Sinteza in vrednotenje 4-izopropil-1,2,4-triazolnih zaviralcev katepsina X

Učakar, Tjaša (author), Hrast, Martina (mentor), Knez, Damijan (comentor)

Katepsin X je ena najkasneje odkritih cisteinskih peptidaz, ki se od ostalih katepsinskih proteaz razlikuje v svoji izključno eksopeptidazni aktivnosti, za katero so odgovorne njene strukturne lastnosti. Najdemo ga v celicah imunskega sistema in v celicah živčevja, njegova aktivnost, ki večinoma temelji na cepitvi C-končnih aminokislinskih ostankov integrinskih receptorjev na celicah imunskega sistema, pa ima pomembno vlogo pri adheziji in migraciji celic. Njegovo delovanje je vpleteno v razvoj številnih bolezni, med najpomembnejšimi pa je razširjanje raka v fazi metastaziranja in razvoj nevrodegenerativnih bolezni. Zaviralci tega encima predstavljajo dobro osnovo za razvoj protitumorskih učinkovin. V predhodnih raziskavah so odkrili zelo učinkovit zaviralec katepsina L-trans-epoksisukcinil-L-levcilamido(4-gvanidino)butan (E-64), ki pa bi zaradi ireverzibilne narave vezave na encim in druge nespecifične tarče v organizmu lahko povzročal številne neželene učinke. Razvoj potencialnih učinkovin gre zato v smeri reverzibilnih zaviralcev. Za spojino z najboljšo zaviralno aktivnostjo se je zaenkrat izkazala spojina Z9. Glede na molekulsko modeliranje in predhodna testiranja spojin je na mestu predpostavka, da je triazolni obroč ključen za vezavo in delovanje encima, z modifikacijo benzodioksanskega dela pa bi lahko učinkovitost še izboljšali. Sintetizirali smo spojine z acetofenonskimi in acetilindolnimi fragmenti, pripravili pa smo tudi spojino, ki ob triazolnem in acetilnem delu molekule vsebuje preprost aliciklični strukturni element. Z reakcijami, ki so v večini primerov potekale v dveh stopnjah, smo sintetizirali 19 spojin, ki smo jih biokemijsko testirali tako, da smo ugotavljali rezidualno aktivnost katepsina X po vezavi potencialnega inhibitorja nanj, ki smo jih redčili do koncentracije 50 µM. Štiri spojine so zavrle encim v enakem koncentracijskem območju kot spojina Z9. Gre za spojine 13, 14, 17 in 29, katerim je skupen heteroatom, sposoben tvorbe vodikove vezi ter hidrofobni del, ki bi s hidrofobnimi interakcijami v hidrofobnem S2 žepu lahko pomenile pomembno strukturno lastnost za uspešno vezavo na encim. Najšibkeje so encim zavirale spojine, ki imajo na fenonski obroč pripete substituente z izrazito polarnimi lastnostmi.

• Language: Slovenian
• Keywords: katepsin X, triazolni zaviralci, protitumorske učinkovine
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-113296
• Publication date in RUL: 19.12.2019

Secondary language

• Language: English
• Title: Synthesis and evaluation of 4-isopropyl-1,2,4-triazole cathepsin X inhibitors

Abstract

Cathepsin X is one of the latest discovered cysteine peptidases, which differs from others in its exclusively exopeptidase acitivity, caused by specific structural characteristics. It is mostly present in immune cells and neuronal system. Its enzymes activity is based on the ability of cleaving C-terminal amino acid residues of integrine receptors in cells of immune system. This mechanism plays an important role in cell adhesion and migration. Cathepsin X is involved in evolution of many diseases but its role is mostly explored in progress of cancer and neurodegenerative diseases. Inhibitors of cathepsin X have very good potential of becoming an effective way of treating tumors. In previous research, a very effective inhibitor L-trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was identified. Due to its irreversible binding into the cathepsin X active site, and due to unspecific binding with other proteins, it can cause side effects. Therefore, the development of new inhibitors is focused on discovering the substances that bind to the enzyme in a specific and reversible manner. Uptill now, compound Z9 is the most effective compound synthetized and evaluated on isolated cathepsin X and in cell-based assays. Based on the results of molecular modelling and previous assays, the triazole seems essential for the inhibitor binding and inhibiting activity. On the other hand, modifications of benzodioxane fragment of compound might improve the binding properties. We synthetized compounds with acetophenone and acetoindole fragments. Additionally, one compound with a simplistic alicyclic fragment instead of aromatic cycle was also prepared. With reactions that consist of two steps, we prepared 19 compounds that were biochemically assayed. In those tests we measured residual activities of the enzyme after binding of potentional inhibitors at 50 µM concentration. .Four compounds demonstrated residual activities in the same concentration range as Z9, namely 13, 14, 17 and 29. Their structure consists of a heteroatom, that could bind into the active site with hydrogen bonds, and a hydrophobic fragment that might bind with hydrophobic interactions to S2 pocket. The highest residual activities was measured for compounds containing polar substituents on aromatic ring.

• Keywords: cathepsin X, triazole inhibitors, antitumor compounds