Evaluation of new cathepsin V inhibitors

Katepsin V je lizosomska cisteinska peptidaza iz družine papainu podobnih cisteinskih peptidaz. Katepsin V je zelo soroden katepsinu L, vendar se, za razliko od katepsina L, ki je v telesu povsod izražen, katepsin V v fizioloških pogojih nahaja le v priželjcu, testisih in epiteliju roženice, pri patoloških procesih pa se izraža tudi v drugih tkivih. Katepsin V je udeležen pri napredovanju raka, pri tem se povečano izraža v tumorjih kolorektalnega raka, raka rektuma in dojk, medtem ko v zdravem črevesu in mlečnih žlezah ni prisoten. Poleg tega ima katepsin V pomembno vlogo v imunskih celicah in protitumornem imunskem odzivu, kjer je pomemben pri aktivaciji cistatina F, ki zavira delovanje citotoksičnih celic. Zaradi udeleženosti pri napredovanju raka, regulacija katepsina V s selektivnimi zaviralci odpira možnosti za razvoj novih terapevtskih pristopov in izboljšanje učinkovitosti imunoterapije raka. V okviru magistrske naloge smo zato želeli identificirati potencialne reverzibilne, selektivne in obenem ne toksične zaviralce katepsina V. V ta namen smo testirali spojine, pridobljene iz virtualnih kemijskih knjižnic, ki so bile izbrane na podlagi molekulskega sidranja v aktivno mesto katepsina V. Njihovo sposobnost zaviranja aktivnosti katepsina V in selektivnost za katepsin V v primerjavi s katepsinom L, smo ovrednotili z metodo encimske kinetike in mikrotermoforeze. Pri tem smo ugotovili, da so najmočnejši zaviralci katepsina V spojine 7, 26 in 28. Spojini 7 in 28 sta selektivna zaviralca katepsina V, spojina 26 pa ni selektivna. Spojinam, smo v nadaljevanju določili vpliv na viabilnost celic in določili koncentracije, pri katerih nimajo toksičnih učinkov na celice. Izkazalo se je, da nobena izmed spojin 7, 26 in 28 ni citotoksična pri koncentracijah manjših od 5 µM. Dodatno smo inhibicijo katepsina V in selektivnost za katepsin V z encimsko kinetiko določili še za spojine, ki so bile iz knjižnic komercialno dostopnih spojin izbrane na podlagi strukturnih lastnosti spojin. Med njimi sta se, na podlagi konstante inhibicije, kot najmočnejša in najbolj selektivna zaviralca katepsina V izkazali spojini 43 in 45. V okviru našega dela smo kot najmočnejši zaviralec katepsina V identificirali piperidin karboksilatni derivat sečnine, spojino 7, ki izkazuje velik potencial za nadaljnji razvoj reverzibilnih, selektivnih in ne toksičnih zaviralcev katepsina V, ki bi se lahko uporabljali kot protitumorne učinkovine.Cathepsin V is a lysosomal cysteine peptidase, a member of the papain-like cysteine peptidase family. It is closely related to cathepsin Lhowever, like cathepsin L it is not ubiquitously expressed while its physiological expression is manly limited to thymus, testis, and corneal epithelium. In pathological processes, it is expressed also in other tissues. It contributes to cancer progression, where its increased expression was observed in colorectal and breast carcinomas, but not in the normal colon or mammary tissue. In addition, cathepsin V plays an important role in immune cells and in antitumor immune response, where it is involved in the activation of cystatin F, which impairs the function of cytotoxic cells. Due to its involvement in cancer progression, targeting this enzyme with selective inhibitors may open new possibilities for improvements in cancer immunotherapy. For this purpose, we tested compounds obtained from virtual chemical libraries, by their molecular docking into the active site of cathepsin V. Their inhibition of cathepsin V activity and selectivity for cathepsin V compared to cathepsin L was evaluated by using enzyme kinetics and microscale thermophoresis. Compounds 7, 26, and 28 were identified as the most potent inhibitors of cathepsin V. Compounds 7 and 28 are selective inhibitors of cathepsin V while compound 26 is a non-selective inhibitor. Best performing compounds were further tested for their effect on cell viability to determine non-cytotoxic compound concentrations. Compounds 7, 26, and 28 did not have cytotoxic effect on cells at concentrations lower than 5 μM. Moreover, additional compounds were selected form commercially available compound libraries based on the structural properties and evaluated for inhibition and selectivity for cathepsin V using enzyme kinetics. Among them, compounds 43 and 45 were the strongest and most selective inhibitors of cathepsin V. In our work, as the most potent cathepsin V inhibitor, we identified the piperidine carboxylate derivative, compound 7. This compound has great potential for further development of reversible, selective, and non-toxic cathepsin V inhibitors that could be used as possible antitumor agents

New insights into the role of cysteine cathepsins in neuroinflammation

Neuroinflammation, which is mediated by microglia and astrocytes, is associated with the progression of neurodegenerative diseases. Increasing evidence shows that activated microglia induce the expression and secretion of various lysosomal cathepsins, particularly during the early stage of neuroinflammation. This trigger signaling cascade that aggravate neurodegeneration. To date, most research on neuroinflammation has focused on the role of cysteine cathepsins, the largest cathepsin family. Cysteine cathepsins are primarily responsible for protein degradation in lysosomeshowever, they also play a role in regulating a number of other important physiological and pathological processes. This review focuses on the functional roles of cysteine cathepsins in the central nervous system during neuroinflammation, with an emphasis on their roles in the polarization of microglia and neuroinflammation signaling, which in turn causes neuronal death and thus neurodegeneration

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy