Determination of the pharmacophore structure, design, synthesis and in vitro evaluation of dual inhibitors of histone deacetylases (HDAC) and Rho-associated protein kinases (ROCK)

[M. Beljkaš]

242 str.

Farmacija - Farmaceutsko-medicinska hemija i strukturna analiza / Pharmacy - Pharmaceutical-medicinal chemistry and structural analysis

Duktalni adenokarcinom pankreasa (pancreatic ductal adenocarcinoma, PDAC) i trostruko-negativni karcinom dojke (triple-negative breast cancer, TNBC) spadaju u tumore sa najlošijom prognozom što naglašava hitnu potrebu za novim terapijskim strategijama u cilju poboljšanja terapijskog ishoda. Epigenetički i kinazni signalni putevi imaju bitnu ulogu u tumorogenezi i progresiji ovih maligniteta. U tom kontekstu, histon-deacetilaze (histone deacetylases, HDAC) i Rho-vezane protein kinaze (Rho-associated coiled-coil kinases, ROCK) se ističu kao relevantni targeti u tretmanu različitih vrsta karcinoma, uključujući TNBC i PDAC. S obzirom na prednosti multitarget terapijskih pristupa, zabilježene sinergističke efekte između HDAC i ROCK inhibitora u in vitro i in silico studijama, kao i njihov značaj u patogenezi ovih tumora, dizajn i razvoj dualnih HDAC/ROCK inhibitora predstavlja inovativnu i obećavajuću strategiju za liječenje ovih agresivnih maligniteta. U ovoj doktorskoj disertaciji istraživanje je sprovedeno u četiri faze. Prvo, primjenom kvantitativnih studija odnosa strukture i aktivnosti (Quantitative structure-activity relationship, QSAR) i molekulskog dokinga, razvijena je farmakofora ROCK1/ROCK2 inhibitora, na osnovu koje su dizajnirani novi ROCK inhibitori. Identifikovan je region fasudila, poznatog ROCK inhibitora, koji nije esencijalan za interakcije sa ROCK enzimima, što je omogućilo njegovu strukturnu modifikaciju i integraciju HDAC farmakofore. Na ovaj način su po prvi put dizajnirani dualni HDAC/ROCK inhibitori. U drugoj fazi istraživanja, dizajnirani ROCK i HDAC/ROCK inhibitori su sintetisani, nakon čega je u trećem segmentu istraživanja ispitana njihova enzimska aktivnost, kao i antitumorska svojstva u dvodimenzionalnim i trodimenzionalnim ćelijskim kulturama TNBC (MDA-MB-231, MDA-MB-468) i PDAC (MiaPaCa-2, Panc-1). Finalno, na osnovu svih prethodnih rezultata, u četvrtoj fazi je uspostavljen detaljan in silico protokol za budući dizajn i optimizaciju strukture dualnih HDAC/ROCK inhibitora. Dati protokol se zasniva na kombinaciji metoda dizajna zasnovanog na strukturi ciljnog mjesta dejstva (Structure-based drug desing, SBDD) i dizajna zasnovanog na strukturi liganda (Ligand-based drug design, LBDD) koje uključuju molekulski doking, 2D-QSAR modele formirane koriščenjem algoritama mašinskog učenja i računanje slobodne energije vezivanja MM-GBSA metodom. Među dizajniranim ROCK inhibitorima, jedinjenje C-19 se izdvojilo po svojoj inhibitornoj aktivnosti na ROCK1 (IC50 = 4,22 μM) i ROCK2 (IC50= 6,13 μM), kao i po citotoksičnom efektu na MiaPaCa-2 i Panc-1 ćelijama (IC50= 19,10 μM i 35,03 μM). Takođe, C-19 je ispoljilo anti-migratorna i anti-invazivna svojstva, uporediva sa fasudilom. Najperspektivnije među sintetisanim HDAC/ROCK dualnim inhibitorima je jedinjenje C-9, koje je pokazalo snažnu inhibiciju ROCK1 (IC50 = 800 nM), ROCK2 (IC50 = 700 nM) i HDAC6 (IC50 = 185 nM). U ispitivanjima citotoksičnosti, C-9 je pokazalo značajno bolju aktivnost od poznatih inhibitora fasudila i tubastatina A, sa IC50 vrijednostima od 5,81 μM za MDA-MB-231, 3,87 μM za MiaPaCa-2 i 19,57 μM za Panc-1. Pored toga, C-9 je demonstrirao visok stepen efikasnosti u 3D tumorskim sferoidnim modelima, inhibirajući formiranje i vijabilnost Panc-1 i MiaPaCa-2 sferoida. Takođe, C-9 je nadmašilo fasudil u smanjenju migracije i invazije MDA-MB-231 i MiaPaCa-2 ćelija, što ga definiše kao obećavajućeg kandidata za dalju prekliničku evaluaciju u tretmanu metastatskih karcinoma. Na osnovu ovih rezultata, C-9 je identifikovan kao vodeći molekul (lead) i njegova struktura je dalje optimizovana u skladu sa prethodno definisanom ROCK farmakoforom, što je dovelo do dizajniranja i sinteze novih HDAC/ROCK dualnih inhibitora. Primjenom metoda SBDD razvijena je serija novih dualnih HDAC/ROCK inhibitora...

Pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC) are among the most aggressive malignancies with extremely poor prognoses, highlighting the urgent need for innovative therapeutic strategies to improve treatment outcomes. Epigenetic mechanisms and kinase signaling pathways play a crucial role in the tumorigenesis and progression of these cancers, with histone deacetylases (HDACs) and Rho-associated protein kinases (ROCKs) emerging as relevant therapeutic targets. Given the advantages of multi-target therapeutic approaches, the observed synergistic effects between HDAC and ROCK inhibitors in in vitro and in silico studies, and their critical roles in the pathogenesis of these tumors, the design and development of dual HDAC/ROCK inhibitors represent an innovative and promising strategy for treating these aggressive malignancies. This research was conducted in four phases. Initially, using quantitative structure-activity relationship (3D-QSAR) studies and molecular docking, a pharmacophore model for ROCK1/ROCK2 inhibitors was developed, leading to the design of novel ROCK inhibitors. A non-essential region of fasudil, a known ROCK inhibitor, was identified by molecular docking study, enabling its structural modification and the incorporation of an HDAC pharmacophore while preserving ROCK inhibitory activity. This approach resulted in the first-ever designed dual HDAC/ROCK inhibitors. In the second phase, the designed ROCK and HDAC/ROCK inhibitors were synthesized, followed by the third phase, which involved evaluating their enzymatic activity and anticancer properties using two-dimensional and three-dimensional cell culture models of TNBC (MDA-MB-231) and PDAC (MiaPaCa-2, Panc-1). Finally, based on all previous findings, the fourth phase established a comprehensive in silico protocol for the future design and structural optimization of dual HDAC/ROCK inhibitors. This protocol integrates structure-based drug design (SBDD) and ligand-based drug design (LBDD) methodologies, incorporating molecular docking, 2D-QSAR models developed using machine learning algorithms, and binding free energy calculations performed using the MM-GBSA approach. Among the designed ROCK inhibitors, compound C-19 demonstrated notable inhibitory activity against ROCK1 (IC50 = 4.22 μM) and ROCK2 (IC50 = 6.13 μM), as well as cytotoxic effects on MiaPaCa-2 and Panc-1 cells (IC50 = 19.10 μM and 35.03 μM). Additionally, C-19 exhibited significant anti-migratory and anti-invasive properties, comparable to fasudil. The most promising of the synthesized HDAC/ROCK dual inhibitors was C-9, which showed potent inhibition of ROCK1 (IC50= 800 nM), ROCK2 (IC50= 700 nM), and HDAC6 (IC50 = 185 nM). In cytotoxicity assays, C-9 demonstrated significantly higher activity than the well-known inhibitors fasudil and tubastatin A, with IC50 values of 5.81 μM for MDA-MB-231, 3.87 μM for MiaPaCa-2, and 19.57 μM for Panc-1. Moreover, C-9 exhibited strong efficacy in 3D tumor spheroid models, effectively inhibiting the formation and viability of Panc-1 and MiaPaCa-2 spheroids. Additionally, C-9 outperformed fasudil in reducing the migration and invasion of MDA-MB-231 and MiaPaCa-2 cells, establishing itself as a promising candidate for further preclinical evaluation in the treatment of metastatic cancers. Based on these results, C-9 was identified as the lead compound, and its structure was further optimized according to the previously defined ROCK pharmacophore model. Structure-based drug design (SBDD) was used to develop a series of novel HDAC/ROCK dual inhibitors...

histon deacetilaze, rho-vezane protein kinaze, dualni inhibitori, racionalni dizajn lijekova, molekulski doking, mašinsko učenje, QSAR, trostruko-negativni tumor dojke, duktalni adenokarcinom pankreasa

histone deacetylases, rho-associated coiled-coil kinases, dual inhibitors, rational drug design, molecular docking, machine learning, QSAR, triple-negative breast cancer, pancreatic ductal adenocarcinoma

615.277.3.074(043.3)

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