ABSTRACT
Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.
Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cysteine/pharmacology , Drug Discovery , High-Throughput Screening Assays , Ligands , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Cyclin-Dependent Kinase 2/metabolism , Cysteine/chemistry , Kinetics , Molecular Structure , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/chemical synthesis , Small Molecule Libraries/analysis , Small Molecule Libraries/chemical synthesisABSTRACT
A kinetic template-guided tethering (KTGT) strategy has been developed for the site-directed discovery of fragments that bind to defined protein surfaces, where acrylamide-modified fragments can be irreversibly captured in a protein-templated conjugate addition reaction. Herein, an efficient and facile method is reported for the preparation of acrylamide libraries from a diverse range of amine fragments using a solid-supported quaternary amine base.
Subject(s)
Acrylamide/chemical synthesis , Acrylamide/chemistry , Amines/chemistry , Catalysis , Combinatorial Chemistry Techniques , Molecular Structure , Quinolines/chemical synthesis , Quinolines/chemistry , Quinolines/pharmacologyABSTRACT
With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.
Subject(s)
Aza Compounds/chemistry , Protein Kinase Inhibitors/chemical synthesis , Spiro Compounds/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Isoquinolines/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinases/metabolism , Protein Structure, Tertiary , Spiro Compounds/chemical synthesis , Spiro Compounds/metabolismABSTRACT
A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology , Crystallography, X-Ray , Drug Design , Drug Evaluation, PreclinicalABSTRACT
5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure-activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic omega-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.
