ABSTRACT
Cellular stress in early mitosis activates the antephase checkpoint, resulting in the decondensation of chromosomes and delayed mitotic progression. Checkpoint with forkhead-associated and RING domains (CHFR) is central to this checkpoint, and its activity is ablated in many tumors and cancer cell lines through promoter hypermethylation or mutation. The interaction between the PAR-binding zinc finger (PBZ) of CHFR and poly(ADP-ribose) (PAR) is crucial for a functional antephase checkpoint. We determined the crystal structure of the cysteine-rich region of human CHFR (amino acids 425-664) to 1.9 Å resolution, which revealed a multizinc binding domain of elaborate topology within which the PBZ is embedded. The PBZ of CHFR closely resembles the analogous motifs from aprataxin-like factor and CG1218-PA, which lie within unstructured regions of their respective proteins. Based on co-crystal structures of CHFR bound to several different PAR-like ligands (adenosine 5'-diphosphoribose, adenosine monophosphate, and P(1)P(2)-diadenosine 5'-pyrophosphate), we made a model of the CHFR-PAR interaction, which we validated using site-specific mutagenesis and surface plasmon resonance. The PBZ motif of CHFR recognizes two adenine-containing subunits of PAR and the phosphate backbone that connects them. More generally, PBZ motifs may recognize different numbers of PAR subunits as required to carry out their functions.
Subject(s)
Adenosine Diphosphate Ribose/chemistry , Cell Cycle Proteins/chemistry , Neoplasm Proteins/chemistry , Adenosine Diphosphate/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray/methods , Humans , Ligands , Mitosis , Molecular Sequence Data , Poly-ADP-Ribose Binding Proteins , Protein Binding , Protein Structure, Tertiary , Surface Plasmon Resonance , Temperature , Ubiquitin-Protein Ligases , Zinc/chemistry , Zinc FingersABSTRACT
The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.
Subject(s)
Aminopyridines/pharmacology , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Discovery , Small Molecule Libraries/pharmacology , Administration, Oral , Aminopyridines/administration & dosage , Aminopyridines/chemistry , Animals , Biological Availability , Caco-2 Cells , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Dogs , Dose-Response Relationship, Drug , Female , Humans , Male , Mice , Models, Molecular , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Rats , Rats, Wistar , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/chemistry , Solubility , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.
Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Drug Evaluation, Preclinical/methods , Luciferases/antagonists & inhibitors , Pyridines/pharmacology , Small Molecule Libraries/pharmacology , Spiro Compounds/pharmacology , Wnt Signaling Pathway/drug effects , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Biological Assay/methods , Biological Availability , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Drug , Humans , Luciferases/metabolism , Mice , Models, Molecular , Molecular Structure , Pyridines/administration & dosage , Pyridines/chemistry , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/chemistry , Spiro Compounds/administration & dosage , Spiro Compounds/chemistry , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
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. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells.
Subject(s)
Aurora Kinase A/antagonists & inhibitors , Aurora Kinase B/antagonists & inhibitors , Drug Design , Imidazoles/chemistry , Imidazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Aurora Kinase A/chemistry , Aurora Kinase A/metabolism , Aurora Kinase B/chemistry , Aurora Kinase B/metabolism , Catalytic Domain , Drug Stability , HCT116 Cells , Humans , Imidazoles/metabolism , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Male , Mice , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , Substrate SpecificityABSTRACT
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.