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Withasomniferol C, a new potential SARS-CoV-2 main protease inhibitor from the Withania somnifera plant proposed by in silico approaches
No full textExploring potent herbal medicine candidates is a promising strategy for combating a pandemic in the present global health crisis. In Ayurveda (a traditional medicine system in India), Withania somnifera (WS) is one of the most important herbs and it has been used for millennia as Rasayana (a type of juice) for its wide-ranging health benefits. WS phytocompounds display a broad spectrum of biological activities (such as antioxidant, anticancer and antimicrobial) modulate detoxifying enzymes, and enhance immunity. Inspired by the numerous biological actions of WS phytocompounds, the present investigation explored the potential of the WS phytocompounds against the SARS-CoV-2 main protease (3CLpro ). We selected 11 specific withanolide compounds, such as withaphysalin, withasomniferol, and withafastuosin, through manual literature curation against 3CLpro . A molecular similarity analysis showed their similarity with compounds that have an established inhibitory activity against the SARS-CoV-2. In silico molecular docking and molecular dynamics simulations elucidated withasomnif- erol C (WS11) as a potential candidate against SARS-CoV-2 3CLpro . Additionally, the present work also presents a new method of validating docking poses using the AlteQ method
Evolucija mozga čovjeka
No full textEvolucija mozga čovjeka započela je prije 4 milijuna godina pojavom reda Australopithecus, najstarijeg potvrđenog izravnog pretka modernog čovjeka. Proučavanjem fosilnih nalaza, potvrđeno je da se mozak hominina (tribus potporodice Homininae koji obuhvaća vrste Homo sapiens, Pan troglodytes i njihove izumrle pretke) povećavao tijekom evolucije. Pokazalo se da sama veličina mozga nije ključna za razvoj kognitivnih sposobnosti, značajan je ponajprije broj i raspored neurona. Tako je čovjek vrsta sa 86 milijardi neurona od čega je 16 milijardi smješteno u moždanoj kori, što predstavlja najviši broj neurona u odnosu na ostale hominine (uključujući izumrle vrste).
U ovom radu obuhvaćeni su mehanizmi i razlozi razvoja mozga suvremenog čovjeka, koji je karakterističan po izvanrednim kognitivnim i bihevioralnim sposobnostima. Objašnjene su metaboličke prepreke koje su nametnute u razvoju mozga primata sa približno 86 milijardi neurona kakav je prisutan u današnjeg čovjeka. Uz prehranu baziranoj na sirovoj hrani kao što su voće i orašasti plodovi, bilo bi nemoguće razviti mozak od 86 milijardi neurona sa tijelom od prosječnih 62 kg bez da se sati hranjenja (unutar 24 sata) povećaju iznad održive vrijednosti (do 8 sati dnevno). Ova metabolička prepreka vjerojatno je savladana termičkom obradom hrane uz pomoć vatre. Termička obrada hrane omogućava bolju i bržu apsorpciju u probavnom sustavu čovjeka, a zahvaljujući mekšoj kozistenciji zahtjeva manje vremena za žvakanje.
Kako bi se ova pretpostavka potkrijepila objašnjena su pravila staničnog skaliranja u primata i uspoređena sa pravilima staničnog skaliranja glodavaca. Pokazalo se da u primata, pa tako i hominina, gustoća neurona ostaje relativno stalna, bez povećanja dimenzija neurona. Gustoća neurona definirana je kao broj neurona u odnosu na ukupan volumen mozga. Gustoća glija stanica približno je konstantna u mozgova sisavaca te ne predstavlja razliku u evoluciji mozga hominina i ostalih sisavaca. Prezentirani su metabolički troškovi za tijelo i mozak današnjeg čovjeka. Uz pomoć metode staničnog skaliranja za neurone, otkriveno je da moždana kora čovjeka sadrži približno 16 milijardi neurona i pretpostavlja se da je upravo to svojstvo koje nas neosporno ističe u odnosu na naše izumrle pretke i ostale primate
Suspected side effects of drugs that inhibit Bruton's tyrosine kinase
