Biochemical evaluation of covalent MurA inhibitors\u27 mechanism of action

UDP-N-acetilglukozamin enolpiruvil transferaza(MurA)je znotrajcelični encim, ki je udeležen v prvi stopnji sinteze peptidoglikana, bistvenega gradnika celične stene bakterij. Encima ne najdemo v človeškemu telesu, zato predstavlja zanimivo in tudi že validirano tarčo za zdravljenje bakterijskih okužb. Protibakterijska učinkovina fosfomicin je trenutno edini znani zaviralec MurA, ki se uporablja v klinični praksi. Kot pri mnogih drugih protibakterijskih učinkovinah se je tudi pri fosfomicinu že razvila bakterijska odpornost, zato potekajo prizadevanja za odkritje novih zaviralcev, ki bi z delovanjem na znotrajcelično stopnjo sinteze peptidoglikana zavrli nastanek bakterijske celične stene in tako delovali baktericidno. V sklopu magistrske naloge smo z različnimi biokemijskimi preskušanji ovrednotili okoli 100 spojin. Spojine smo pridobili iz različnih virov. V večini primerov je šlo za majhne elektrofilne fragmente, za katere smo pri preskušanjih želeli ugotoviti ali uspešno zavirajo encim MurA in ali se na encim vežejo kovalentno. Ključna ugotovitev naloge je bila določitev spojin primernih za nadaljnje raziskovanje in optimizacijo do morebitnih protibakterijskih učinkovin. Rezidualno aktivnost pod 50 % smo določili okoli 70 spojinam. Tem smo z dodatnimi testi določili tudi vrednosti IC50. Glede na vrednost IC50 je najmočnejši zaviralec spojina 51, 4-nitrobenzen-1-tiol, z vrednostjo 0,125 ?M. Ireverzibilen način zaviranja smo določali s testom časovne odvisnosti vezave in redčenja. Ugotovili smo, da ireverzibilen način vezave nakazuje 14 spojin. Pri delu vrednotenih spojin (29–52) smo želeli določiti tudi kinetične parametre značilne za ireverzibilen tip zaviranja. Zaradi velike reaktivnosti spojin nam to ni uspelo. Pri spojinah, ki niso nakazovale ireverzibilnega delovanja smo preko reverzibilne encimske kinetike s hitrostjo reakcij ugotavljali vrsto vezave. Pri spojini 36 smo določili kompetitivni reverzibilni način vezave na encim. Spojin 53–101 nismo kinetično vrednotili. Določili pa smo, da so med temi za nadaljnja preskušanja ireverzibilnosti najprimernejše spojine 58, 59, 60, 64, 85 in 90.UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an enzyme that participates in the first committed step in the synthesis of the essential bacterial cell wall building block – peptidoglycan. Because of its absence in human body, the enzyme represents an interesting and already validated target for antibacterial agents. The antibacterial agent fosfomycin is currently the only known MurA inhibitor used in clinical practice. As with many antibacterials, the bacterial resistance to fosfomycin is already present. This only highlights the importance for the discovery of new bactericidal inhibitors acting on the intercellular stages of peptidoglycan synthesis. In this master thesis, around 100 compounds with different biochemical assays were evaluated. Compounds were acquired from various sources. Most of them were small electrophilic fragments, which were tested for their ability to inhibit MurA enzyme and whether their action of inhibition was covalent. The main aim of this master thesis was the determination of suitable compounds for further investigation and optimization into potential antibacterial agents. Residual activity lower than 50% was determined for around 70 compounds. Those compounds were eligible for determination of IC50 value. The most potent compound according to IC50 value is compound 51, 4-nitrobenzene-1-thiol with IC50 value of 0,125 ?M. The irreversible mode of action was tested through time dependency and dilution assay. 14 compounds showed potential irreversible inhibition mode. We evaluated part of the compounds (29 – 52) with kinetic parameters characteristic for irreversible inhibitors. The evaluation was unsuccessful due to high reactivity of the compounds. Fragments that did not indicate any features of irreversible action, the mode of binding to enzyme was determined with reversible enzyme kinetics and reaction rates. Compound 36 indicated competitive reversible binding mode. Compounds 53 – 101 were not evaluated with kinetic constants, but we still managed to determine compounds 58, 59, 60, 64, 85 and 90 as most promising for further investigation of irreversible binding mode