drive cancer cell proliferation. Hormone-based treatments such as selective estrogen receptor modulators (SERMs) and aromatase inhibitors prevent estrogen signalling through the ERα, but in many cases resistance to these therapies develops. FOXA1 is a pioneer factor for ERα that is required to facilitate ERα binding to its target genes and its expression is considered to be a defining gene that characterizes ERα positive luminal breast cancers. Here we show that FOXA1 is an essential gene in ERα positive, tamoxifen resistant and aromatase-resistant breast cancer cell lines suggesting inhibitors that target the FOXA1/ERα pathway could have broad utility in these settings. To this end we performed a cell-based screen to identify novel small molecule regulators of FOXA1/ERα pathway activity. Our novel cell-based assay allowed us to screen over 180, 000 discrete compounds in a high through-put manner. Counter screens and phenotypic profiling in FOXA1 positive and negative breast cancer cell lines were used to remove non-specific compounds and direct ERα modulators. Second batches of confirmed actives were profiled through the screening cascade and by using ChIP assays we have observed a reduction in FOXA1 binding at the chromatin that is commensurate with a similar loss of ERα binding, demonstrating for the first time pharmacological modulation of FOXA1/ERα occupancy at the chromatin level. Citation Format: jon roffey, Aurore Lejeune, Michelle Barnard, Julie Stock, Ai Ching Wong, Jenny McKelvie, Kelly Holmes, Jason Carroll, Stephen Myatt. The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2015-LB-125

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)–binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.

A high throughput chemical screening campaign has led to the identification of 3-aminobenzo[b]thiophene-2-carboxamides as LIMK1 inhibitors. Evolution of bicyclic hits to the tricyclic 4-aminobenzothieno[3,2-d]pyrimidine, using a traditional medicinal chemistry SAR guided approach, resulted in a significant increase in potency. Further elaboration has seen the 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidine emerge as a LIMK1 inhibitor lead candidate.