No full textZa liječenje različitih vrsta limfoma B limfocita otkriveni su lijekovi koji inhibiraju Brutonovu tirozin kinazu (BTK) odnosno enzim koji potiče malignu proliferaciju B limfocita. Njenom inhibicijom blokiraju se signalni putevi koji su bitni u nastajanju malignih promjena. U ovu skupinu lijekova ubrajaju se ibrutinib, akalabrutinib i zanubrutinib. Ovaj diplomski rad donosi analizu svih prijava sumnji na nuspojave lijekova koji inhibiraju BTK, koje su Agenciji za lijekove i medicinske proizvode (HALMED) prijavljene u razdoblju od 1. siječnja 2015. godine do 31. prosinca 2021. godine. Prijave su analizirane s obzirom na: spol, dob, struku prijavitelja, ozbiljnost, kriterij ozbiljnosti, klasifikaciju organskih sustava Medicinskog rječnika za regulatorne poslove (MedDRA), učestalost prijave i lijekove u istodobnoj primjeni. U promatranom razdoblju zaprimljene su 123 prijave sa ukupno 220 nuspojava. Najveći broj prijava zabilježen je za muški spol (65,9%) i za dobnu skupinu od 65 do 74 godina (28,5%). Najveći broj nuspojava prijavili su liječnici, a veliki broj nuspojava ocijenjen je ozbiljnima (69,9%). Od ukupno 220 nuspojava najveći broj njih prema klasifikaciji organskih sustava pripada u SOC infekcije i infestacije (17,7%), SOC poremećaji kože i potkožnog tkiva (10%) te SOC srčani poremećaji (8,2%), a najčešće su prijavljivane fibrilacija atrija, sepsa i pneumonija. Navedeni podaci o nuspojavama lijekova koji inhibiraju BTK u skladu su s podacima na europskoj i svjetskoj razini. Broj prijava sumnji na nuspojave, kako lijekova koji inhibiraju BTK tako i ostalih lijekova, raste svake godine. To je rezultat kvalitetnog rada HALMEDA i drugih svjetskih agencija na promicanju svjesnosti o važnosti prijavljivanja sumnji na nuspojave lijekova. Potrebni su daljnji napori u unaprjeđenju sigurnosti lijekova koji inhibiraju BTK kako bi se osiguralo sigurno i učinkovito liječenje pacijenata sa što manje štetnih nuspojava.Drugs that inhibit Bruton's tyrosine kinase (BTK), which is an enzyme that promotes malignant proliferation of B lymphocytes, have been discovered for the treatment of different types of B lymphocyte lymphoma. Inhibition of this enzyme blocks the signaling pathways that are important in the development of malignant changes. This group of drugs includes ibrutinib, acalabrutinib and zanubrutinib. This thesis provides an analysis of all suspected reports of adverse reactions to drugs that inhibit BTK, which were reported to the Agency for Medicinal Products and Medical Devices (HALMED) in the period from January 1, 2015 to December 31, 2021. Reports were analyzed considering: gender, age, reporter qualification, seriousness, seriousness criteria, Medical Dictionary of Regulatory Affairs (MedDRA) organ system classification, the most common adverse reactions and concomitant medications. In the observed period, 123 reports were received with a total of 220 adverse reactions. The highest number of reports was recorded for males (65.9%) and for the age group 65 to 74 years (28.5%). The highest number of adverse reactions was reported by doctors, and a large number of them were assessed as serious (69.9%). Out of a total of 220 adverse reactions, the highest number of them according to the classification of organ systems belongs to SOC infections and infestations (17.7%), SOC disorders of the skin and subcutaneous tissue (10%) and SOC heart disorders (8.2%) and the most commonly reported were atrial fibrillation, sepsis and pneumonia. The data in this thesis are in accordance with the data at European and global level. The number of reports of suspected adverse reactions, both for drugs that inhibit BTK and for other drugs, is higher every year. This is the result of the quality work of HALMED and other world agencies, to raise awareness on the importance of reporting suspected adverse drug reactions. Further efforts are needed to improve the safety of BTK-inhibiting drugs to ensure safe and effective treatment of patients with as few adverse reactions as possible
Kontrola prometa trombocitnih receptora glikoproteina Ib-IX I integrina αIIbβ3
