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The recently declared global pandemic of a new human coronavirus called SARS-CoV-2, which causes respiratory tract disease COVID-19, has reached worldwide resonance and global efforts are being made to look for possible cures. Sophisticated molecular docking software, as well as available protein sequence and structure information, offer the ability to test the inhibition of two important targets of SARS-CoV-2, furin (FUR) enzyme, and spike glycoprotein, or spike protein (SP), that are key to host cell adhesion and hijacking. The potential inhibitory effect and mechanism of action of acid–base forms of different antiviral drugs, dominant at physiological pH, chloroquine (CQ), hydroxychloroquine (HCQ), and cinanserin (CIN), which have been shown to be effective in the treatment of SARS-CoV-2 virus, is reported with the special emphasis on their relative abundances. On the other hand, the potential inhibitory effect of the dominant acid–base forms of quercetin (Q) and its oxidative metabolite 2- (3, 4-dihydroxybenzoyl)-2, 4, 6-trihydroxy-3(2H) benzofuranone (BZF), which are constituents of traditional food products believed to exhibit antiviral effects, was also examined. The undertaken study includes the determination of the major energy contributions to the binding energy as well as in-depth analysis of amino acid residues at the active pocket and possible interactions. The approach that we propose here may be an additional strategy for combating the deadly virus by preventing the first step of the virus replication cycle. Preliminary research has shown that the investigated compounds exert an inhibitory effect against the SARS-CoV-2 furin enzyme and spiked glycoprotein through different acid–base forms. These investigations may be helpful in creating potential therapeutic agents in the fight against the SARS-CoV-2 virus. On the other hand, the results we predicted in this computational study may be the basis for new experimental in vitro and in vivo studies.

Predmet istraživanja ove doktorske teze je in vitro određivanje antiradikalske aktivnosti odabranih neurotranmitera, njihovih prekursora i metabolita, a u cilju uspostavljanja odnosa na liniji struktura-antiradikalska aktivnost. Rezultati su upotpunjeni teorijskim proračunima kako bi se dobila potpuna slika parametara važnih za aktivnost prema radikalima od interesa, ali odredili termodinamički i kinetički najpovoljniji mehanizmi. Veliki broj spoljašnjih faktora utiče na povećanje koncentracije slobodnih radikala u organizmu pri čemu nastaje oksidacioni stres. U ovom procesu se ireverzibilno oštećuju biološki značajni molekuli i to dejstvom reaktivnih vrsta kiseonika, azota, hlora i sumpora. U literaturi je pokazano da su slobodni radikali direktno ili indirektno povezani sa razvojem neurodegenerativnih bolesti (Parkinsonove, Alchajmerove, šizofrenije i amiotrofične lateralne skleroze). Aktivnost može biti izražena ili kroz oksidaciju biološki molekula ili formiranje polimernih struktura, odnosno plakova. Većina ispitivanih egzogenih antioksidanasa ne može preći krvno-moždanu barijeru, tako da se ne mogu direktno koristiti za lečenje neurodegenerativnih bolesti. Zbog toga se sve veći značaj pridaje molekulima koji su prisutni u organizmu. Po svojoj strukturi, većina neurotransmitera pripada klasi kateholamina, odnosno poseduju kateholnu grupu koja je i čest strukturni parametar dobrih antioksidanasa. Dopamin, norepinefrin, epinefrin, njihov prekursor L-DOPA i dopaminski metabolit DOPAC poseduju ovu strukturnu jedinicu. Zamenom jedne hidroksilne grupe metoksigrupom nastaju 3-metoksitiramin, homovanilinska kiselina, vanililbademova kiselina (vanillylmandelic acid – VMA), značajni metaboliti neurotransmitera koji se eksperimentalno određuju prilikom dijagnoze bolesti povezanih sa promenom koncentracije dopamina. Tiramin, tirozin i oktopamin imaju hidroksilnu grupu u ppoložaju u odnosu na alifatični niz dok fenilalanin i feniletilamin u strukturi nemaju hidroksilne grupe na aromatičnom prstenu. Tri hidroksilne grupe na aromatičnom prstenu v su prisutne kod 6-hidroksidopamina. Svi ovi molekuli su uključeni u istraživanje kako bi se eksperimentalno i teorijski odredili