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Proc Natl Acad Sci U S A. Jun 25, 1996; 93(13): 6687–6692.
PMCID: PMC39087

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) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, 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 mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 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.

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  • Wang KK, Villalobo A, Roufogalis BD. Calmodulin-binding proteins as calpain substrates. Biochem J. 1989 Sep 15;262(3):693–706. [PMC free article] [PubMed]
  • Croall DE, DeMartino GN. Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev. 1991 Jul;71(3):813–847. [PubMed]
  • Saido TC, Sorimachi H, Suzuki K. Calpain: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J. 1994 Aug;8(11):814–822. [PubMed]
  • Iizuka K, Kawaguchi H, Yasuda H. Calpain is activated during hypoxic myocardial cell injury. Biochem Med Metab Biol. 1991 Dec;46(3):427–431. [PubMed]
  • Siman R, Noszek JC. Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo. Neuron. 1988 Jun;1(4):279–287. [PubMed]
  • Lee KS, Frank S, Vanderklish P, Arai A, Lynch G. Inhibition of proteolysis protects hippocampal neurons from ischemia. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7233–7237. [PMC free article] [PubMed]
  • Azuma M, David LL, Shearer TR. Cysteine protease inhibitor E64 reduces the rate of formation of selenite cataract in the whole animal. Curr Eye Res. 1991 Jul;10(7):657–666. [PubMed]
  • Wang KK, Yuen PW. Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol Sci. 1994 Nov;15(11):412–419. [PubMed]
  • Arlinghaus L, Mehdi S, Lee KS. Improved posthypoxic recovery with a membrane-permeable calpain inhibitor. Eur J Pharmacol. 1991 Dec 10;209(1-2):123–125. [PubMed]
  • Brorson JR, Manzolillo PA, Miller RJ. Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells. J Neurosci. 1994 Jan;14(1):187–197. [PubMed]
  • Hong SC, Goto Y, Lanzino G, Soleau S, Kassell NF, Lee KS. Neuroprotection with a calpain inhibitor in a model of focal cerebral ischemia. Stroke. 1994 Mar;25(3):663–669. [PubMed]
  • Bartus RT, Baker KL, Heiser AD, Sawyer SD, Dean RL, Elliott PJ, Straub JA. Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage. J Cereb Blood Flow Metab. 1994 Jul;14(4):537–544. [PubMed]
  • Bartus RT, Hayward NJ, Elliott PJ, Sawyer SD, Baker KL, Dean RL, Akiyama A, Straub JA, Harbeson SL, Li Z, et al. Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration. Stroke. 1994 Nov;25(11):2265–2270. [PubMed]
  • Aoki K, Imajoh S, Ohno S, Emori Y, Koike M, Kosaki G, Suzuki K. Complete amino acid sequence of the large subunit of the low-Ca2+-requiring form of human Ca2+-activated neutral protease (muCANP) deduced from its cDNA sequence. FEBS Lett. 1986 Sep 15;205(2):313–317. [PubMed]
  • Buroker-Kilgore M, Wang KK. A Coomassie brilliant blue G-250-based colorimetric assay for measuring activity of calpain and other proteases. Anal Biochem. 1993 Feb 1;208(2):387–392. [PubMed]
  • Wang KK, Villalobo A, Roufogalis BD. Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease. Arch Biochem Biophys. 1988 Feb 1;260(2):696–704. [PubMed]
  • Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. [PubMed]
  • Wang KK, Roufogalis BD, Villalobo A. Characterization of the fragmented forms of calcineurin produced by calpain I. Biochem Cell Biol. 1989 Oct;67(10):703–711. [PubMed]
  • Sasaki T, Kikuchi T, Yumoto N, Yoshimura N, Murachi T. Comparative specificity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic fluorogenic substrates. J Biol Chem. 1984 Oct 25;259(20):12489–12494. [PubMed]
  • Raser KJ, Posner A, Wang KK. Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents. Arch Biochem Biophys. 1995 May 10;319(1):211–216. [PubMed]
  • Sakihama T, Kakidani H, Zenita K, Yumoto N, Kikuchi T, Sasaki T, Kannagi R, Nakanishi S, Ohmori M, Takio K, et al. A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6075–6079. [PMC free article] [PubMed]
  • Yang HQ, Ma H, Takano E, Hatanaka M, Maki M. Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of mu-calpain large subunit. J Biol Chem. 1994 Jul 22;269(29):18977–18984. [PubMed]
  • Takano E, Ma H, Yang HQ, Maki M, Hatanaka M. Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains. FEBS Lett. 1995 Mar 27;362(1):93–97. [PubMed]
  • Saido TC, Shibata M, Takenawa T, Murofushi H, Suzuki K. Positive regulation of mu-calpain action by polyphosphoinositides. J Biol Chem. 1992 Dec 5;267(34):24585–24590. [PubMed]
  • Wang KK, Posner A, Hajimohammadreza I. Total protein extraction from cultured cells for use in electrophoresis and western blotting. Biotechniques. 1996 Apr;20(4):662–668. [PubMed]
  • Rosser BG, Powers SP, Gores GJ. Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach. J Biol Chem. 1993 Nov 5;268(31):23593–23600. [PubMed]
  • Gusovsky F, Daly JW. Maitotoxin: a unique pharmacological tool for research on calcium-dependent mechanisms. Biochem Pharmacol. 1990 Jun 1;39(11):1633–1639. [PubMed]
  • Hajimohammadreza I, Probert AW, Coughenour LL, Borosky SA, Marcoux FW, Boxer PA, Wang KK. A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death. J Neurosci. 1995 May;15(5 Pt 2):4093–4101. [PubMed]
  • Koh JY, Choi DW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods. 1987 May;20(1):83–90. [PubMed]
  • Caner H, Collins JL, Harris SM, Kassell NF, Lee KS. Attenuation of AMPA-induced neurotoxicity by a calpain inhibitor. Brain Res. 1993 Apr 2;607(1-2):354–356. [PubMed]
  • Parkes C, Kembhavi AA, Barrett AJ. Calpain inhibition by peptide epoxides. Biochem J. 1985 Sep 1;230(2):509–516. [PMC free article] [PubMed]
  • Siman R, Baudry M, Lynch G. Brain fodrin: substrate for calpain I, an endogenous calcium-activated protease. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3572–3576. [PMC free article] [PubMed]
  • Seubert P, Ivy G, Larson J, Lee J, Shahi K, Baudry M, Lynch G. Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies. Brain Res. 1988 Sep 6;459(2):226–232. [PubMed]
  • Bartus RT, Dean RL, Cavanaugh K, Eveleth D, Carriero DL, Lynch G. Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain. J Cereb Blood Flow Metab. 1995 Nov;15(6):969–979. [PubMed]
  • Bahr BA, Tiriveedhi S, Park GY, Lynch G. Induction of calpain-mediated spectrin fragments by pathogenic treatments in long-term hippocampal slices. J Pharmacol Exp Ther. 1995 May;273(2):902–908. [PubMed]
  • Meldrum B, Garthwaite J. Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci. 1990 Sep;11(9):379–387. [PubMed]
  • Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med. 1994 Mar 3;330(9):613–622. [PubMed]
  • Kishimoto H, Simon JR, Aprison MH. Determination of the equilibrium dissociation constants and number of glycine binding sites in several areas of the rat central nervous system, using a sodium-independent system. J Neurochem. 1981 Oct;37(4):1015–1024. [PubMed]

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