Sinteza kinoksalinskih in kinolinskih kovalentnih zaviralcev kaspaze-1

Fricelj, Tanja (author), Knez, Damijan (mentor), Meden, Anže (comentor)

Staranje, poškodbe, okužbe in bolezni so dejavniki, odgovorni za sprožitev nevrovnetja, ki ima ključno vlogo pri razvoju nevrodegenerativnih bolezni, kot sta Alzheimerjeva in Parkinsonova bolezen. V normalnih, fizioloških pogojih mikroglija in astrociti ščitijo nevrone, vendar pa lahko ob aktivaciji zaradi različnih dejavnikov sproščajo več provnetnih molekul – citokinov in reaktivnih kisikovih zvrsti, kar pripelje do poškodbe nevronov. V tem procesu so pomembni tudi vnetni proteinski kompleksi, inflamasomi, ki aktivirajo proteolitične encime, imenovane kaspaze. Kaspaze, razdeljene na vnetne in apoptotične, so ključne za sprožitev vnetnih odzivov in celično smrt. Kaspaza-1, ki je ena izmed najbolj raziskanih kaspaz, sproži vnetje z aktivacijo citokinov IL-1β in IL-18 ter cepitvijo gasdermina D, kar vodi v programirano celično smrt (piroptoza). Zaradi vloge kaspaze-1 pri vnetnih in apoptotičnih procesih so zaviralci kaspaze-1 potencialno uporabni v terapiji nevrodegenerativnih bolezni. V okviru magistrske naloge smo sintetizirali analoge literaturno opisanih reverzibilnih zaviralcev kaspaze-1, pri katerih se v S4 vezavni žep vežejo aromatski fragmenti, kot sta kinoksalin ter kinolin. Na njihovo ogrodje smo preko distančnikov pritrdili različne elektrofilne bojne glave in preverili učinkovitost zaviranja encimske aktivnosti človeške kaspaze-1. Pri sintezi smo najprej na aromatsko jedro pripeli distančnik, v večini primerov γ-aminobutanojsko kislino, nato pa še 2-aminoetil-N-metilamin. Na pripravljene aminske derivate smo nato v reakcijah tvorbe amidne vezi, N-alkiliranja ter N-aciliranja pripeli različne elektrofilne bojne glave. Po potrditvi identitete in čistosti končnih derivatov smo za 18 analogov preverili sposobnost zaviranja encimske aktivnost človeške kaspaze-1 v biokemijskem testu z uporabo fluorogenega substrata. Kaspazo-1 je bolj ali manj učinkovito zaviralo pet derivatov, najmočnejša zaviralca sta bila karbamoil fluorid 25 (rezidualna aktivnost pri 100 µM = 16,9 %) ter vinilsulfonamid 34 (rezidualna aktivnost pri 100 µM = 2,6 %). Na odsotnost zaviralne aktivnosti večine pripravljenih analogov po vsej verjetnosti vplivata dolžina distančnika med elektrofilom in aromatskim delom molekule ter dejstvo, da pri molekulah ni prisotne ustrezne funkcionalne skupine za tvorbo interakcij z vezavnim žepom S1, ki bi elektrofilni center postavila v bližino katalitičnega Cys285.

• Language: Slovenian
• Keywords: Nevrovnetje, inflamasom, kaspaza-1, kovalentni zaviralci, kinolin, kinoksalin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-164883
• Publication date in RUL: 15.11.2024

Secondary language

• Language: English
• Title: Synthesis of quinoxaline- and quinoline-based covalent caspase-1 inhibitors

Abstract

Aging, injuries, infections, and diseases are factors responsible for triggering neuroinflammation, which plays a crucial role in the development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Under normal physiological conditions, microglia and astrocytes protect neurons, but when activated by various factors, they can increase the release of pro-inflammatory molecules – cytokines and reactive oxygen species, leading to neuronal damage. In this process, inflammatory protein complexes called inflammasomes, which activate proteolytic enzymes known as caspases, are also important. Caspases, divided into inflammatory and apoptotic, are of key importance for triggering inflammatory responses and cell death. Caspase-1, one of the most studied caspases, triggers inflammation by activating cytokines IL-1β and IL-18 and cleaving gasdermin D, leading to a form of cell death called pyroptosis. Due to the importance of caspase-1 in inflammatory and apoptotic processes, there is potential for the use of caspase-1 inhibitors in the therapy of neurodegenerative diseases. We synthesized analogues of literature-described reversible caspase-1 inhibitors, where aromatic fragments such as quinoxaline and quinoline bind to the S4 binding pocket. Various electrophilic warheads were attached to the aromatic scaffold via linkers, and their inhibition of human caspase-1’s was evaluated. Linkers, in most cases γ-aminobutyric acid combined with aminoethyl-N-methylamine, were firstly attached to the aromatic core. Various electrophilic warheads were then added onto the amine derivatives through amide bond formation, N-alkylation, and N-acylation. After confirming the identity and purity of the final compounds, the inhibitory potencies of 18 analogues against human caspase-1 were determined in a biochemical assay using a fluorogenic substrate. Five derivatives inhibited caspase-1, the most potent were carbamoyl fluoride 25 (residual activity at 100 µM = 16.9%) and vinyl sulfonamide 34 (residual activity at 100 µM = 2.6%). The lack of inhibition for the majority of synthesized compounds can be explained by the distance of the linker connecting the electrophile and aromatic part of the molecule, and the absence of an appropriate functional group for interactions with the S1 binding pocket, which would position the electrophilic centre near the catalytic Cys285.

• Keywords: Neuroinflammation, inflammasome, caspase-1, covalent inhibitors, quinoline, quinoxaline