JOURNAL OF NEUROCHEMISTRY
| 2014 | 130 | 161–164
doi: 10.1111/jnc.12732
University of North Dakota School of Medicine and Health Sciences, Department of Basic Sciences, Grand Forks, North Dakota Read the full article ‘Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis’ on page 268.
Multiple sclerosis (MS) is an inflammatory and demyelinating disease (Stadelmann et al. 2011) that affects over 2.3 million people worldwide. The etiology of MS remains unknown but is thought to be the result of multiple genetic, environmental, and immunological factors (Ascherio and Munger 2007; Milo and Kahana 2010). The disease targets the CNS resulting in the loss of myelin and the formation of demyelinating plaques (Compston and Coles 2002). The loss of myelin within the plaque results in a decrease in synapse density and an increase in neuronal cell loss (Noseworthy et al. 2000). All of which, can have a profound effect on motor function and can induce deficits in learning, memory, and cognitive function (Noseworthy et al. 2000). Therefore, developing new orally available therapeutic strategies to reduce or slow the progression of demyelination in MS is of great importance to people who suffer from this disease. While the etiology of the disease remains unknown, demyelinating lesions based on plaque morphology, myelin protein expression, oligodendrocyte pathology, and the presence of complex deposition have provided valuable information regarding the pathology of the disease (Lucchinetti et al. 1996). Types I and II, based on the presence of T-cell and macrophage infiltration or Ig deposition and complement activation, respectively, suggests that the demyelination is secondary to an autoimmune reaction against the host’s myelin. Interestingly, plaque Types III and IV do not show hallmarks of immune system activation but rather are classified by mature oligodendrocyte cell death or oligodendrocyte degeneration. All of which suggests that MS pathology may be secondary to an autoimmune reaction or consist of a more complex sequelae of events that disrupts oligodendrocyte health and function (Kornek and Lassmann 2003). Despite the understanding of the pathological differences in MS, only a handful of FDA-approved drugs are available to treat people suffering from the disease. Available therapies focus on reducing CNS inflammation or involve the
use of disease modifying agents that act to slow disease progression (Rice 1999; Menge et al. 2008) all of which treat the physical symptoms associated with the disease (Palumbo and Bosetti 2013). These include corticosteroids and nonsteroidal anti-inflammatory drugs used to reduce CNS inflammation and ease the severity of demyelinating attacks or the use of disease modifying drugs that work by suppressing or altering the body’s immune response. The later drug therapies are based on the premise that MS is in large part secondary to an autoimmune-mediated disorder. It is without argument both strategies have increased the quality of life for those who suffer from MS by reducing symptoms of the disease and disease severity. Trager et al. (2014) on the other hand, have adapted a strategy using an orally available inhibitor of calpain (SNJ-1945) in which they demonstrate the reduction of the development of clinical signs in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE). In this manuscript, the authors show that the oral administration of SNJ-1945 inhibits both the consolidation of the peripheral immune response and reduces central gliosis and neuronal damage. Inhibiting cellular proteolysis and attenuating peptide signaling represents an interesting and novel approach to reduce injury progression in this model. Furthermore, targeting calpainmediated proteolysis and the subsequent downstream peptide signaling inhibited by this orally available compound may offer alternative therapies to help reduce plaque formation and disease severity in patients suffering from MS. Received March 16, 2014; revised manuscript received April 1, 2014; accepted April 2, 2014. Address correspondence and reprint requests to Thad A. Rosenberger, University of North Dakota School of Medicine and Health Sciences, Department of Basic Sciences, 501 North Columbia Road, Grand Forks, North Dakota 58203. E-mail: [email protected] Abbreviations used: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 161--164
