Oxidative folding and structural analyses of a Kunitz-related inhibitor and its disulfide intermediates: functional implications.

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase β with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa.

Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta.

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date.

Assessing experimental models in myocardial injury: lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion.

Background: Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-alpha-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases mu- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-alpha.

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude.

Calpain inhibitor A-558693 in experimental focal cerebral ischemia in rats.

Objectives: Calpains are intracellular proteases, which are activated in various cerebral injuries. We studied the expression of mu-calpain in a model of focal cerebral ischemia/reperfusion and the efficacy of the calpain inhibitor A-558693.

Methods: A transient occlusion of the middle cerebral artery was produced in male Wistar rats by using the suture model with 3 hours of ischemia and 24 hours of reperfusion.

Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor.

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits.