FARMACIA, 2010, Vol.58, 2 136 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 3.4.22.17) are multidomain cytosolic enzymes that catalyze the limited degradation of proteins involved in key biological processes, such as cytoskeletal