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The calpain family of calcium-dependent serine proteases when activated promote stress-induced cell death by proteolytic cleavage of a number of cellular (Ferreira et al. 2010) and myelin proteins (Schaecher et al. 2001; Medveczky et al. 2006). Over 12 calpain isoforms exist in eukaryotes that exhibit calcium dependence, non-lysosomal expression, and whose enzymatic activity is optimal at neutral pH (Cuerrier et al. 2005). Although their activity was first found in brain and skeletal muscle it is now recognized that they are ubiquitously expressed throughout the body but are closely associated with brain dysfunction (Wang et al. 1996; Huang and Wang 2001). Interestingly, the relatively non-specific substrate preference of the calpain family of proteases and their non-lysosomal expression suggests that these enzymes maintain a calcium-dependent regulatory role in maintaining intracellular protein activity and are involved in peptide signaling as opposed to being solely a digestive protease (Huang and Wang 2001). The two most characterized isoforms that are highly expressed in brain, calpain I, and II, differ in their dependence on calcium for activity. In this regard, the calpain I isoform requires lM levels of calcium to induce activity, whereas calpain II requires near mM levels (Cuerrier et al. 2005). All known isoforms of calpain consist of multidomain enzymes that are structurally heterodimeric (Huang and Wang 2001). The proteolytic active site is situated at the interface between a papain-like domain I and II, which together form the proteolytic core of the enzyme (Cuerrier et al. 2005). These domains contain two calcium-binding sites whose occupation is necessary for the rearrangement of the catalytic center into an active conformation that promote substrate hydrolysis (Moldoveanu et al. 2004). Because of the complexity of cellular calcium control the development of calpain inhibitors has focused on formulating relevant compounds that bind the catalytic center of the different calpain isoforms that disrupts catalysis or competes for available substrate (Lubisch et al. 2003). Irreversible inhibitors are available that are based on a reactive dipeptide backbone that is capable of entering and covalently binding the catalytic moiety of the enzyme (Sasaki et al. 1990). Reversible calpain inhibitors are designed based on reactive moieties that mimic substrate transition states and have focused on modifying the dipeptide backbone to contain aldehyde- or ketone-like moieties that reversibly bind to the catalytic center of the enzyme (Crawford et al. 1988). The development of reversible inhibitors provides more therapeutically relevant applications, however, because of the chemical structure of the compound these compounds are problematic with respect to stability, increased non-specific metabolism, and selectivity (Cuerrier et al. 2006) because of non-specific binding to nucleophilic amino acids and reactive plasma thiol groups that decrease bioavailability (Cuerrier et al. 2006). With regard to SNJ-1945, this non-specific metabolism is reduced by “masking” the reactive aldehyde
using peptidyl a-ketoamide functionality (Li et al. 1993; Harbeson et al. 1994). This modification increased the membrane permeability of the calpain inhibitor and its resistance to non-specific plasma esterase activity while maintaining reasonable solubility in water (Cuerrier et al. 2006) that improved bioavailability (Shirasaki et al. 2006). Furthermore, the peptidyl a-ketoaminde provided good CNS penetration that exceeded the IC50 of calpain I and II (86– 190 mM) out to 4 h following a single oral doses ranging between 50 and 120 mg/kg (Oka et al. 2006; Suzuki et al. 2014). Thus, given the potential impact that calpain-mediated proteolysis has in many human diseases and CNS disorders (Huang and Wang 2001; Liu et al. 2008) the development of orally available calpain inhibitor with positive pharmacokinetic properties offers a very unique opportunity to uncover the role that peptide signaling and calcium-induced proteolysis has in many disorders. In this regard, it is well accepted that immune and inflammatory reactions found with MS and EAE can directly increase the activity and expression of calpain that is paralleled by an increase in the calpain-mediated degradation of axonal neurofilament protein and a 96 kD myelin associated glycoprotein (Shields and Banik 1999). In addition, calpain activation is known to induce the proteolytic processing of p35 to p25 that coordinates with cyclin dependent 5 kinase forming a complex that contributes to neuronal cell death via the hyperphosphorylation of tau protein in a model of chemical induced hypoxia (BarrosMinones et al. 2013). Similar calpain-dependent peptide signaling resulting in the formation of a deleterious p25/ cyclin-dependent 5 kinase complex have been demonstrated using a model of alpha-synuclein-induced calcium dysregulation (Czapski et al. 2013) and the peroxide-induced cleavage of glycogen synthase kinase-3 beta (Feng et al. 2013). Furthermore, glutamate toxicity results in a calpaindependent truncation Src protein that results in the inactivation of Akt signaling which results in neuronal cell death (Hossain et al. 2013). All of which suggest that the calciumdependent activation of calpain can directly alter cellular signaling by degrading receptors and protein targets as well as increase downstream peptide signaling that can further disrupt neuronal-glial homeostasis (Mo et al. 2013). Given the unknown etiology of MS and the complexity of plaque types found in the disease suggests that the modulation of immune or inhibition of inflammatory pathways may not reveal the complete nature of the disease. Thus, developing a more anomalous therapeutic strategy that targets calciuminduced protein degradation and peptide signaling may provide wider therapeutic platform that more closely address the pathology associated with the disease. In this regard, it is known that calpain expression and activity is increased in EAE (Shields and Banik 1999; Shields et al. 1999; Zheng and Bizzozero 2011) and that calpain inhibition reduces injury progression in this model
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 161--164
Review
(Shields et al. 1999; Schaecher et al. 2001). More importantly, the orally available calpain inhibitor SNJ-1945 (50 mg/kg) was able to achieve this level of protection in this EAE model (Trager et al. 2014) despite subjecting the animals to much lower doses of SNJ-1945 than described in other in vivo models of neural injury (Oka et al. 2006; Koumura et al. 2008). Because the EAE model utilized in this study was induced by inoculating mice with guinea pig myelin basic protein (0.4 mg) resulting in an immunemediated demyelinating injury mimicking the Type I MS plaque characteristic (Palumbo and Bosetti 2013), suggests that the predominating effect of treatment may be the result of inhibiting the peripheral immune response and in turn attenuation of the induction of central demyelination. Nevertheless, given the fact that oral SNJ-1945 was able to reduce injury progression in this model suggests that this drug may be beneficial in reducing other type of MS pathology not directly associated with T-cell and macrophage infiltration. Furthermore, the use of SNJ-1945 may provide a powerful tool to determine the role calpainmediated proteolysis and subsequent peptide signaling has in the central demyelinating events in other animal models of MS that are more closely associated with Types II-IV demyelinating MS plaques. Regardless, therapeutically targeting calpain-mediated proteolysis and the subsequent peptide signaling represents a clear deviation from current therapeutic theory which may provide a useful tool in the treatment of MS as well as provide a greater understanding of the molecular mechanisms involved in demyelination found in MS.
Acknowledgments and conflicts of interest disclosure The author declares no conflict of interest.
References Ascherio A. and Munger K. L. (2007) Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann. Neurol. 61, 288–299. Barros-Minones L., Martin-de-Saavedra D., Perez-Alvarez S. et al. (2013) Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate. Biochim. Biophys. Acta 1832, 705–717. Compston A. and Coles A. (2002) Multiple sclerosis. Lancet 359, 1221–1231. Crawford C., Mason R. W., Wikstrom P. and Shaw E. (1988) The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B. Biochem. J. 253, 751–758. Cuerrier D., Moldoveanu T. and Davies P. L. (2005) Determination of peptide substrate specificity for mu-calpain by a peptide librarybased approach: the importance of primed side interactions. J. Biol. Chem. 280, 40632–40641. Cuerrier D., Moldoveanu T., Inoue J., Davies P. L. and Campbell R. L. (2006) Calpain inhibition by alpha-ketoamide and cyclic
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hemiacetal inhibitors revealed by X-ray crystallography. Biochemistry 45, 7446–7452. Czapski G. A., Gassowska M., Wilkaniec A., Cieslik M. and Adamczyk A. (2013) Extracellular alpha-synuclein induces calpain-dependent overactivation of cyclin-dependent kinase 5 in vitro. FEBS Lett. 587, 3135–3141. Feng Y., Xia Y., Yu G., Shu X., Ge H., Zeng K., Wang J. and Wang X. (2013) Cleavage of GSK-3beta by calpain counteracts the inhibitory effect of Ser9 phosphorylation on GSK-3beta activity induced by H(2)O(2). J. Neurochem. 126, 234–242. Ferreira S. M., Lerner S. F., Brunzini R., Reides C. G., Evelson P. A. and Llesuy S. F. (2010) Time course changes of oxidative stress markers in a rat experimental glaucoma model. Invest. Ophthalmol. Vis. Sci. 51, 4635–4640. Harbeson S. L., Abelleira S. M., Akiyama A., Barrett R., 3rd, Carroll R. M., Straub J. A., Tkacz J. N., Wu C. and Musso G. F. (1994) Stereospecific synthesis of peptidyl alpha-keto amides as inhibitors of calpain. J. Med. Chem. 37, 2918–2929. Hossain M. I., Roulston C. L., Kamaruddin M. A. et al. (2013) A truncated fragment of Src protein kinase generated by calpainmediated cleavage is a mediator of neuronal death in excitotoxicity. J. Biol. Chem. 288, 9696–9709. Huang Y. and Wang K. K. (2001) The calpain family and human disease. Trends Mol. Med. 7, 355–362. Kornek B. and Lassmann H. (2003) Neuropathology of multiple sclerosis-new concepts. Brain Res. Bull. 61, 321–326. Koumura A., Nonaka Y., Hyakkoku K., Oka T., Shimazawa M., Hozumi I., Inuzuka T. and Hara H. (2008) A novel calpain inhibitor, ((1S)-1 ((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino) carbonyl)-3-met hylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester, protects neuronal cells from cerebral ischemia-induced damage in mice. Neuroscience 157, 309–318. Li Z., Patil G. S., Golubski Z. E., Hori H., Tehrani K., Foreman J. E., Eveleth D. D., Bartus R. T. and Powers J. C. (1993) Peptide alphaketo ester, alpha-keto amide, and alpha-keto acid inhibitors of calpains and other cysteine proteases. J. Med. Chem. 36, 3472–3480. Liu J., Liu M. C. and Wang K. K. (2008) Calpain in the CNS: from synaptic function to neurotoxicity. Sci. Signal. 1, re1. Lubisch W., Beckenbach E., Bopp S. et al. (2003) Benzoylalaninederived ketoamides carrying vinylbenzyl amino residues: discovery of potent water-soluble calpain inhibitors with oral bioavailability. J. Med. Chem. 46, 2404–2412. Lucchinetti C. F., Bruck W., Rodriguez M. and Lassmann H. (1996) Distinct patterns of multiple sclerosis pathology indicates heterogeneity on pathogenesis. Brain Pathol. 6, 259–274. Medveczky P., Antal J., Patthy A., Kekesi K., Juhasz G., Szilagyi L. and Graf L. (2006) Myelin basic protein, an autoantigen in multiple sclerosis, is selectively processed by human trypsin 4. FEBS Lett. 580, 545–552. Menge T., Weber M. S., Hemmer B. et al. (2008) Disease-modifying agents for multiple sclerosis: recent advances and future prospects. Drugs 68, 2445–2468. Milo R. and Kahana E. (2010) Multiple sclerosis: geoepidemiology, genetics and the environment. Autoimmun. Rev. 9, A387–A394. Mo M., Hoang H. T., Schmidt S., Clark R. B. and Ehrlich B. E. (2013) The role of chromogranin B in an animal model of multiple sclerosis. Mol. Cell. Neurosci. 56, 102–114. Moldoveanu T., Jia Z. and Davies P. L. (2004) Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites. J. Biol. Chem. 279, 6106–6114. Noseworthy J. H., Lucchinetti C., Rodriguez M. and Weinshenker B. G. (2000) Multiple sclerosis. N. Engl. J. Med. 343, 938–952. Oka T., Walkup R. D., Tamada Y., Nakajima E., Tochigi A., Shearer T. R. and Azuma M. (2006) Amelioration of retinal degeneration and
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proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl) amino)carbonyl)-3-me thylbutyl)carbamic acid 5-methoxy-3oxapentyl ester. Neuroscience 141, 2139–2145. Palumbo S. and Bosetti F. (2013) Alterations of brain eicosanoid synthetic pathway in multiple sclerosis and in animal models of demyelination: role of cyclooxygenase-2. Prostaglandins Leukot. Essent. Fatty Acids 89, 273–278. Rice G. P. (1999) Treatment of secondary progressive multiple sclerosis: current recommendations and future prospects. BioDrugs 12, 267–277. Sasaki T., Kishi M., Saito M., Tanaka T., Higuchi N., Kominami E., Katunuma N. and Murachi T. (1990) Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins. J. Enzym. Inhib. 3, 195–201. Schaecher K. E., Shields D. C. and Banik N. L. (2001) Mechanism of myelin breakdown in experimental demyelination: a putative role for calpain. Neurochem. Res. 26, 731–737. Shields D. C. and Banik N. L. (1999) Pathophysiological role of calpain in experimental demyelination. J. Neurosci. Res. 55, 533–541. Shields D. C., Schaecher K. E., Saido T. C. and Banik N. L. (1999) A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc. Natl Acad. Sci. USA 96, 11486–11491.
Shirasaki Y., Yamaguchi M. and Miyashita H. (2006) Retinal penetration of calpain inhibitors in rats after oral administration. J. Ocul. Pharmacol. Ther. 22, 417–424. Stadelmann C., Wegner C. and Bruck W. (2011) Inflammation, demyelination, and degeneration - recent insights from MS pathology. Biochim. Biophys. Acta 1812, 275–282. Suzuki R., Oka T., Tamada Y., Shearer T. R. and Azuma M. (2014) Degeneration and dysfunction of retinal neurons in acute ocular hypertensive rats: involvement of calpains. J. Ocul. Pharmacol. Ther. 30, 419–428. Trager N., Smith A., Wallace Iv G., Azuma M., Inoue J., Beeson C., Haque A. and Banik N. L. (2014) Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis. J. Neurochem. 130, 268–279. Wang K. K., Nath R., Posner A. et al. (1996) An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective. Proc. Natl Acad. Sci. USA 93, 6687–6692. Zheng J. and Bizzozero O. A. (2011) Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis. J. Neurochem. 117, 143–153.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 161--164
| 2014 | 130 | 161–164
doi: 10.1111/jnc.12732
University of North Dakota School of Medicine and Health Sciences, Department of Basic Sciences, Grand Forks, North Dakota Read the full article ‘Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis’ on page 268.
Multiple sclerosis (MS) is an inflammatory and demyelinating disease (Stadelmann et al. 2011) that affects over 2.3 million people worldwide. The etiology of MS remains unknown but is thought to be the result of multiple genetic, environmental, and immunological factors (Ascherio and Munger 2007; Milo and Kahana 2010). The disease targets the CNS resulting in the loss of myelin and the formation of demyelinating plaques (Compston and Coles 2002). The loss of myelin within the plaque results in a decrease in synapse density and an increase in neuronal cell loss (Noseworthy et al. 2000). All of which, can have a profound effect on motor function and can induce deficits in learning, memory, and cognitive function (Noseworthy et al. 2000). Therefore, developing new orally available therapeutic strategies to reduce or slow the progression of demyelination in MS is of great importance to people who suffer from this disease. While the etiology of the disease remains unknown, demyelinating lesions based on plaque morphology, myelin protein expression, oligodendrocyte pathology, and the presence of complex deposition have provided valuable information regarding the pathology of the disease (Lucchinetti et al. 1996). Types I and II, based on the presence of T-cell and macrophage infiltration or Ig deposition and complement activation, respectively, suggests that the demyelination is secondary to an autoimmune reaction against the host’s myelin. Interestingly, plaque Types III and IV do not show hallmarks of immune system activation but rather are classified by mature oligodendrocyte cell death or oligodendrocyte degeneration. All of which suggests that MS pathology may be secondary to an autoimmune reaction or consist of a more complex sequelae of events that disrupts oligodendrocyte health and function (Kornek and Lassmann 2003). Despite the understanding of the pathological differences in MS, only a handful of FDA-approved drugs are available to treat people suffering from the disease. Available therapies focus on reducing CNS inflammation or involve the
use of disease modifying agents that act to slow disease progression (Rice 1999; Menge et al. 2008) all of which treat the physical symptoms associated with the disease (Palumbo and Bosetti 2013). These include corticosteroids and nonsteroidal anti-inflammatory drugs used to reduce CNS inflammation and ease the severity of demyelinating attacks or the use of disease modifying drugs that work by suppressing or altering the body’s immune response. The later drug therapies are based on the premise that MS is in large part secondary to an autoimmune-mediated disorder. It is without argument both strategies have increased the quality of life for those who suffer from MS by reducing symptoms of the disease and disease severity. Trager et al. (2014) on the other hand, have adapted a strategy using an orally available inhibitor of calpain (SNJ-1945) in which they demonstrate the reduction of the development of clinical signs in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE). In this manuscript, the authors show that the oral administration of SNJ-1945 inhibits both the consolidation of the peripheral immune response and reduces central gliosis and neuronal damage. Inhibiting cellular proteolysis and attenuating peptide signaling represents an interesting and novel approach to reduce injury progression in this model. Furthermore, targeting calpainmediated proteolysis and the subsequent downstream peptide signaling inhibited by this orally available compound may offer alternative therapies to help reduce plaque formation and disease severity in patients suffering from MS. Received March 16, 2014; revised manuscript received April 1, 2014; accepted April 2, 2014. Address correspondence and reprint requests to Thad A. Rosenberger, University of North Dakota School of Medicine and Health Sciences, Department of Basic Sciences, 501 North Columbia Road, Grand Forks, North Dakota 58203. E-mail: [email protected] Abbreviations used: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 161--164