strukturni parametri, broj i vrsta supstituenata, kao i krajnje grupe alifatičnog niza značajni za antiradikalsku aktivnost. The research topic of this doctoral dissertation is in vitro determination of antiradical activity of selected neurotransmitters, their precursors and metabolites, with the main goal of establishment of the structure-antiradical activity relationship. The results incorporate theoretical calculations in order to obtain a full picture of parameters important for activity towards radicals of interest, but also to determine the thermodynamically and kinetically most favorable mechanism. A large number of external factors influences the increase in the concentration of free radicals which leads to oxidative stress. In this process, the biologically important molecules are irreversibly damaged by the activity of reactive oxygen, nitrogen, chlorine and sulfur species. It is shown in the literature that free radicals are directly or indirectly connected to the development of neurodegenerative diseases (Parkinson, Alzheimer, schizophrenia, amyotrophic lateral sclerosis). The activity of the reactive species is expressed through oxidation of biologically important molecules or the formation of polymeric structures, plaque. The most of investigated exogenous antioxidants cannot cross the blood-brain barrier, therefore they cannot be used for the treatment of neurodegenerative diseases. Because of this, there is an increased interest in the naturally occurring molecules in an organism. Structurally, the majority of neurotransmitters are catecholamines, which means that they possess catechol moiety, the common structural motif of good antioxidants. Dopamine, norepinephrine, epinephrine, their precursor L-DOPA and dopamine metabolite DOPAC have this group. When one of the hydroxyl groups is exchanged with methoxy group the following molecules are obtained: 3-methoxytyramine, homovanillic acid and vanillylmandelic acid. These molecules are experimentally determined in the diagnosis of diseases resulting from a change in the dopamine concentration. Tyramine, tyrosine, and octopamine have one hydroxyl group in p-position with respect to the aliphatic chain. Phenylalanine and phenylethylamine do not possess any hydroxyl groups viii attached to the aromatic ring. There are three hydroxyl groups in the structure of 6- hydroxydopamine. All of these molecules are included in the investigation so experimentally and theoretically important structural parameters like number and type of substituents and end groups of the aliphatic chain are determined.

Poslednjih godina 1, 2, 4-triazol-3-tioni privlače dosta pažnje zahvaljujući njihovim biološkim osobinama. Petočlani prsten ovih jedinjenja je strukturni deo brojnih farmakološki zanimljivih jedinjenja koja pokazuju antidepresivne, antiinflamatorne, antibakterijske i antikancerogene osobine. U našem prethodnom radu1 kombinovali smo fenolne farmakofore sa 1, 2, 4-triazol-3-tionom u cilju dobijanja novih jedinjenja sa boljim antioksidativnim osobinama. Na osnovu dobijenih rezultata u toj studiji može se reći da je SPLET dominantan mehanizam antioksidativnog delovanja u metanolu. Na dalje je ispitivana mogućnost triazola koji su pokazali najbolje antioksidativne osobine, da neutrališu tri slobodno radikalske vrste (izo- propoksil radikal, vinil peroksil radikal i trihlormetilperoksil radikal). Ravnotežne geometrije svih oblika ispitivanih jedinjenja, kao i slobodno radikalskih vrsta koje učestvuju u reakciji izračunate su na B3LYP-D3/6- 311++G(d, p) nivou teorije, u metanolu i benzenu. Ovi rastvarači korišćeni su da bi se ispitao uticaj polarne i nepolarne sredine. Dobijeni rezultati ukazuju da sva tri slobodna radikala mogu biti neutralisana dejstvom ispitivanih triazola, ali je pitanje koji je mehanizam antioksidativnog delovanja dominantan. SET-PT mehanizam je najmanje verovatan reakcioni put antioksidativnog delovanja. U metanolu, prva dva ispitivana radikala mogu biti neutralisana i HAT i SPLET mehanizmom. U benzenu je dominatan SPLET mehanizam. Rezultati ostvareni za trihlormetilperoksil radikal ukazuju da ovaj radikal može biti inaktiviran reakcijom HAT mehanizma u metanolu, dok su u benzenu HAT i SPLET konkurentni mehanizmi.

