7-Substituirani derivati nitroksolina kot potencialni inhibitorji katepsina B

magistrsko delo

Jana Čimžar (Avtor), Bogdan Štefane (Mentor), Jurij Lah (Član komisije za zagovor), Jurij Svete (Član komisije za zagovor)

Povzetek

V magistrskem delu smo proučevali nove inhibitorje katepsina B. Delo je sestavljeno iz načrtovanja, sinteze in testiranja prej omenjenih inhibitorjev. Encim katepsin B ima eksopeptidazno in endopeptidazno aktivnost. Prva opravlja pomembne fiziološke funkcije, z drugo pa povezujejo patološka stanja. V zadnjih študijah se je kot inhibitor endopeptidazne aktivnosti katepsina B izkazala uveljavljena zdravilna učinkovina nitroksolin. V povezavi z novejšimi raziskavami so odkrili izboljšan nitroksolinski inhibitor katepsina B. Izboljšan inhibitor ima elektrofilno stransko verigo na mestu 7 nitroksolina. V magistrskem delu smo z molekulskim sidranjem s programskim paketom Schrödinger poiskali nove 7- in/ali 8-substituirane derivate nitroksolina, ki bi lahko inhibitorno delovali na endopeptidazno aktivnost katepsina B. Načrtovani inhibitorji so imeli večinoma v strukturi molekule vključeno rahlo elektrofilno mesto, s katerim bi se v aktivnem mestu encima lahko kovalentno povezali z aminokislinskim ostankom Cys29. Rezultate molekulskega sidranja smo razvrščali na podlagi vrednosti Glide Score in MM-GBSA dG Bind. Rezultate smo primerjali z rezultati referenčnih spojin, torej nitroksolina in izboljšanega 7-substituiranega nitroksolinskega inhibitorja. V nadaljevanju smo sintetizirali in okarakterizirali 13 spojin, ki so v okviru molekulskega sidranja pokazale najobetavnejše rezultate. Večino spojin smo sintetizirali s Suzuki-Miyaura reakcijo pripajanja, pri kateri smo kot reagente uporabili trifluoroborate ali estre borovih kislin. Na koncu smo načrtovane inhibitorne lastnosti sintetiziranih spojin preverili z encimskimi testi. Z merjenjem fluorescence smo opazovali kinetiko encimske pretvorbe ustreznega substrata. Potencialne inhibitorne lastnosti spojin v povezavi z endopeptidazno aktivnostjo človeškega katepsina B smo najprej opazovali s preliminarnimi testi. Zatem smo s titracijskimi krivuljami s programom GraphPad Prism spojinam določili normalizirane IC50 vrednosti. IC50 Vrednost smo določili tudi referenčni spojini nitroksolin. Sedem spojin je imelo bistveno nižje IC50 vrednosti od IC50 vrednosti referenčnega nitroksolina. V magistrskem delu smo izvedli tudi preliminarne teste inhibicije endopeptidazne aktivnosti človeškega katepsina L, da smo preverili selektivnost sintetiziranih potencialnih inhibitorjev.

Ključne besede

nitroksolin;protirakavo delovanje;katepsin B;molekulsko sidranje;Suzuki-Miyaura reakcija pripajanja;encimski testi;encimska kinetika;magistrska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2020
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[J. Čimžar]
UDK:	547.831:577.15(043.2)
COBISS:	27013379
Št. ogledov:	510
Št. prenosov:	172
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	7-Substituted nitroxoline derivatives as potential inhibitors of cathepsin B
Sekundarni povzetek:	In the master’s thesis, we have been developing new cathepsin B inhibitors. The work consisted of design, synthesis and testing of beforementioned inhibitors. Enzyme cathepsin B has exopeptidase and endopeptidase activity. The first one is involved in important physiological functions, whereas the second is connected to the pathological conditions. In recent studies, an established active pharmaceutical ingredient nitroxoline has been shown as the inhibitor of cathepsin’s B endopeptidase activity. An overview of recent research shows on discovery of improved nitroxoline inhibitor of cathepsin B. This inhibitor has an electrophilic side chain in the position 7 of nitroxoline. In the master’s thesis, we have been searching for new 7- and/or 8-substituted nitroxoline’s derivatives, which could inhibit cathepsin’s B endopeptidase activity. The new compounds were designed, using molecular docking Schrödinger program package in the initial phase of development. Designed inhibitors mostly included in the structure of the molecule mildly electrophilic part, which could covalently bind to the amino acid residue Cys29 in the active site of the enzyme. The results of molecular docking were ranked by the values of Glide Score and MM-GBSA dG Bind. The results have been compared to the results of reference compounds, i.e. nitroxoline and improved nitroxoline’s inhibitor. In the second phase of the development, 13 compounds with most promised results were synthesised and characterised. Most of the compounds have been synthesised through the Suzuki-Miyaura coupling reaction, where trifluoroborates and boronate esters have been used as reagents. In the end, the designed inhibitory properties of synthesised compounds have been tested with the enzyme tests. We have been observing the kinetics of the enzyme conversion of an appropriate substrate by measuring fluorecence. Firstly, the potent inhibitory properties in terms of human cathepsin’s B endopeptidase activity have been observed through preliminary tests. Afterwards, the normalised IC50 values of compounds have been determined with titration curves using the GraphPad Prism program. The IC50 value of the reference compound nitroxoline has also been determined. The IC50 values of seven compounds were much lower than the IC50 value of the reference nitroxoline. In the master’s thesis also preliminary tests of human cathepsin’s L endopeptidase activity inhibition have been performed to check the selectivity of synthesised potent inhibitors.
Sekundarne ključne besede:	cathepsin B;nitroxoline;molecular docking;Suzuki-Miyaura cross-coupling reactions;enzyme tests;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	1000375
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Kemija
Strani:	111 str.
ID:	12003874