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The calpain family of calcium-dependent serine proteases when activated promote stress-induced cell death by proteolytic cleavage of a number of cellular (Ferreira et al. 2010) and myelin proteins (Schaecher et al. 2001; Medveczky et al. 2006). Over 12 calpain isoforms exist in eukaryotes that exhibit calcium dependence, non-lysosomal expression, and whose enzymatic activity is optimal at neutral pH (Cuerrier et al. 2005). Although their activity was first found in brain and skeletal muscle it is now recognized that they are ubiquitously expressed throughout the body but are closely associated with brain dysfunction (Wang et al. 1996; Huang and Wang 2001). Interestingly, the relatively non-specific substrate preference of the calpain family of proteases and their non-lysosomal expression suggests that these enzymes maintain a calcium-dependent regulatory role in maintaining intracellular protein activity and are involved in peptide signaling as opposed to being solely a digestive protease (Huang and Wang 2001). The two most characterized isoforms that are highly expressed in brain, calpain I, and II, differ in their dependence on calcium for activity. In this regard, the calpain I isoform requires lM levels of calcium to induce activity, whereas calpain II requires near mM levels (Cuerrier et al. 2005). All known isoforms of calpain consist of multidomain enzymes that are structurally heterodimeric (Huang and Wang 2001). The proteolytic active site is situated at the interface between a papain-like domain I and II, which together form the proteolytic core of the enzyme (Cuerrier et al. 2005). These domains contain two calcium-binding sites whose occupation is necessary for the rearrangement of the catalytic center into an active conformation that promote substrate hydrolysis (Moldoveanu et al. 2004). Because of the complexity of cellular calcium control the development of calpain inhibitors has focused on formulating relevant compounds that bind the catalytic center of the different calpain isoforms that disrupts catalysis or competes for available substrate (Lubisch et al. 2003). Irreversible inhibitors are available that are based on a reactive dipeptide backbone that is capable of entering and covalently binding the catalytic moiety of the enzyme (Sasaki et al. 1990). Reversible calpain inhibitors are designed based on reactive moieties that mimic substrate transition states and have focused on modifying the dipeptide backbone to contain aldehyde- or ketone-like moieties that reversibly bind to the catalytic center of the enzyme (Crawford et al. 1988). The development of reversible inhibitors provides more therapeutically relevant applications, however, because of the chemical structure of the compound these compounds are problematic with respect to stability, increased non-specific metabolism, and selectivity (Cuerrier et al. 2006) because of non-specific binding to nucleophilic amino acids and reactive plasma thiol groups that decrease bioavailability (Cuerrier et al. 2006). With regard to SNJ-1945, this non-specific metabolism is reduced by “masking” the reactive aldehyde
using peptidyl a-ketoamide functionality (Li et al. 1993; Harbeson et al. 1994). This modification increased the membrane permeability of the calpain inhibitor and its resistance to non-specific plasma esterase activity while maintaining reasonable solubility in water (Cuerrier et al. 2006) that improved bioavailability (Shirasaki et al. 2006). Furthermore, the peptidyl a-ketoaminde provided good CNS penetration that exceeded the IC50 of calpain I and II (86– 190 mM) out to 4 h following a single oral doses ranging between 50 and 120 mg/kg (Oka et al. 2006; Suzuki et al. 2014). Thus, given the potential impact that calpain-mediated proteolysis has in many human diseases and CNS disorders (Huang and Wang 2001; Liu et al. 2008) the development of orally available calpain inhibitor with positive pharmacokinetic properties offers a very unique opportunity to uncover the role that peptide signaling and calcium-induced proteolysis has in many disorders. In this regard, it is well accepted that