benzothiophene and steroidal derivatives of aminoquinoline

Botulinum neurotoksini su najjaĉi poznati prirodni otrovi i izazivaĉi botulizma – potencijalno smrtonosne neuroparalitiĉke bolesti. U poslednje vreme, sve veći broj studija je usmeren ka pronalaţenju inhibitora botulinum neurotoksina serotipa A (BoNT/A) aktivnih unutar ćelije, jer terapija antitelima ima uspeha jedino pre nego što toksin uĊe u neuron. U okviru ove doktorske disertacije izvršena je sinteza i detaljno ispitivanje inhibitorne aktivnosti novih steroidnih i benzo[b]tiofenskih derivata 4-aminohinolina prema kratkom nizu (BoNT/A LC) i holotoksinu BoNT/A. U istraţivanju je korišćen proteolitiĉki in vitro esej i ćelijski esej u motornim neuronima razvijenim iz embrionalnih matiĉnih ćelija miša (mES-MN). Dodatno, molekulsko modelovanje i uklapanje novih derivata u aktivno mesto enzima izvršeno je korišćenjem programa Schr dinger Suite 2016-4. U in vitro proteolitiĉkom eseju, sintetisana jedinjenja su ostvarila do 85% inhibicije BoNT/A LC pri koncentraciji 20 μM, dok su IC50 vrednosti bile u opsegu 0,7– 10,2 μM. U preintoksikacionom modelu u motornim neuronima razvijenim iz embrionalnih matiĉnih ćelija miša (mES-MN) novi derivati su vršili zaštitu proteina SNAP-25i do 88%, u niskim mikromolarnim koncentracijama i u dozno-zavisnom reţimu. Najaktivniji derivati su testirani u postintoksikacionom modelu, u kome se jedinjenja dodaju ćelijskoj kulturi 30 ili 60 minuta posle holotoksina. U oba modela je uoĉena korelacija procenta zaštite SNAP-25 i primenjene koncentracije jedinjenja. Jedinjenje 17 (JK141) je pokazalo 99% zaštite SNAP-25 kada se administrira 30 minuta posle BoNT/A...Botulinum neurotoxins are the most poisonous (biological) substances known and causative agents of botulism – serious and potentially fatal neuroparalytic illness. Recently, the majority of efforts have focused on identification of botulinum neurotoxin serotype A (BoNT/A) inhibitors with intracellular activity, because antibody-based treatments are successful only before toxin enters a neuron. In this doctoral dissertation synthesis and detailed evaluation of inhibitory potencies of new steroidal and benzo[b]thiophene 4-aminoquinoline derivatives against BoNT/A light chain (LC) and full length BoNT/A is reported. Both in vitro proteolytic assay and cell-based assay using mouse embryonic stem cell derived motor neurons (mES-MNs) were employed. To rationalize the inhibitory potencies of the new derivatives, structure-based docking simulations were performed using Schr dinger Suite 2016-4 and the modules therein. Using in vitro HPLC-based assay, the newly synthesized molecules have shown BoNT/A LC inhibition up to 85% at 20 μM and IC50 values ranging from 0.7–10.2 μM. Compounds tested during BoNT/A challenge in mES-MNs in preintoxication model were found to protect SNAP-25 proteinii by up to 88% at low μM concentrations and in dose-dependent manner. The most effective derivatives were also tested in a postexposure model, where compounds were added 30 or 60 minutes following holotoxin administration. In both pre- and postintoxication models, dose-dependent behavior was observed. Compound 17 (JK141) showed 99% of SNAP-25 cleavage protection when administrated 30 minutes after BoNT/A..