Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation.

The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function.

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737).

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells.

Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B.

CCT241533 is a potent and selective inhibitor of CHK2 that potentiates the cytotoxicity of PARP inhibitors.

CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533.

Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange.

The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain.

The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors.

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties.