INHIBITION OF A CALCIUM-DEPENDENT CYSTEINE PROTEASE BY DOXYCYCLINE

Transcription

1 136 INHIBITION OF A CALCIUM-DEPENDENT CYSTEINE PROTEASE BY DOXYCYCLINE ADRIAN C. NICOLESCU 1 *, CONSTANTIN MIRCIOIU 2 1 Queen s University, Faculty of Health Sciences, Department of Biochemistry, 18 Stuart Street, Kingston, Ontario, K7L 3N6, Canada 2 UMF Carol Davila, Faculty of Pharmacy, 6 Traian Vuia Street, Bucharest, Romania *corresponding author: nicolesc@queensu.ca Abstract The activation of calcium-dependent cysteine proteases (calpains) due to an uncontrolled increase in the cellular calcium influx can lead to aberrant degradation of cellular proteins and cell death. The inhibitory effect of doxycycline on other proteases, but not calpains, has been reported. We discovered that doxycycline significantly inhibits murine calpain 2 at concentrations 30 µm. An increase in the calcium ion concentration did not affect this inhibitory effect, suggesting that doxycycline does not act by simply chelating the calcium ions. The analysis of the amino acid sequences of murine calpain 2 and human calpains 1 and 2 suggests that doxycycline is likely to have similar effects on human calpains. These results extend the non-antimicrobial activity of doxycycline to a different family of proteases, and could be used to design compounds potentially useful for modulating the activity of calpains. Rezumat Activarea cistein-proteazelor dependente de calciu (calpaine) datorată unei creşteri necontrolate a influxului celular de calciu poate conduce la degradarea aberantă a proteinelor celulare şi la moartea celulelor. Efectul inhibitor al doxiciclinei asupra altor proteaze, însă nu asupra calpainelor, este documentat. Noi am evidenţiat că doxiciclina inhibă semnificativ calpaina 2 murină la concentraţii 30 µm. O creştere a concentraţiei ionilor de calciu nu a afectat acest efect inhibitor, sugerând că doxiciclina nu acţionează prin simpla complexare a ionilor de calciu. Analiza secvenţelor de aminoacizi ai calpainei 2 murine şi ai calpainelor 1 şi 2 umane sugerează că doxiciclina ar avea efect similar asupra calpainelor umane. Aceste rezultate extind acţiunea non-antimicrobiană a doxiciclinei la o altă clasă de proteaze şi pot fi folosite pentru conceperea de compuşi cu utilitate potenţială în modularea activităţii calpainelor. Keywords: cysteine proteases, doxycycline, enzyme inhibition Introduction Calcium-dependent cysteine proteases or calpains (EC ) are multidomain cytosolic enzymes that catalyze the limited degradation of proteins involved in key biological processes, such as cytoskeletal

2 137 remodeling, signal transduction, cell differentiation, embryonic development, apoptosis, necrosis, vesicular protein transport, and cell migration [1-3]. The best characterized calpains, calpain 1 and calpain 2, are abundantly expressed in most tissues and consist of an isoform-specific 80 kda subunit (with 60% identity between calpain 1 and calpain 2) that contains the protease core, and a common 28 kda subunit. Both enzymes have several calcium-binding sites, which allosterically affect the enzyme activity. The function of calpains must be tightly regulated since aberrant activation of calpains, following cells inability to regulate calcium influx to the cytoplasm, can result in uncontrolled protein degradation and irreversible cell damage. Enhanced calpain activity has been associated with various pathologies, including myocardial infarction, heart failure, hypertension, atrial fibrillation, as well as stroke, brain trauma, Alzheimer s and Huntington s diseases, Duchenne muscular dystrophy, liver dysfunction, cataract, and certain types of cancer [2, 4, 5]. Since the overactivation of calpains has been linked to the development of various pathological conditions, calpains represent important targets for pharmacological inhibition. Doxycycline, a broad-spectrum antibiotic, has been shown to inhibit at sub-antimicrobial concentrations other calcium-dependent proteases, such as matrix metalloproteases [6, 7, 11], supposedly by interfering with the enzyme s calcium binding sites [6, 8]. The present work hypothesized that doxycycline inhibits calpains and tested the effect of doxycycline on murine calpain 2 activity. Materials and methods Materials. All reagents were of analytical grade and unless otherwise specified were purchased from Sigma-Aldrich (Oakville, ON). Murine recombinant calpain 2 was a generous gift from Dr. Peter L. Davies from Queen s University, Kingston, Ontario. Kinetic analysis of calpain 2 activity. The hydrolysis of 30 µm PLFAER fluorogenic substrate (prepared in 1.8 % v/v DMSO) by 0.1 µm calpain 2 in 50 mm Tris ph 7.6, containing 1 mm dithiothreitol (DTT), was measured at 25 C in a plate reader-based protocol in the presence or absence of doxycycline. Calpeptin, a known inhibitor of calpains, was used as a positive control to validate the assay. To prevent possible aggregation

3 138 of calpain 2 at high calcium concentrations, the assay buffer was supplemented with 150 mm NaCl. Assays were performed in a volume of 120 µl per well in black polystyrene half-area plates (Corning, NY). Fluorescence associated with the cleavage product was measured (λ ex 335 nm, λ em 595 nm, cut-off filter 475 nm) every 8 s for 30 min in a SPECTRAmax Gemini XPS (Molecular Devices, Sunnyvale, CA) plate reader. The rate of product formation in each well was determined through linear regression of the experimental data (r 2 > 0.99). Appropriate lag times, to preclude data prior to equilibration at 25 C, and end times, to preclude data following a loss of linearity, were entered manually prior to linear regression to obtain initial rates. The initial rates in the presence of inhibitors were normalized to the initial rates for the vehicle controls. All experiments were performed in triplicates and the concentration of doxycycline required to produce 50 % inhibition (IC 50 ) was determined from the nonlinear fit of data using GraphPad Prism version 4.03 (GraphPad Software, San Diego, CA). Sequence alignment. The amino-acid sequences for the large subunit of murine calpain 2, and human calpain 2 and human calpain 1 were obtained from the NCBI protein data base. Multiple amino-acid sequence alignment was performed by using the program Clustal W [9]. Statistical analysis. All data are presented as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way ANOVA followed by Newman-Keuls post hoc test. Two-tailed P values < 0.05 were considered statistically significant. Results and discussion The present study demonstrates, in a cell-free system, that doxycycline inhibits murine calpain 2 at moderate micromolar concentrations and that an excess in calcium ions concentration does not significantly affect this inhibitory effect. The ability of the kinetic assay to provide reliable results was tested with calpeptin, a non-specific inhibitor of calpains. The level of inhibition obtained for calpeptin (Figure 1) was consistent with previously reported calpeptin inhibitory concentrations [10].

4 139 Figure 1 Calpeptin inhibition of PLFAER fluorogenic substrate degradation by murine calpain 2 in the presence of 1 mm CaCl 2. Data are means ± SD of triplicates. * Statistically significant difference compared to vehicle control, P < 0.05, one-way ANOVA, where PLFAER is a fluorescence resonance energy transfer based substrate. Doxycycline inhibited the calcium-dependent activation of murine calpain 2 in a concentration-dependent manner (Figure 2 and table I). The inhibitory effect of doxycycline became significant at 30 µm (approx. 18% inhibition), and was substantial (approx. 65% inhibition) at 100 µm. Figure 2 Doxycycline inhibition of PLFAER fluorogenic substrate degradation by murine calpain 2 in the presence of 1 mm CaCl 2. Data are means ± SD of triplicates. * Statistically significant difference compared to vehicle control, P < 0.05, one-way ANOVA. Doxycycline is known to chelate divalent metal ions, including calcium. Therefore, the inhibitory effect of doxycycline on calpain 2 was also investigated in the presence of a large excess of calcium ions. The

5 140 presence of 10 mm CaCl 2 in the reaction mixture did not reduce significantly the inhibitory effect of doxycycline on calpain 2. Under these conditions, doxycycline inhibited calpain 2 in a concentration-dependent manner (Figure 3 and table I), and at similar inhibitory levels to those observed in the presence of 1 mm CaCl 2, suggesting that the inhibitory effect of doxycycline is not simply due to a complexation reaction. Figure 3 Doxycycline inhibition of PLFAER fluorogenic substrate degradation by murine calpain 2 in the presence of 10 mm CaCl 2. Data are means ± SD of triplicates. * Statistically significant difference compared to vehicle control, P < 0.05, one-way ANOVA. Table I Initial rates for doxcycline inhibition of murine calpain 2 and calpain [doxycycline] (µm) Initial rates ± SD (min -1 ) 1 mm CaCl 2 10 mm CaCl ± ± ± ± ± ± ± ± 4.2 The calculated inhibitory potency of doxycycline was 72 µm (Figure 4) in the presence of either 1 mm or 10 mm CaCl 2, further emphasizing an interaction of doxycycline with calpain that is not significantly affected by calcium. Interestingly, doxycycline also inhibited

6 141 the activity of the calpain protease core at comparable levels as the fulllength enzyme (data not shown). The calpain protease core (containing calpain s domains 1 and 2 and the catalytic site) requires for its activation two calcium ions, which potentially may be targeted by molecules interfering with the protein s calcium-binding sites. Figure 4 Concentration-dependent effect of doxycycline on murine calpain 2 activity. Data represent means ± SD of triplicates and were fitted to sigmoidal (variable slope) curves The analysis of the amino acid sequence alignment indicates an overall identity of 93% conservation between the murine and human calpains 2, as well as a 96% conservation of the calcium-binding residues (Figure 4). There is also a 61% amino acid conservation between murine calpain 2 and human calpain 1, and a 88% conservation of the calciumbinding residues between these proteins (Figure 4). This analysis predicts that doxycycline is likely to have similar inhibitory effects on human calpains. Using matrix metalloprotease 7, it has been suggested that doxycycline inhibits matrix metalloproteases by binding to the structural calcium- and zinc-binding sites of these proteins and, thus, destabilizing the proteins native structure [6]. A similar scenario can be proposed for the observed inhibition of calpain 2 by doxycyclin. Additionally, the proximity

7 142 of aromatic amino acid residues to calcium-binding sites may further stabilize the binding of doxycycline to the protein. murcpn MAGIAMKLAKDREAAEGLGSHERAIKYLNQDYETLRNECLEAGALFQDPS 50 humcpn MAGIAAKLAKDREAAEGLGSHERAIKYLNQDYEALRNECLEAGTLFQDPS 49 humcpn1 MSEEIITPVYCTGVSAQVQKQRARELGLGRHENAIKYLGQDYEQLRVRCLQSGTLFRDEA 60 murcpn2 FPALPSSLGFKELGPYSSKTRGIEWKRPTEICADPQFIIGGATRTDICQGALGDSWLLAA 110 humcpn2 FPAIPSALGFKELGPYSSKTRGMRWKRPTEICADPQFIIGGATRTDICQGALGDCWLLAA 109 humcpn1 FPPVPQSLGYKDLGPNSSKTYGIKWKRPTELLSNPQFIVDGATRTDICQGALGDCWLLAA 120 murcpn2 IASLTLNEEILARVVPLDQSFQENYAGIFHFQFWQYGEWVEVVVDDRLPTKDGELLFVHS 170 humcpn2 IASLTLNEEILARVVPLNQSFQENYAGIFHFQFWQYGEWVEVVVDDRLPTKDGELLFVHS 169 humcpn1 IASLTLNDTLLHRVVPHGQSFQNGYAGIFHFQLWQFGEWVDVVVDDLLPIKDGKLVFVHS 180 murcpn2 AEGSEFWSALLEKAYAKINGCYEALSGGATTEGFEDFTGGIAEWYELRKPPPNLFKIIQK 230 humcpn2 AEGSEFWSALLEKAYAKINGCYEALSGGATTEGFEDFTGGIAEWYELKKPPPNLFKIIQK 229 humcpn1 AEGNEFWSALLEKAYAKVNGSYEALSGGSTSEGFEDFTGGVTEWYELRKAPSDLYQIILK 240 murcpn2 ALEKGSLLGCSIDITSAADSEAVTYQKLVKGHAYSVTGAEEVESSGSLQKLIRIRNPWGQ 290 humcpn2 ALQKGSLLGCSIDITSAADSEAITFQKLVKGHAYSVTGAEEVESNGSLQKLIRIRNPWGE 289 humcpn1 ALERGSLLGCSIDISSVLDMEAITFKKLVKGHAYSVTGAKQVNYRGQVVSLIRMRNPWGE 300 murcpn2 VEWTGKWNDNCPSWNTVDPEVRANLTERQEDGEFWMSFSDFLRHYSRLEICNLTPDTLTC 350 humcpn2 VEWTGRWNDNCPSWNTIDPEERERLTRRHEDGEFWMSFSDFLRHYSRLEICNLTPDTLTS 349 humcpn1 VEWTGAWSDSSSEWNNVDPYERDQLRVKMEDGEFWMSFRDFMREFTRLEICNLTPDALKS 360 murcpn2 DSYKKWKLTKMDGNWRRGSTAGGCRNYPNTFWMNPQYLIKLEEEDED-DEDG--ERGCTF 407 humcpn2 DTYKKWKLTKMDGNWRRGSTAGGCRNYPNTFWMNPQYLIKLEEEDED-EEDG--ESGCTF 406 humcpn1 RTIRKWNTTLYEGTWRRGSTAGGCRNYPATFWVNPQFKIRLDETDDP-DDYGDRESGCSF 419 murcpn2 LVGLIQKHRRRQRKMGEDMHTIGFGIYEVPEELTGQTNIHLSKNFFLTTRARERSDTFIN 467 humcpn2 LVGLIQKHRRRQRKMGEDMHTIGFGIYEVPEELSGQTNIHLSKNFFLTNRARERSDTFIN 466 humcpn1 VLALMQKHRRRERPFGRDMETIGFAVYEVPPELVGQPAVHLKRDFFLANASRARSEQFIN 479 murcpn2 LREVLNRFKLPPGEYVLVPSTFEPHKNGDFCIRVFSEKKADYQTVDDEIEANI-EEIEAN 526 humcpn2 LREVLNRFKLPPGEYILVPSTFEPNKDGDFCIRVFSEKKADYQAVDDEIEANL-EEFDIS 525 humcpn1 LREVSTRFRLPPGEYVVVPSTFEPNKEGDFVLRFFSEKSAGTVELDDQIQANLPDEQVLS 539 murcpn2 EEDIGDGFRRLFAQLAGEDAEISAFELQTILRRVLAKREDIKSDGFSIETCKIMVDMLDE 586 humcpn2 EDDIDDGVRRLFAQLAGEDAEISAFELQTILRRVLAKRQDIKSDGFSIETCKIMVDMLDS 585 humcpn1 EEEIDENFKALFRQLAGEDMEISVKELRTILNRIISKHKDLRTKGFSLESCRSMVNLMDR 599 murcpn2 DGSGKLGLKEFYILWTKIQKYQKIYREIDVDRSGTMNSYEMRKALEEAGFKLPCQLHQVI 646 humcpn2 DGSGKLGLKEFYILWTKIQKYQKIYREIDVDRSGTMNSYEMRKALEEAGFKMPCQLHQVI 645 humcpn1 DGNGKLGLVEFNILWNRIRNYLSIFRKFDLDKSGSMSAYEMRMAIESAGFKLNKKLYELI 659 murcpn2 VARFADDELIIDFDNFVRCLVRLEILFKIFKQLDPENTGTIQLDLISWLSFSVL- 700 humcpn2 VARFADDQLIIDFDNFVRCLVRLETLFKIFKQLDPENTGTIELDLISWLCFSVL- 699 humcpn1 ITRYSEPDLAVDFDNFVCCLVRLETMFRFFKTLDTDLDGVVTFDLFKWLQLTMFA 714 Figure 5 Amino acid sequence alignment of murine calpain 2, human calpains 1 and 2. Calciumbinding residues are colored in red and highlighted in yellow. Tryptophan residues in the proximity of the calcium-binding sites are highlighted in green.