immune and inflammatory reactions found with MS and EAE can directly increase the activity and expression of calpain that is paralleled by an increase in the calpain-mediated degradation of axonal neurofilament protein and a 96 kD myelin associated glycoprotein (Shields and Banik 1999). In addition, calpain activation is known to induce the proteolytic processing of p35 to p25 that coordinates with cyclin dependent 5 kinase forming a complex that contributes to neuronal cell death via the hyperphosphorylation of tau protein in a model of chemical induced hypoxia (BarrosMinones et al. 2013). Similar calpain-dependent peptide signaling resulting in the formation of a deleterious p25/ cyclin-dependent 5 kinase complex have been demonstrated using a model of alpha-synuclein-induced calcium dysregulation (Czapski et al. 2013) and the peroxide-induced cleavage of glycogen synthase kinase-3 beta (Feng et al. 2013). Furthermore, glutamate toxicity results in a calpaindependent truncation Src protein that results in the inactivation of Akt signaling which results in neuronal cell death (Hossain et al. 2013). All of which suggest that the calciumdependent activation of calpain can directly alter cellular signaling by degrading receptors and protein targets as well as increase downstream peptide signaling that can further disrupt neuronal-glial homeostasis (Mo et al. 2013). Given the unknown etiology of MS and the complexity of plaque types found in the disease suggests that the modulation of immune or inhibition of inflammatory pathways may not reveal the complete nature of the disease. Thus, developing a more anomalous therapeutic strategy that targets calciuminduced protein degradation and peptide signaling may provide wider therapeutic platform that more closely address the pathology associated with the disease. In this regard, it is known that calpain expression and activity is increased in EAE (Shields and Banik 1999; Shields et al. 1999; Zheng and Bizzozero 2011) and that calpain inhibition reduces injury progression in this model
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 161--164
Review
(Shields et al. 1999; Schaecher et al. 2001). More importantly, the orally available calpain inhibitor SNJ-1945 (50 mg/kg) was able to achieve this level of protection in this EAE model (Trager et al. 2014) despite subjecting the animals to much lower doses of SNJ-1945 than described in other in vivo models of neural injury (Oka et al. 2006; Koumura et al. 2008). Because the EAE model utilized in this study was induced by inoculating mice with guinea pig myelin basic protein (0.4 mg) resulting in an immunemediated demyelinating injury mimicking the Type I MS plaque characteristic (Palumbo and Bosetti 2013), suggests that the predominating effect of treatment may be the result of inhibiting the peripheral immune response and in turn attenuation of the induction of central demyelination. Nevertheless, given the fact that oral SNJ-1945 was able to reduce injury progression in this model suggests that this drug may be beneficial in reducing other type of MS pathology not directly associated with T-cell and macrophage infiltration. Furthermore, the use of SNJ-1945 may provide a powerful tool to determine the role calpainmediated proteolysis and subsequent peptide signaling has in the central demyelinating events in other animal models of MS that are more closely associated with Types II-IV demyelinating MS plaques. Regardless, therapeutically targeting calpain-mediated proteolysis and the subsequent peptide signaling represents a clear deviation from current therapeutic theory which may provide a useful tool in the treatment of MS as well as provide a greater understanding of the molecular mechanisms involved in demyelination found in MS.
Acknowledgments and conflicts of interest disclosure The author declares no conflict of interest.
References Ascherio A. and Munger K. L. (2007) Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann. Neurol. 61, 288–299. Barros-Minones L., Martin-de-Saavedra D., Perez-Alvarez S. et al. (2013) Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate. Biochim. Biophys. Acta 1832, 705–717. Compston A. and Coles A. (2002) Multiple sclerosis. Lancet 359, 1221–1231. Crawford C., Mason R. W., Wikstrom P. and Shaw E. (1988) The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B. Biochem. J. 253, 751–758. Cuerrier D., Moldoveanu T. and Davies P. L. (2005) Determination of peptide substrate specificity for mu-calpain by a peptide librarybased approach: the importance of primed side interactions. J. Biol. Chem. 280, 40632–40641. Cuerrier D., Moldoveanu T., Inoue J., Davies P. L. and Campbell R. L. (2006) Calpain inhibition by alpha-ketoamide and cyclic
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