Mechanistic insight into the anticarcinogenic and neuroprotective activity of natural polyphenolic compounds

Namen doktorske disertacije je z uporabo inovativnih računalniških pristopov preučiti in pojasniti slabo raziskane molekularne mehanizme antikarcinogenega in nevroprotektivnega delovanja naravnih polifenolnih spojin v vlogi preprečevalcev nastanka raka ter v vlogi zaviralcev napredovanja karcinogeneze in Alzheimerjeve bolezni. V prvem delu doktorske disertacije smo z uporabo kvantomehanskih simulacij obravnavali polifenol [6]-gingerol in tripeptid glutation v vlogi lovilcev devetih genotoksičnih kemijskih karcinogenenov ter tako kot prvi omogočili dragocen vpogled v pripadajoče geometrije reaktantov in prehodnih stanj, ki potrjujejo veljavnost predpostavljenega SN2 reakcijskega mehanizma. Predstavljena kvantnomehanska metodologija ima velik potencial, da postane standardno orodje za napovedovanje kemozaščitnega potenciala številnih polifenolnih spojin v vlogi lovilcev kemijskih karcinogenov epoksidnega tipa. V drugem delu doktorske disertacije smo s kvantnomehansko obravnavo ksantohumola iz hmelja in njegovih derivatov izoksantohumola, 8-prenilnaringenina in 6-prenilnaringenina v vlogi lovilcev aflatoksin B1 ekso-8,9-epoksida razkrili povezavo med strukturo in antikarcinogeno aktivnostjo izbranih prenilflavonoidov ter potrdili veljavnost predpostavljenega SN2 reakcijskega mehanizma. Znižanje z aflatoksinom B1 inducirane citotoksičnosti in genotoksičnosti je bilo potrjeno tudi in vitro na celicah človeškega hepatoma HepG2 po dodatku ksantohumola. V tretjem delu doktorske disertacije smo razvili popolnoma nov protokol za inverzno molekulsko sidranje, s pomočjo katerega je mogoče identificirati zožen nabor najbolj verjetnih tarčnih človeških proteinov izbrane polifenolne spojine. Na podlagi afinitete izbranega polifenola kurkumina za vezavo na specifične človeške proteine, ki sodelujejo v onkogenih in nevrodegenerativnih signalnih kaskadah, smo kot prvi uspešno razkrili mehanizme njegovega antikarcinogenega in nevroprotektivnega delovanja. Predstavljeni protokol inverznega molekulskega sidranja ima velik potencial v farmacevtskih aplikacijah, saj gre za univerzalen pristop, s pomočjo katerega lahko predvidimo proteinske tarče in pojasnimo že opažene ter napovemo nove potencialne biološke učinke izbranih spojin vodnic. V četrtem delu doktorske disertacije smo z uporabo molekulskega sidranja, simulacij molekulske dinamike in prostoenergijskih izračunov preučevali štiri prehranske polifenole kot potencialne inhibitorje encima fosfodiesteraza 4D, ki predstavlja validirano terapevtsko tarčo za zdravljenje Alzheimerjeve bolezni. Izračunane vezavne proste energije potrjujejo, da kurkumin, [6]-gingerol, kapsaicin in resveratrol predstavljajo potencialne inhibitorje encima fosfodiesteraza 4D, pri čemer ima kurkumin največji inhibitorni potencial. S kombiniranim računalniškim pristopom smo tako prvič dobili mehanistični vpogled v povezavo med strukturo in nevroprotektivnim delovanjem štirih preučevanih polifenolov, ki je eksperimentu nedosegljiv. V petem delu doktorske disertacije smo predstavili ekstrakcijske, destilacijske in karakterizacijske metode za pridobivanje visoko kvalitetnih bioaktivnih ekstraktov, eteričnih olj in posameznih polifenolnih spojin iz rožmarina ter metode za določanje njihovega antioksidativnega, antimikrobnega, protivnetnega in antikarcinogenega potenciala. S temeljitim pregledom obstoječih raziskav in z usmeritvami za nadaljnje raziskave smo postavili temelje za uspešne aplikacije ekstraktov, eteričnih olj ter številnih polifenolnih spojin iz rožmarina v farmacevtski in prehranski industriji. Trdno verjamemo, da v doktorski disertaciji predstavljeni in silico pristopi in razkriti inhibitorni mehanizmi tvorijo izhodišče za hitrejši in cenejši razvoj novih funkcionalnih prehranskih dopolnil in zdravil za preprečevanje in zdravljenje raka ter Alzheimerjeve bolezni s potencialno širšim terapevtskim oknom in manjšimi