8 143 The activation of calpains by calcium ions involves conformational changes in the calpain s stucture, changes that are necessary for the proper alignment of the catalytic triad. Conceivably, doxycycline binding to calcium-coordinating amino acids in calpains prevents these conformational changes required for enzymatic activity. Conclusions The present study demonstrates for the first time the inhibitory effect of doxycycline on murine calpain 2. Considering the overall amino acid conservation between the murine calpain 2 and human calpains 1 and 2, it is likely that doxycycline would similarly inhibit human calpains. The development of molecules that inhibit calpains at sites away from the catalytic cleft represents a viable alternative for drug discovery. The inhibition mechanism of calpains by doxycyline may involve allosteric sites and, thus, doxycycline may provide an example and research tool for designing more specific calpain inhibitors with potential use in therapy. Acknowledgements The authors are thankful to Drs. Peter L. Davies and Michael Neisham for the use of the research infrastructure. Adrian Nicolescu is a recipient of a Heart and Stroke Foundation of Canada research fellowship. In loving memory of Dr. Maria Ivona Poenaru ( ). References 1. Goll D.E., Thompson V.F., Li H., Wei W., Cong J., The calpain system, Physiological Reviews, 2003, 83, Zatz M., Starling A., Calpains and disease, New England Journal of Medicine, 2005, 352, Franco S.J., Huttenlocher A., Regulating cell migration: calpains make the cut, Journal of Cell Science, 2005, 118, Nixon R.A., The calpains in aging and aging-related diseases, Ageing Research Reviews, 2003, 2, Perrin C., Vergely C., Rochette L., Calpains and cardiac diseases, Annales de Cardiologie et d'angeiologe (Paris), 2004, 53, Garcia R.A., Pantazatos D.P., Gessner C.R., Go K.V., Woods V.L. Jr., Villarreal F.J., Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry, Molecular Pharmacology, 2005, 67, Nicolescu A.C., Holt A., Kandasamy A.D., Pacher P., Schulz R., Inhibition of matrix metalloproteinase-2 by PARP inhibitors, Biochemical and Biophysical Research Communications, 2009, 387, Penescu M. et al, The application of mass spectrometry for identifing modern biochemical markers of nephropathies, Farmacia, 2009, 57(6),

9 Thompson J.D., Higgins D.G., T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research, 1994, 22, Tsujinaka T., Kajiwara Y., Kambayashi J., Sakon M., Higuchi N., Tanaka T., Mori T., Synthesis of a new cell penetrating calpain inhibitor (calpeptin), Biochemical and Biophysical Research Communications, 1988, 153, Habor A., Peroxisome proliferator activated receptors, Farmacia, 2010, 58(1), Manuscript received: July 28 th 2009