An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Abstract

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) K; values for ,u-and m-calpains of 0.21 ,uM and 0.37 ,IM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of ,u-calpain was found to interact with PD150606. In low micromolar range, PD150606 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.Calpain (EC 3.4.22.17) is a class of cytosolic cysteine protease that is activated by elevated intracellular calcium (1-3). Interest in this class of enzymes has grown substantially in recent years because it has been implicated in the pathophysiology of several degenerative conditions; including cerebral ischemia, myocardial ischemia, and cataract (4-8). The unifying features of these pathological conditions are that calcium serves as a trigger for cellular injury and that calpain may represent a crucial mediator of the degenerative response. Uncontrolled activation of calpain leads to cytoskeletal protein (e.g., spectrin) breakdown, degradation of many receptor proteins (e.g., epidermal growth factor receptor), and enzyme systems (e.g., protein kinase C and calmodulin-dependent kinases) and consequently cell death (1,8). Several studies have shown that peptidic inhibitors of calpain protect cells in models of ischemic, hypoxic and/or excitotoxic neuronal injury (6, 9-13). However, a clear interpretation of the role of calpain in these studies has been hampered by the lack of selectivity of these peptidic inhibitors (8).The predominant forms of calpain in mammalian tissues are ,u-calpain and m-calpain, requiring low and high micromolar calcium, respectively, for in vitro activation. Both of these isoforms are heterodimers in which the large subunit (80 kDa) contains a distinct cysteine protease domain and a calcium-binding domain with four helix-loop-helix (EF-hand) structures (14 MATERIALS AND METHODSProtease Assays and Kinetic Studies. The calpain microplate assay was carried out as described (15). Briefly, a mixture containing 0.5 mg/ml casein, 20 mM DTT, 50 mM Tris-HCl (pH 7.4), 0.01 unit purified ,u-calpain (human erythrocytes) or m-calpain (r...

Oncogenic ras-induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells

“…The mammalian cal- (45).) To confirm the possibility that ras down-regulates Beclin-1 in a calpain-dependent manner we treated the ras-transformed cells with PD150606, a small molecule calpain inhibitor that is structurally unrelated to ALLM (46). We found that, similar to what was observed with ALLM, treatment with PD150606 does result in the up-regulation of Beclin-1 in these cells (Fig.…”

Calcium, Calpain, and Calcineurin in Low-Frequency Depression of Transmitter Release

“…Extracellular drug application. Stock solutions were prepared in DMSO or H 2 O and were dissolved before use in 2 ml saline in the static bath and mixed thoroughly to obtain final concentrations of 100 M N-acethyl-Lleucyl-L-leucyl-L-norleucinal (N-acetyl-Leu-Leu-Norleu-al, calpain inhibitor I, ALLN, AcLLnL-CHO) (Sigma Aldrich); 100 M 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD 150606) inhibitor, which specifically blocks the Ca 2ϩ binding site of calpain and noncompetitively blocks calpain activity by binding to the Ca 2ϩ -binding domain of calpain (Wang et al, 1996a); 100 M control 3-phenyl-2-mercaptopropionic acid (PD 145305) (Calbiochem); 100 M carbobenzoxyvalinyl-phenylalaninal (MDL-28170) (Enzo Life Sciences), inhibitor of calpain I and II; 50 M 1,2-bis(o-aminophenoxy)ethane-N, N,NЈ,NЈ-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) (Calbiochem); and tetra(acetoxymethylester)-AM (EGTA-AM) (Invitrogen) at DMSO concentration Ͻ0.1-0.2% (v/v). To load cells with the Ca 2ϩ buffer, EGTA-AM plus 1% Pluronic F-127 was applied at final concentration of 50 M in saline containing 0.2% DMSO for 30 min, followed by an additional 30 min incubation to permit complete de-esterification (Zhong and Zucker, 2004).…”

“…Therefore, we sought alternative mechanisms that could cause hysteresis in activation of calcineurin. One possibility is activation of the Ca 2ϩ -dependent protease calpain, which partially cleaves the regulatory domain of calcineurin to give constitutively active phosphatase (Wang et al, 1989).…”

Retinoid-induced activation of NF-κB in APL cells is not essential for granulocytic differentiation, but prolongs the life span of mature cells

“…The possible implication of calpain in ATRA-induced IkBa degradation was tested by using a calpain inhibitor, PD150606, which blocks its calcium-binding site (Wang et al, 1996). Addition of this inhibitor to ATRA-treated cells resulted in an increased level of IkBa expression as compared to its expression in cells treated with ATRA alone (Figure 2d and e), while the inactive analogue, PD145305, had no effect (Figure 2e).…”