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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Novel Calpain InhibitorsZoltán Bánóczi 1 , Levente E. Dókus 1 , Ágnes Tantos 2 , Attila Farkas 2 ,Péter Tompa 2 , Péter Friedrich 2 , and Ferenc Hudecz 1,31 Research Group of Peptide Chemistry, Eötvös L. University Hungarian Academy ofSciences, Budapest, Hungary; 2 Institute of Enzymology, Biological Research Center,Hungarian Academic of Sciences, Budapest, Hungary; 3 Department of Organic Chemistry,Eötvös L. University, Budapest 112 POB 32, H-1518, HungaryIntroductionCalpains are intracellular cysteine proteases and are of considerable interest due to theirimplication in numerous physiological events. Besides physiological functions, they play akey role in some well-studied pathological processes. The overactivation of calpains, whichresults in the disorder in Ca 2+ homeostasis, increases the degradation of the enzymesubstrates and could contribute to the development of the Alzheimer and/or Huntingtondiseases and also to death of nervous cells caused by traumatic brain injury, spinal cordinjury [1].One of the main methods to study the calpain function is to inhibit the enzyme.Unfortunately, there is only one specific calpain inhibitor of native origin known, thecalpastatin protein. Other inhibitors, mentioned in the literature, are not specific. Use ofthese inhibitors might confuse the interpretation of the results [2]. The use of calpastatin orits region B as specific inhibitors, is compromised by limited cell-penetration. The calpaininhibition may be important not only in the functional studies, but also in blocking ofcalpain overactivation in different diseases. These claims require more selective inhibitors,and trigger intensive research in this field.Results and DiscussionOur aim is to develop new, specific and cell-permeable calpain inhibitor(s), to be used foranalysis of calpain function under different conditions and also for the development ofdrugs to treat different calpain dependent diseases. For this, our plan was to synthesizeazapeptide inhibitors based on the calpain substrate (TPLKSPPPSPR) described by us [3].In these new inhibitors, the Lys residue after which the calpain cleaves the substrate wasreplaced by azaglycine (aGly) moiety. Besides this azapeptide, which is similar to thesubstrate sequence (TPLaGlySPPPSPR), shorter peptides were prepared (TPLaGlySPPPSand TPLaGlySP) to study the role of the amino acids at the C-terminal in inhibition. Forexamining the importance of amino acids in the position P 2 and P 3 aGlySPPPS,TPVaGlySPPPS, TPTaGly-NH 2 -S(Bzl)PPPS(Bzl) MBHA-resin SPPPS, TSLaGlySPPPS andTWLaGlySPPPS azapeptidesBoc-NH-NH 2 , CDI in DMFwere synthesized based on thepreference matrix [3]. TheBoc-NH-NH-CO-NH-S(Bzl)PPPS(Bzl)33% TFA/DCMNH 2 -NH-CO-NH-S(Bzl)PPPS(Bzl)MBHA-resinMBHA-resinazapeptides containing aGly wereproduced by solid phase peptidesynthesis on MBHA resin usingBoc/Bzl strategy (Figure 1). The1. Boc-Leu-OHazaglycine moiety was incorporatedwith the reaction of 1,1’-2. Boc-Pro-OH3. Boc-Thr(Bzl)-OHcarbonyldiimidazole (CDI) and4. Ac 2 OBoc-hydrazide. The cleavedAc-T(Bzl)PL-NH-NH-CO-NH-S(Bzl)PPPS(Bzl)HF/p-cresoleMBHA-resin peptides were purified by RP-HPLC and were characterized byanalytical RP-HPLC and ESI-MSAc-TPL-NH-NH-CO-NH-SPPPS-NH 2(Table 1).Figure 1. Outline of a typical synthesis of azapeptide inhibitorsThe inhibitory effect ofFig. 1. Outline of a typical synthesis of azapeptide peptides was determined ininhibitors.calpain buffer (10 mM HEPES,150 mM NaCl, 1 mM EDTA, 5mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM -mercaptoethanol, pH606