Vrednotenje substituiranih psoralenov kot zaviralcev LMP7 podenote imunoprotesoma

Rančnik, Pia (author), Gobec, Stanislav (mentor), Gobec, Martina (co-mentor)

Proteasom je multikatalitični kompleks, ki homeostazo celice vzdržuje preko odstranjevanja nepravilno zvitih in poškodovanih proteinov ter drugih signalnih molekul, ki nastanejo tekom različnih celičnih procesov. Preoteasom obstaja v več oblikah, a sta med pomembnejšimi standardni in imunoproteasom. Slednji ima pomembno vlogo predvsem v oblikovanju imunskega odziva. Povezujejo ga s številnimi patološkimi stanji (avtoimunskimi, rakavimi in nevrodegenerativnimi obolenji), zato s svojimi katalitičnimi podenotami (MECL-1, LMP2 in LMP7) predstavlja vedno bolj zanimivo farmakološko tarčo. Tekom magistrske naloge smo izvedli 5-stopenjsko sintezo potencialnega zaviralca LMP7 podenote i-proteasoma ter ga skupaj s še 47 drugimi, predhodno sintetiziranimi analogi psoralenskega skeleta biokemijsko ovrednotili. Najprej smo s Pechmannovo kondenzacijo med rezorcinolom in dietil-2-acetilsukcinatom sintetizirali osnovni kumarinski skelet. Nato smo na mestu 7-kumarinskega skeleta hidroksilni skupini z reakcijo nukleofilne substitucije pripeli 2-bromoacetofenon in dobili predhodnico psoralenskega skeleta, katero smo z reakcijo kondenzacije in posledično ciklizacijo pretvorili do slednjega. V zadnjih dveh stopnjah smo aktivirano karboksilno skupino pretvorili v amidno skupino, kamor smo vezali klorokarbonilsulfenil klorid in dobili končno spojino z 1,3,4-oksatiazol-2-onom kot elektrofilom. V sklopu biokemijskega vrednotenja smo sprva pri 10 μM koncentracijah spojin določili rezidualno aktivnost LMP7 katalitične podenote i-proteasoma. Na podlagi rezidualne aktivnosti smo izbrali 8 spojin z najmočnejšimi zaviralnimi lastnostmi in jih podrobneje ovrednotili s parametroma encimske kinetike Ki in IC50. Za vse spojine so bile vrednosti v nizko mikormolarnem območju. Ugotovili smo, da je za zaviralno aktivnost i-proteasoma v terminalnem delu psoralenskega skeleta pomemben sukcimidni ester ali oksatiazolon za ireverzibilne zaviralce in karboksilna skupina za reverzibilne, čeprav slednja ni izkazala močnejših zaviralnih sposobnosti. Med funkcionalnih skupinami na furanskem obroču psoralenskega skeleta so najmočneje zavirale aromatske spojine in sicer spojine, ki imajo 2,5-dimetiltiofenski, furanski in nesubstituiran benzenov obroč. Dobljeni podatki RANČNIK, Pia: Vrednotenje substituiranih psoralenov kot zaviralcev LMP7 podenote imunoproteasoma. predstavljajo dobro osnovo za nadaljnje načrtovanje in sintezo zaviralcev LMP7 podenote i-proteasoma, ki bodo imeli izboljšane zaviralne lastnosti.

• Language: Slovenian
• Keywords: proteasom proteini zaviralci imunoproteasoma psoralen sintezni postopki
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [P. Rančnik]
• Year: 2016
• Number of pages: IV, 54 f.
• PID: 20.500.12556/RUL-81508
• UDC: 543:616-097(043.3)
• COBISS.SI-ID: 4045169
• Publication date in RUL: 11.04.2016

Secondary language

• Language: English
• Title: Evaluation of substituted psoralenes as inhibitors of the LMP7 subunit of immunoproteasome

Abstract

The most significant function of proteasome is to maintain proteins homeostasis with removal of misfolded and damaged proteins, which occurs in different cellular processes. His subtype, immunoproteasome, is preferentially formed under impact of immunomodulatory cytokines. Antigene presentation to cytotoxic T lymphocytes, their proliferation and differation as well as regulation of cytokines production and other immune functions of immunoproteasome with his catalytic subunits represent more and more interesting pharmacological target. Its involvment in disease progression is linked with specific autoimmune, malignant and neurodegenerative diseases. Within this masters degree we successfully synthesized a potential inhibitor of the LMP7 subunit of the immunoproteasome in 5 reaction steps. The biochemical evaluation was completed with this and also other 47 psoralen analogues that have previously been synthesized. Starting with Pechamann condensation, reaction of diethyl acetosuccinate with resorcinol was carried out, yielding 7-hydroxycoumarin ring. The precursor of psoralen skeleton was synthesized by nucleophile substitution where hidroxyl group was replaced by 2- bromoacetophenone. Afterwards, cyclisation to form psoralen skeleton has been achieved by condensation. In the last two steps carboxyl group was modified to amide group and chlorocarbonylsulfenyl chloride was added to obtain the final compound with 1,3,4- oxathiazolon as an electrophile. Determination of residual activity was carried out. 8 compounds with good inhibitory properties were identified. For these compounds parameters of enzyme kinetics (Ki in IC50) were examined in detail. The key structural requirements significant for the immunoproteasome inhibition of the psoralen analogues are succinimide ester or oxathiazolone which are essential for irreversibile inhibitors and carboxylic acid for reversibile inhibitors, however the last one does not show strong inhibitory properties. Among the functional groups on the furane on the psoralen skeleton, the aromatic compounds such as 2,5-dimethyl tiofen, furan and unsubstituted phenyl group showed the strongest inhibitory properties. These and other compounds based on psoralen skeleton represent a topic of further structural investigations and improvement of inhibition of the immunoproteasome subunit LMP7.

• Keywords: proteasome proteins inhibitors of immunoproteasome psoralen