ABSTRACT
Human ß-nerve growth factor (ß-NGF) and its associated receptor, human tropomyosin receptor kinase A (hTrkA), have been demonstrated to be key factors in the perception of pain. However, efficacious small molecule therapies targeting the intracellularly located hTrkA kinase have not been explored thoroughly for pain management. Herein, we report the pharmacological properties of a selective hTrkA allosteric inhibitor, 1. 1 was shown to be active against the full length hTrkA, showing preferential binding for the inactive kinase, and was confirmed through the X-ray of hTrkA···1 bound complex. 1 was also found to inhibit ß-NGF induced neurite outgrowth in rat PC12 cells. Daily oral administration of 1 improved the joint compression threshold of rats injected intra-articularly with monoiodoacetate over a 14-day period. The efficacy of 1 in a relevant chronic pain model of osteoarthritis coupled with in vitro confirmation of target mediation makes allosteric hTrkA inhibitors potential candidates for modulating pain.
ABSTRACT
Light-activated inhibition of cathepsin activity was demonstrated in a cell-based assay. Inhibitors of cathepsin K, Cbz-Leu-NHCH2 CN (2) and Cbz-Leu-Ser(OBn)-CN (3), were caged within the complexes cis-[Ru(bpy)2 (2)2 ]Cl2 (4) and cis-[Ru(bpy)2 (3)2 ](BF4 )2 (5) (bpy=2,2'-bipyridine) as 1:1 mixtures of Δ and Λ stereoisomers. Complexes 4 and 5 were characterized by (1) H NMR, IR, and UV/Vis spectroscopies and electrospray mass spectrometry. Photochemical experiments confirm that 4 releases two molecules of 2 upon exposure to visible light for 15 min, whereas release of 3 by 5 requires longer irradiation times. IC50 determinations against purified cathepsinâ K under light and dark conditions with 4 and 5 confirm that inhibition is enhanced from 35- to 88-fold, respectively, upon irradiation with visible light. No apparent toxicity was observed for 4 in the absence or presence of irradiation in bone marrow macrophage (BMM) or PC3 cells, as determined by MTT assays, at concentrations up to 10 µM. Compound 5 is well tolerated at lower concentrations (<1 µM), but does show growth-inhibitory effects at higher concentrations. Confocal microscopy experiments show that 4 decreases intracellular cathepsin activity in osteoclasts with light activation. These results support the further development of caged nitrile-based inhibitors as chemical tools for investigating spatial aspects of proteolysis within living systems.
Subject(s)
Cathepsin K/antagonists & inhibitors , Coordination Complexes/chemistry , Enzyme Inhibitors/chemistry , Light , Ruthenium/chemistry , 2,2'-Dipyridyl/chemistry , Animals , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Cathepsin K/metabolism , Cell Line , Cell Survival/drug effects , Coordination Complexes/chemical synthesis , Coordination Complexes/toxicity , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Mice , Microscopy, ConfocalABSTRACT
Cumyl ester is an optimal C-terminal protecting group for glycine benzophenone imine in asymmetric alkylation reactions catalyzed by Cinchona chiral phase-transfer catalysts. High levels of enantioselectivity have been obtained (up to 94% ee) with this substrate, which provides an attractive alternative to the analogous tert-butyl ester. N-terminal imines and the C-terminal esters can be cleaved from alkylation products by hydrogenolysis, while maintaining acid-labile side chain protecting groups.
Subject(s)
Benzophenones/chemistry , Glycine/analogs & derivatives , Glycine/chemistry , Imines/chemistry , Alkylation , Esters , Molecular Structure , StereoisomerismABSTRACT
A novel method for caging protease inhibitors is described. The complex [Ru(II)(bpy)(2)(1)(2)](PF(6))(2) (2) was prepared from the nitrile-based peptidomimetic inhibitor Ac-Phe-NHCH(2)CN (1). (1)H NMR, UV-vis, and IR spectroscopic and mass spectrometric data confirmed that 2 equiv of inhibitor 1 bind to Ru(II) through the nitrile functional group. Complex 2 shows excellent stability in aqueous solution in the dark and fast release of 1 upon irradiation with visible light. As a result of binding to the Ru(II) center, the nitriles of complex 2 are caged, and 2 does not act as a potent enzyme inhibitor. However, when 2 is irradiated, it releases 1, which inhibits the cysteine proteases papain and cathepsins B, K and L up to 2 times more potently than 1 alone. Ratios of the IC(50) values in the dark versus in the light ranged from 6:1 to 33:1 for inhibition by 2 against isolated enzymes and in human cell lysates, confirming that a high level of photoinduced enzyme inhibition can be obtained using this method.
Subject(s)
2,2'-Dipyridyl/chemistry , Cysteine Proteinase Inhibitors/chemistry , Light , Nitriles/chemistry , Organometallic Compounds/chemistry , Ruthenium/chemistry , Cysteine Proteases/metabolism , Models, Molecular , Organometallic Compounds/chemical synthesis , PeptidomimeticsABSTRACT
A divergent method for incorporating polypyridyl ligands into peptides is reported. Three N-Fmoc unnatural amino acids (1-3) that contain varying linkers between the alpha-carbon and a 2-(hydroxymethyl)pyridyl group were synthesized in enantioenriched form. These amino acids were used as anchors for incorporating multidentate ligands onto a peptide chain in a site-specific fashion. Multiple peptide-ligand conjugates were synthesized from single precursors by solution- or solid-phase methods. Peptides containing more than one metal-binding unit can be produced by this method.