stranskimi učinki.This doctoral dissertation aims to examine and elucidate the poorly understood molecular mechanisms of anticarcinogenic and neuroprotective effects of natural polyphenols as chemopreventive agents as well as in the role of suppressors of carcinogenesis and Alzheimer\u27s disease, using innovative computational approaches. In the first part of the doctoral dissertation, we examined polyphenol [6]-gingerol and the tripeptide glutathione as scavengers of nine ultimate genotoxic chemical carcinogens. Using quantum mechanical simulations, we obtained valuable insights into the proposed SN2 reaction mechanisms and the geometries of the corresponding reactants and transition states. The presented quantum-mechanical methodology has a high potential to become a standard tool for predicting the chemopreventive potential of various polyphenolic compounds as scavengers of chemical carcinogens of the epoxy type. In the second part of the doctoral dissertation, we investigated xanthohumol and its derivatives isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin as scavengers of aflatoxin B1 exo-8,9-epoxide. Using quantum mechanical simulations, we discovered the connection between the structure and the anticarcinogenic activity of the studied prenylflavonoids, as well as confirmed the validity of the proposed SN2 reaction mechanism. Reduction in aflatoxin B1-induced cytotoxicity and genotoxicity was also confirmed in vitro in human hepatocellular carcinoma HepG2 cell line after the addition of xanthohumol. In the third part of the doctoral dissertation, we developed a novel inverse molecular docking protocol, which identifies a narrow set of prioritized human protein targets of a selected polyphenolic compound. We were the first to successfully uncover the molecular mechanisms of curcumin\u27s anticarcinogenic and neuroprotective activities, based on its binding affinity to specific human proteins involved in oncogenic and neurodegenerative signaling cascades. Moreover, the developed protocol possesses a high potential in pharmaceutical applications, as it represents a universal approach that can be used to explain the already observed as well as predict new potential biological effects of the studied lead compounds. In the fourth part of the doctoral dissertation, we performed molecular docking, molecular dynamics simulations, and binding free energy calculations to investigate inhibitory mechanisms of four dietary polyphenols against phosphodiesterase 4D, which represents a high-interest therapeutic target for the treatment of Alzheimer’s disease. The calculated binding free energies proved that curcumin, [6]-gingerol, capsaicin, and resveratrol represent potential inhibitors of phosphodiesterase 4D, with curcumin exhibiting the highest inhibitory potency. With the combined computational approach, we for the first time obtained a mechanistic insight into the connection between the structure and neuroprotective activities of the four studied polyphenols, which is unattainable using laboratory experiments. In the fifth part of the doctoral dissertation, we presented extraction, distillation, and characterization methods for obtaining high-quality bioactive extracts, essential oils, and major bioactive polyphenols from rosemary as well as methods for determining their antioxidant, antimicrobial, anti-inflammatory, and anticarcinogenic potentials. With a comprehensive review of existing research and guidelines for further research, we laid the foundation for successful applications of extracts, essential oils, and numerous bioactive polyphenols from rosemary in the pharmaceutical and food industries. We firmly believe that presented in silico approaches as well as uncovered inhibitory mechanisms form a solid basis for faster and cheaper development of novel functional dietary supplements and drugs for the prevention and treatment of cancer and Alzheimer\u27s disease with a potentially wider therapeutic window and fewer side effects