No full textPlatelets are small anucleate blood cells, produced from their precursor cells megakaryocytes, and have a primary role in haemostasis; following tissue trauma and exposure of the subendothelial matrix, a coordinated series of events enables the formation of a platelet plug, and central to this process are the platelet surface receptors. Two major receptors are the glycoprotein (GP) Ib-V-IX complex, composed of GPIbα, GPIbβ, GPIX and GPV, and integrin αIIbβ3, composed of αIIb and β3, that bind collagen bound von Willebrand factor and fibrinogen, respectively. These receptors are responsible for initiating, driving, and amplifying essential platelet function and therefore mediating haemostasis. However, the mechanisms by which platelets control their surface receptor expression remains unclear. Here, the role of clathrin mediated endocytosis (CME) proteins was investigated using both a pharmacological and genetic approach. We show that upon activation of mouse platelets, individual receptors and subunits are regulated independently, and when activated via GPVI but not the PAR receptors, inhibiting dynamin-2 and clathrin impairs this. We also show that the activity of the small GTPase Rab5 differentially regulates different receptors in the human megakaryocytic cell line Dami. Taken together, we provide a role for CME proteins in the regulation of platelet receptor trafficking.Trombociti su male krvne stanice bez jezgre, proizvedene iz svojih prekursorskih stanica megakariocita, koje imaju primarnu ulogu u hemostazi; nakon traume tkiva i izlaganja subendotelnog matriksa, koordinirani niz događaja omogućuje stvaranje trombocitnog čepa, a središnji dio ovog procesa čine površinski receptori trombocita. Dva glavna receptora su kompleks glikoproteina (GP) Ib-V-IX, sastavljen od GPIbα, GPIbβ, GPIX i GPV, i integrin αIIbβ3, sastavljen od αIIb i β3, koji vežu von Willebrandov faktor vezan za kolagen odnosno fibrinogen. Ti su receptori odgovorni za pokretanje i pojačavanje funkcije trombocita i stoga posreduju u hemostazi. Međutim, mehanizmi kojima trombociti kontroliraju ekspresiju svojih površinskih receptora ostaju nejasni. U ovom radu istražili smo ulogu proteina klatrinom posredovane endocitoze (CME) koristeći farmakološki i genetski pristup. Pokazujemo da se nakon aktivacije mišjih trombocita pojedinačni receptori i podjedinice reguliraju neovisno, a kada se aktiviraju putem GPVI, ali ne i PAR receptora, farmakološka inhibicija dinamina-2 i klatrina to onemogućuje. Također, pokazujemo da aktivnost male GTPaze Rab5 diferencijalno regulira različite receptore u ljudskoj megakariocitnoj staničnoj liniji Dami. U zaključku, ovo istraživanje pruža uvid u ulogu CME proteina u regulaciji prometa trombocitnih receptora
Utjecaj parenja i izolacije na preferencijalnu konzumaciju metamfetamina kod D. melanogaster
No full textMany molecular mechanisms are involved in the reward system of the brain. Natural rewards include food, mating and socialization. Artificial rewards include psychostimulative substances, such as methamphetamine (METH). Dopaminergic reward system of fruit fly in Drosophila melanogaster can be stimulated by both natural and artificial rewards. Reward system in D. melanogaster is mainly regulated by signal molecules dopamine (DA) and neuropeptide F (NPF) signalling. Positive social experiences are rewarding and increase the levels of DA and NPF in D. melanogaster. Sexual deprivation in D. melanogaster has led to an increased preference for ethanol and is explained through the mechanism of NPF signalling. Negative social experiences lead to increased aggression, lack of sleep, disturbed food intake and mating. It causes many epigenetic changes with the emphasis on DNA methylation. Social isolation increases the preference for psychostimulative substances in many species and in flies an increase in preference to ethanol post social isolation was discovered so far.
The aim of this thesis is to analyse the effect of different social experiences on the preferential consumption of METH in D. melanogaster. I analysed the effect of isolation/grouping and its duration, as well as, sexual deprivation, which has not yet been done in D. melanogaster.
I included four different groups of flies – isolated mated and virgin and grouped mated and virgin. The FlyCafe experiment allowed the self – administration of METH over the three days of the experiment. Isolated flies show higher METH preference and the preference decreases as the duration of isolation increases. Group housed flies show aversion to METH and this aversion increases with the duration of grouping. Social isolation shows the dominant effect on preference compared to sexual deprivation. The highest change in preference, with regards to duration of isolation/grouping, is present in group housed and mated flies. It indicates the importance of the mating duration in regards to the peak mating capacity in flies.
Future experiments should analyse the long-lasting effects of social isolation and sexual deprivation on preferential consumption of METH, to more precisely define environmental effect on the motivation for METH consumption.Mnogi molekularni mehanizmi su uključeni u nagrađujući sustav u mozgu. Prirodne nagrade uključuju hranu, parenje i socijalizaciju. Umjetne nagrade uključuju psihostimulativna sredstva, poput metamfetamina (METH). Dopaminergični nagrađujući sustav vinske mušice u Drosophila melanogaster može se poticati prirodnim i umjetnim nagradama. Nagrađujući sustav u D. melanogaster većinski je reguliran signalizirajućim molekulama dopamina (DA) i neuropeptid F (NPF) signalizacijom. Pozitivna socijalna iskustva su nagrađujuća te povećavaju razinu DA i NPF u mušica. Deprivacija parenja je u D. melanogaster dovela do povećane preference za etanol te je objašnjena mehanizmom NPF signalizacije. Negativna socijalna iskustva dovode do povećane agresije, manjka sna, poremećaja hranjenja te parenja. Dolazi do mnoštva epigenetičkih promjena s naglaskom na promjene u metilaciji DNA. Socijalna izolacija povećava preferencu ka psihostimulativnim sredstivma u raznih vrsta, a u mušica je do sada otkriveno povećanje u preferenci ka etanolu kao posljedica socijalne izolacije.
Cilj ovog rada je ispitati utjecaj različitih socijalnih iskustava na preferencijalnu konzumaciju METH-a u D. melanogaster. Ispitan je efekt trajanja izolacije/grupiranja, kao i utjecaj parenja, što do sada nije bilo ispitano u D. melanogaster.
U eksperimenut su uključene četiri skupine D. melanogaster – izolirane parene i neparene te grupirane parene i neparene. FlyCafe eksperimentom omogućena im je samoadministracija METH-a kroz tri dana eksperimenta. Izolirane mušice pokazuju veću preferencu ka METH-u te je preferenca niža sa dužim periodom izolacije. Grupirane mušice pokazuju averziju ka METH-u te se averzija povećava s duljinom grupiranja. Socijalna izolacija pokazuje dominantni utjecaj na preferencu u usporedbi s deprivacijom parenja. Najveća promjena u preferenci s obzirom na duljinu izolacije/grupiranja je prisutna kod grupiranih i parenih mušica. To ukazuje na značaj duljine parenja s obzirom na vrhunac sposobnosti parenja u mušica.
Budući eksperimenti trebali bi ispitati dugotrajniji utjecaj socijalne izolacije i deprivacije parenja na preferencijalnu konzumaciju METH-a, kako bi se preciznije definirao okolinski utjecaj na motivaciju za konzumiranjem METH-a
Assessment of antioxidative and cyto/genoprotective properties of chestnut honey against UVB radiation in vitro
No full textUltraljubičasto B (UVB) zračenje izaziva niz štetnih učinaka u stanicama kao što su izravna oštećenja DNA, oksidacijski stres, poremećaji signalnih puteva, upalni odgovor te fotokancerogeneza. Sve intenzivnije se istražuju različiti prirodni spojevi koji pružaju zaštitu od štetnog djelovanja UV zračenja. Med je u potpunosti prirodan i nutritivan proizvod koji sadrži flavonoide i fenole s antioksidacijskim učinkom. Ultraljubičasto B zračenje je dokazano genotoksičan agens, pa stoga hipoteza iz koje proizlazi istraživanje je da bi tretiranje limfocita medom iz ekološkog uzgoja moglo povećati stopu preživljenja te sniziti razinu primarnih oštećenja DNA i razinu oksidacijskog stresa nakon ozračivanja. U tu svrhu korišten je med od kestena s visokim udjelom polifenola za koji je utvrđena dobra antioksidacijska aktivnost. Istraživanje je provedeno u uvjetima in vitro na limfocitima i plazmi periferne krvi zdrave muške osobe. Provedena su dva modela izlaganja stanica i pune krvi: (I) pred-tretman medom u trajanju od 2 h, nakon kojega je slijedilo ozračivanje UVB zračenjem i dodatnih 1 h inkubacije stanica/krvi na 37 °C, (II) tretman koji je uključivao ozračivanje UVB zračenjem i zatim inkubaciju stanica/krvi s medom u trajanju od 2 h na 37 °C. Ispitani su učinci meda u koncentracijama koje odgovaraju dozama dnevno prihvatljivog unosa meda prehranom i deset puta većoj koncentraciji meda od dnevno prihvatljive doze. Razina primarnih oštećenja limfocitne DNA, kao i protektivni učinak meda, procijenjena je pomoću komet-testa u alkalnim uvjetima, dok su u uzorcima plazme određeni markeri oksidacijskog stresa. Med od kestena pokazao je dobre citoprotektivne i genoprotektivne te antioksidacijske učinke nakon izlaganja limfocita i pune krvi UVB zračenju. Povoljan učinak meda bio je izraženiji nakon tretmana ozračenih uzoraka. Optimalnom koncentracijom meda pokazala se ona od 1 mg/mL, dok su najmanja (0,2157 mg/mL) i najveća (10 mg/mL) koncentracija pokazale prooksidacijsko djelovanje u mjerenju razine markera oksidacijskog stresa.Ultraviolet B (UVB) radiation causes a number of harmful effects in cells including direct DNA damage, oxidative stress, signalling pathway disorders, inflammatory response and photocancerogenesis. Great