Funkcionalizovane nanočestice imaju veliki potencijal u oblasti dostave antitumorskih lekova do željenog tkiva. One mogu da menjaju farmakokinetički i farmakodinamički profil lekova, čime se povećava njihov terapeutski indeks. Međutim, uprkos ostvarenom napretku i potencijalno velikim prednostima nad klasičnom terapijom, samo mali broj tretmana raka zasnovanih na nanočesticama je odobren. Do sada su dizajnirane mnogobrojne inovativne, multifunkcionalne i kompleksne nanočestice. Ali, to se ne odražava u dizajnu niti broju klinički odobrenih nanosistema. Dizajn nanosistema mora biti kompleksan kako bi se savladale sve prepreke u organizmu koje sam lek ne može da prevaziđe, međutim, to značajno otežava njihovu karakterizaciju i predviđanje njihove efikasnosti. Sam eksperimentalni pristup rešavanju ovog problema je dugotrajan i skup. Takođe, zbog neophodne prostorne i vremenske rezolucije za ispitivanje nekih aspekata njihovog fiziološkog efekta, eksperimentalni pristup u nekimslučajevima tehnički nije moguć.Opšti cilj ove doktorske disertacije je razvijanje modela odabranih nanosistema i računarsko simuliranje njihove strukture i fiziološkog efekta.Dva glavna, specifična cilja ovog istraživanja bila su:1. Izvođenje teorijskih proračuna u cilju dobijanja korisnih strukturnih i energetskih podataka o ispitivanim funkcionalizovanim nanočesticama zlata;2. Modelovanje i simuliranje njihovog fiziološkog efekta.Za istraživanja su korišćeni modeli koji obuhvataju više vremenskih i prostornih skala (nivo molekula - molekulska dinamika, nivo ćelije - stohastičke reakciono-difuzione rekacije, nivo tumora - modelovanje zasnovano na agentima). Opšti zaključak ovog istraživanja je da je teorijski pristup komplementaran eksperimentima, jer se na ovaj način mogu detaljnoispitati aspekti koje je nemoguće eksperimentalno testirati. Simulacije na više različitih nivoa mogu povezati različite vremenske i prostorne rezolucije čime se može dati preliminarna procena efikasnosti nanonosača u fiziološkim uslovima. Ovaj pristup je znatno brži i zahteva mnogo manje resursa od isključivo eksperimentalnog pristupa što može dovesti do povećanja uspešnosti i brzine dovođenja novih nanosistema u kliničku praksu., Functionalized nanoparticles hold great potential as antitumor drug delivery carriers. They can change the pharmacokinetic and pharmacodynamic profile of carried drugs, which increases their therapeutic index. However, despite the advances and potentially great advantages over conventional therapy, only a small number of nanoparticle-based cancer treatments have been approved. The nanosystem design must be complex to overcome all the obstacles in the body. However, this significantly complicates their characterization as well as the prediction of their efficacy. Applying only the experimental approach to solve this problem is time-consuming and expensive. Also, due to the spatial and temporal resolution necessary to investigate some aspects of the physiological effect, an experimental approach, in some cases, is technically not possible.The general goal of this thesis is to develop models of selected nanosystems, and simulate their structure and physiological effect. The two main, specific objectives of this research were: 1. Obtaining useful structural and energetic data of the examined functionalized gold nanoparticles by theoretical calculations; 2. Modeling and simulation of their physiological effect. Models used in this thesis include several temporal and spatial scales (molecule level - molecular dynamics, cell level - stochastic reaction-diffusion reactions, tumor level - agent-based modeling). The general conclusion of this research is that the theoretical approach is complementary to experiments. This way we can obtain detailed information on aspects that are impossible to test experimentally. Simulations of several different levels can link different temporal and spatial resolutions, which can provide a preliminary assessment of the nanosystem’s efficacy in physiological conditions. This approach is much faster and requires much less resources than the purely experimental approach, which can increase efficiency and speed of bringing new nanosystems into clinical practice.

