Search

Človeška katepsina K in S sta člana družine papainu podobnih peptidaz z zelo podobnima terciarnima strukturama. V človeškem telesu imata številne fiziološke vloge, pri čemer je najpomembnejša biološka vloga katepsina K razgradnja kolagenskih vlaken kostnega tkiva, medtem ko ima katepsin S zelo pomembno fiziološko vlogo pri regulaciji predstavitve antigenov. Katepsina K in S morata biti ustrezno regulirana, saj njuni povečani encimski aktivnosti lahko prispevata k napredovanju številnih bolezni. Glede na to, da so nekateri inhibitorji katepsinov K in S dosegli klinične faze razvoja, ta dva encima predstavljata pomembni potencialni terapevtski tarči za razvoj zdravilnih učinkovin. Ciljanje evolucijsko manj ohranjenih alosteričnih mest bi lahko omogočilo bolj selektivno inhibicijo, ki bi povzročala manj stranskih učinkov, kar bi tako predstavljalo alternativni način za terapevtske namene. Hiperbolični inhibitor katepsina K, metil [(3RS)-2,5-dioksopirolidin-3-il]glicinat (3a), smo podrobneje okarakterizirali in pokazali, da je selektiven na katepsin K glede na človeške katepsine B, L, S in V. Glede na to, da je 3a imel šibek inhibitorni učinek tudi na katepsin S, smo na njegovi osnovi zasnovali in pripravili potencialne inhibitorje katepsinov K in S, ki bi se z večjo afiniteto in bolj selektivno vezali na posamezen encim. Tistim z največjo potentnostjo smo določili njihove točnejše vrednosti afinitet ter mehanizme delovanja. Namen raziskovalnega dela je bil tudi, da bi na osnovi superpozicije terciarnih struktur človeških katepsinov K in S in na osnovi potencialnih alosteričnih poti človeškega katepsina K, predvidenih s simulacijami molekularne dinamike, načrtali in pripravili mutantne oblike teh dveh encimov ter identificirali aminokislinske ostanke, ki so pomembni za njuno alosterično regulacijo. Načrtali in pripravili smo spojine s substituenti na dveh mestih za diverzifikacijo sukcinimidnega ogrodja spojine 3a. Med njimi smo spojino metil [(3R)-2,5-dioksopirolidin-3-il]-L-treoninat (R-3c) okarakterizirali kot hiperbolični inhibitor katepsina S ter ji z rentgensko strukturno analizo določili absolutno (2S,3R,3'R)-konfiguracijo. Nato smo načrtali in pripravili ciklične derivate spojine R-3c ter med štirimi derivati spojino (3’RS)-3-{[(1S,2R)-2- hidroksicikloheksil]amino}pirolidin-2,5-dion ((1S,2R)-7) identificirali in okarakterizirali kot hiperbolični in selektivni inhibitor katepsina S z največjo potentnostjo. Z meritvami encimske kinetke smo pokazali, da hiperbolični inhibitorji katepsina K oz. S, in sicer spojine 3a, R-3c in (1S,2R)-7, delujejo po podobnih mehanizmih kot alosterična efektorja katepsina K. Na ta način smo identificirali in okarakterizirali prva malomolekulska hiperbolična inhibitorja katepsina S (R-3c in (1S,2R-7). Poleg tega smo potrdili del hipoteze raziskovalnega dela, saj smo dokazali, da spojina (1S,2R)-7 selektivno inhibira katepsin S glede na katepsin K. Mehanizmi delovanja, ki smo jih določili tem spojinam, so v skladu z rezultati testiranj spojin na razgradnjo makromolekulskih substratov s katepsinoma K in S. Hiperbolični inhibitorji 3a, R-3c in (1S,2R)- 7 imajo potencial za nadaljnji razvoj do spojin vodnic, uporabnih v raziskovalne namene. Načrtali in pripravili smo mutantne oblike katepsinov K in S z ostanki zamenjanimi z ostankom alanina v predvideni alosterični poti in v alosteričnem mestu katepsina K ter v homolognem mestu katepsina S. Pokazali smo, da sta ostanka v alosterični poti N202 in N208 pomembna za alosterično komunikacijo katepsina K med alosteričnim mestom in mestom S2. Human cathepsins K and S are members of the protein family of cysteine cathepsins with very similar tertiary structures. They have numerous physiological roles in human body, particularly the most important biological role of cathepsin K is degradation of collagen fibre in bone tissue, whereas cathepsin S plays a very important physiological role in regulating antigen presentation. Cathepsins K and S have to be sufficiently regulated since their increased enzyme activities could contribute to the progression of numerous diseases. Considering certain inhibitors of individual cathepsins K and S have reached clinical trials, these two enzymes represent important potential therapeutic targets for drug design. Targeting evolutionary less conserved allosteric sites could enable more selective inhibition, which could cause less side effects and could therefore represent an alternative approach for therapeutic purposes. After detailed characterization of hyperbolic inhibitor of cathepsin K, methyl [(3RS)-2,5- dioxopyrrolidin-3-yl]glycinate (3a), it was shown that it is selective against cathepsin K over human cathepsins B, L, S in V. Since 3a displayed weak inhibitory effect for cathepsin S, as well, we designed and prepared potential inhibitors of cathepsins K and S based on 3a, which would bind with higher affinities and more selectively to individual enzyme. For the most potent inhibitors, more accurate values of affinities and mechanisms of action were determined. The aim of the research work was also to design and prepare mutant forms of enzymes based on superposition of tertiary structures of human cathepsins K and S and based on potential allosteric pathways of human cathepsin K, predicted by molecular dynamic simulations, as well, and to identify residues important for their allosteric regulation. We designed and prepared compounds with substituents at two sites for diversification of the succinimide scaffold of compound 3a. Among them, methyl [(3R)-2,5-dioxopyrrolidin-3-yl]-L-threoninate (R-3c) was characterized as a hyperbolic inhibitor of cathepsin S and its absolute (2S,3R,3'R)-configuration was determined by X-ray structural analysis. Then, we designed and prepared cyclic derivatives of the compound R-3c and among four derivatives, (3’RS)-3- {[(1S,2R)-2- hydroxycyclohexyl]amino}pyrrolidine-2,5-dione ((1S,2R)-7) was identified and characterized as the most potent hyperbolic selective inhibitor of cathepsin S. It was shown by enzyme kinetics that allosteric modifiers of cathepsin K or S, compounds 3a, R-3c and (1S,2R)- 7, act via similar mechanisms as allosteric effectors of cathepsin K NSC13345 in NSC94914. Therefore, the first small-molecule allosteric effectors of cathepsin S (R-3c and (1S,2R)-7) were identified and characterized. Apart from that, one part of the hypotheses of our research work was confirmed since it was shown that the compound (1S,2R)-7 selectively inhibits cathepsin S over cathepsin K. The mechanisms of action, which were determined for the compounds, are consistent with the results of compound screening on degradation of macromolecular substrates via cathepsins K and S. Hyperbolic inhibitors 3a, R-3c in (1S,2R)-7 have the potential to be used for further development to lead compounds, which could be used for research purposes. Mutant forms of cathepsins K and S with residues substituted by alanine residue in predicted allosteric pathway and allosteric site of cathepsin K as well as its homologous site of cathepsin S were designed and prepared. We have shown that residues in predicted allosteric pathway N202 and N208 are important for allosteric communication in cathepsin K between allosteric site and site S.