effort is invested into the investigation of various natural compounds that can provide protection from the harmful effects of UVB radiation. Honey is a completely natural and nutritive product that contains flavonoids and phenols with antioxidant effect. Ultraviolet B radiation has been proven to be genotoxic, therefore the initial hypothesis of the study was that treating lymphocytes with honey can increase the survival rate and decrease primary DNA damage as well as the oxidative stress levels after exposure to irradiation. For this purpose, we used chestnut honey, which has been proven to have high ratios of polyphenols and good antioxidant activity. The study was conducted in vitro on the lymphocytes and peripheral blood plasma of a healthy male. There were two models of cells and whole blood exposure: (I) 2 hours of pretreatment with honey followed by exposure to UVB irradiation and an additional 1 hour incubation of cells/whole blood at 37 °C, (II) treatment including exposure to UVB irradiation and incubation of cells/whole blood with honey for 2 hours at 37 °C. The effects of the honey were analyzed at concentrations that correspond to the daily acceptable honey intake dose and a 10 times larger concentration than the daily acceptable dose. The primary damage of lymphocyte DNA, as well as the protective effect of honey, was evaluated using the alkaline comet assay, while the plasma samples were used to determine markers of oxidative stress. Chestnut honey showed good cytoprotective and genoprotective, as well as antioxidative effect after exposure of lymphocytes and whole blood to UVB irradiation. The beneficial effect of honey was more pronounced after the treatment of irradiated samples. The optimal concentration of honey was shown to be 1 mg/mL, while the lowest (0,2157 mg/mL) and the largest (10 mg/mL) concentrations showed a prooxidative effect in measuring oxidative stress markers
Kiralne stacionarne faze
No full textSkup podataka, uglavnom kvantno-kemijskih računa (optimizacija, računa energija i simulacija UV i CD spektara), koji su rezultirali objavom tri rada:
1) Knezevic, A.; Novak, J.; Bosak, A.; Vinkovic, M.; Structural isomers of saligenin-based β2-agonists: synthesis and insight into the reaction mechanism, Organic & Biomolecular Chemistry, 2020, 18, 9675-9688
2) Knezevic, A.; Novak, J.; Vinkovic, V.; New Brush-Type Chiral Stationary Phases for Enantioseparation of Pharmaceutical Drugs, Molecules, 2019, 24, 823
3)Knezevic, A.; Novak, J.; Pescitelli, G; Vinkovic, V.; Determination of the Absolute Configuration of ( S )- N -(1-Aryl-allyl)-3,5-dinitrobenzamides and Their Elution Order on Brush-Type Chiral Stationary Phases, European Journal of Organic Chemistry, 2018, 3982-399
Implementation and validation of Raman technique for substance identification
No full textRazvijena je i validirana Raman metoda za identifikaciju male organske molekule sukladno smjernicama Međunarodne konferencije za harmonizaciju ICH Q2 (R1). U sklopu razvoja i validacije Raman metode za identifikaciju ulaznih supstancija Benzoyl peroxide, hydrous i Clindamycin hydrochloride definirani su kriteriji koji dokazuju vjerodostojnost i prikladnost korištenja Raman metode. Kriteriji prihvatljivosti metode za identifikaciju ispitali su se kroz parametar selektivnosti, kojim je testirana specifičnost, a ujedno i otpornost Raman metode za identifikaciju ulazne sirovine.
Kriteriji prihvatljivosti za pozitivan i negativan test definirani su prema uputama proizvođača Thermo Scientific za uređaj TruScan RM Analyzer. Rezultati p-vrijednosti svakog od analiziranog uzorka (Benzoyl peroxide, hydrous i Clindamycin hydrochloride) za pozitivan test veći su od 0.1, stoga su sirovine uspješno zadovoljile kriterij prihvatljivosti (p≥0.1). Kreiranim metodama za ispitane uzorke uspješno je identificirana i potvrđena vrsta materijala. Negativnim testom (testom selektivnosti baze podataka) potvrdilo se da kreirane metode razlikuju supstanciju koja se ispituje od ostalih sirovina slične kemijske strukture. Postupkom uvođenja i validacije utvrdila se prikladnost Raman metode za identifikaciju ulaznih supstancija Benzoyl peroxide, hydrous i Clindamycin hydrochloride.Raman spectroscopy method for the identification of a small organic molecule was developed and validated in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use ICH Q2 (R1). The criteria were defined as a part of the development and validation of Raman method for identification of substances Benzoyl peroxide, hydrous and Clindamycin hydrochloride to prove the reliability and suitability of the use of Raman spectroscopy. The acceptance criteria of the proposed identification method were examined through the selectivity parameter that tests the specificity and robustness of the Raman method for identifying the substance.