Funkcionalizovane nanočestice imaju veliki potencijal u oblasti dostave antitumorskih lekova do željenog tkiva. One mogu da menjaju farmakokinetički i farmakodinamički profil lekova, čime se povećava njihov terapeutski indeks. Međutim, uprkos ostvarenom napretku i potencijalno velikim prednostima nad klasičnom terapijom, samo mali broj tretmana raka zasnovanih na nanočesticama je odobren. Do sada su dizajnirane mnogobrojne inovativne, multifunkcionalne i kompleksne nanočestice. Ali, to se ne odražava u dizajnu niti broju klinički odobrenih nanosistema. Dizajn nanosistema mora biti kompleksan kako bi se savladale sve prepreke u organizmu koje sam lek ne može da prevaziđe, međutim, to značajno otežava njihovu karakterizaciju i predviđanje njihove efikasnosti. Sam eksperimentalni pristup rešavanju ovog problema je dugotrajan i skup. Takođe, zbog neophodne prostorne i vremenske rezolucije za ispitivanje nekih aspekata njihovog fiziološkog efekta, eksperimentalni pristup u nekim slučajevima tehnički nije moguć. Opšti cilj ove doktorske disertacije je razvijanje modela odabranih nanosistema i računarsko simuliranje njihove strukture i fiziološkog efekta. Dva glavna, specifična cilja ovog istraživanja bila su: 1. Izvođenje teorijskih proračuna u cilju dobijanja korisnih strukturnih i energetskih podataka o ispitivanim funkcionalizovanim nanočesticama zlata; 2. Modelovanje i simuliranje njihovog fiziološkog efekta. Za istraživanja su korišćeni modeli koji obuhvataju više vremenskih i prostornih skala (nivo molekula - molekulska dinamika, nivo ćelije - stohastičke reakciono-difuzione rekacije, nivo tumora - modelovanje zasnovano na agentima). Opšti zaključak ovog istraživanja je da je teorijski pristup komplementaran eksperimentima, jer se na ovaj način mogu detaljno ispitati aspekti koje je nemoguće eksperimentalno testirati. Simulacije na više različitih nivoa mogu povezati različite vremenske i prostorne rezolucije čime se može dati preliminarna procena efikasnosti nanonosača u fiziološkim uslovima. Ovaj pristup je znatno brži i zahteva mnogo manje resursa od isključivo eksperimentalnog pristupa što može dovesti do povećanja uspešnosti i brzine dovođenja novih nanosistema u kliničku praksu. Functionalized nanoparticles hold great potential as antitumor drug delivery carriers. They can change the pharmacokinetic and pharmacodynamic profile of carried drugs, which increases their therapeutic index. However, despite the advances and potentially great advantages over conventional therapy, only a small number of nanoparticle-based cancer treatments have been approved. The nanosystem design must be complex to overcome all the obstacles in the body. However, this significantly complicates their characterization as well as the prediction of their efficacy. Applying only the experimental approach to solve this problem is time-consuming and expensive. Also, due to the spatial and temporal resolution necessary to investigate some aspects of the physiological effect, an experimental approach, in some cases, is technically not possible. The general goal of this thesis is to develop models of selected nanosystems, and simulate their structure and physiological effect. The two main, specific objectives of this research were: 1. Obtaining useful structural and energetic data of the examined functionalized gold nanoparticles by theoretical calculations; 2. Modeling and simulation of their physiological effect. Models used in this thesis include several temporal and spatial scales (molecule level - molecular dynamics, cell level - stochastic reaction-diffusion reactions, tumor level - agent-based modeling). The general conclusion of this research is that the theoretical approach is complementary to experiments. This way we can obtain detailed information on aspects that are impossible to test experimentally. Simulations of several different levels can link different temporal and spatial resolutions, which can provide a preliminary assessment of the nanosystem’s efficacy in physiological conditions. This approach is much faster and requires much less resources than the purely experimental approach, which can increase efficiency and speed of bringing new nanosystems into clinical practice.