ABSTRACT
The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.
Subject(s)
Cathepsins/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Nitriles/chemistry , Catalytic Domain , Dipeptides/chemistry , Drug Design , Humans , Models, Chemical , Molecular Conformation , Nitriles/classification , Peptides/chemistry , Piperidines/chemistry , Pyrrolidines/chemistry , Structure-Activity RelationshipABSTRACT
The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.
Subject(s)
MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/chemistry , Mitogen-Activated Protein Kinases/physiology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/physiology , Alternative Splicing , Animals , Calorimetry , Catalysis , Fluorescein-5-isothiocyanate/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/physiology , Kinetics , MAP Kinase Signaling System/genetics , Mice , Mitogen-Activated Protein Kinase 14 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/genetics , Peptide Fragments/physiology , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary/genetics , Spectrometry, Fluorescence , Structure-Activity Relationship , Surface Plasmon Resonance , Thermodynamics , UltracentrifugationABSTRACT
The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.
