ABSTRACT
OBJECTIVE: TGF-beta plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. METHODS AND RESULTS: The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-beta and activin-induced Smad2/3 phosphorylation and TGF-beta-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle alpha-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. CONCLUSIONS: These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.
Subject(s)
Azabicyclo Compounds/pharmacology , Carotid Artery Injuries/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Activin Receptors, Type I/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Administration, Oral , Animals , Azabicyclo Compounds/administration & dosage , Azabicyclo Compounds/metabolism , Binding Sites , Carotid Artery Injuries/pathology , Carotid Artery Injuries/physiopathology , Cell Line , Fibroblasts/drug effects , Fibroblasts/pathology , Fibrosis , Humans , Male , Myoblasts, Smooth Muscle/drug effects , Myoblasts, Smooth Muscle/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Transforming Growth Factor-beta Type I , Transforming Growth Factor beta/physiologyABSTRACT
We describe the discovery, using shape-based virtual screening, of a potent, ATP site-directed inhibitor of the TbetaRI kinase, an important and novel drug target for fibrosis and cancer. The first detailed report of a TbetaRI kinase small molecule co-complex confirms the predicted binding interactions of our small molecule inhibitor, which stabilizes the inactive kinase conformation. Our results validate shape-based screening as a powerful tool to discover useful leads against a new drug target.