Acceptance criteria were defined for positive and negative test according to the manufacturer's instructions Thermo Scientific for TruScan RM Analyzer. p-value results each of the analysed samples (Benzoyl peroxide, hydrous and Clindamycin hydrochloride) in Positive test was higher than 0.1 and passed the acceptance criteria (p≥0.1). Methods created for the above-mentioned samples correctly confirmed the type of the raw material. Negative test (database selectivity test) confirmed that the created method correctly distinguishes tested substances from other raw materials with a similar chemical structure. Introduction and validation procedure determined the suitability of the proposed Raman spectroscopy method for Benzoyl peroxide, hydrous and Clindamycin hydrochloride identification
Computational study of the monoamine oxidase B mechanism-based irreversible inhibitors
No full textPozadina: Monoamin oksidaza B (MAO-B) flavoenzim je odgovoran za metabolizam endogenih i egzogenih amina, uključujući monoaminske neutransmitere čija je narušena homeostaza implicirana kod razvitka mnogih neurodegenerativnih oboljenja. MAO-B predstavlja osnovnu farmakološku metu za liječenje Alzheimerove i Parkinsonove bolesti. Komercijalni lijekovi, selegilin i razagilin, administriraju se uz dijetalne restrikcije te, u većim dozama, gube selektivnost što rezultira učestalijim nuspojavama većeg intenziteta. Stoga, postoji pritisak tržišta za razvitkom novih, mehanizmom temeljenih inhibitora MAO-B enzima. Rezultati: Inovativni pristup razvijen je za dizajn novih inhibitora te uključuje spajanje temeljnih aromatskih struktura s propargilaminskom jezgrom koja je prisutna kod komercijalno dostupnih lijekova koji ciljaju MAO enzime. Metodom molekulskog pristajanja i potom simulacijama molekulske dinamike, dobiven je povoljniji termodinamički profil vezanja novodizajniranog liganda N-metil-N-propargil-2-indol (42Me) na aktivno mjesto MAO-B. Optimalniji kinetički profili inhibitorne aktivnosti spoja 42Me okarakteriziran je mehanističkim DFT izračunima na klaster modelu MAO-B enzima. Sprovođenjem populacijske analize liganda, potvrđen je mehanizam hidridne apstrakcije prvog koraka inhibicijske reakcije na temelju referentnog liganda koji ima dobro poznat mehanizam vezanja. Zaključak: Dobiveni rezultati ukazuju na to da je novodizajnirani ligand 42Me kinetički te termodinamički povoljniji propargilaminski inhibitor MAOB (MAOI) od postojećih lijekova, selegilina i razagilina. Reakcija inhibicije MAO-B enzima s kandidatom 42Me efikasija je 69x naspram selegilina, odnosno 622x od razagilina. Primijenjen dizajn propargilaminskih MAOI pokazao se uspješnim te će dobiveni rezultati pomoći u daljnjem razvoju inhibitora poboljšanih kinetičkih i termodinamičkih parametara.Background: Monoamine oxidase B (MAO-B) is a flavoenzyme responsible for the metabolism of endogenic and exogenic amines such as monoamine neurotransmitters whose disturbed homeostasis is implicated in the wide range of neurodegenerative pathogenesis. MAO-B represents a primary pharmacological target for the treatment of Alzheimer's and Parkinson's disease. Commercial drugs, selegiline, and rasagiline, are administrated with dietary restrictions and in high doses are associated with more frequent and greater intensity side effects. There is a constant market pressure for the development of new, mechanism-based MAO-B inhibitors with more favorable pharmacokinetic profiles.
Results: An innovative approach was developed for the drug design which involves joining of the scaffolds with propargylamine core which is present in commercial drugs which target MAO enzymes. A more favorable thermodynamic profile was obtained for the newly designed N-methyl-Npropargyl-2-indol (42Me) using methods of script based molecular docking and molecular dynamics simulations. A more optimal kinetic profile of the inhibitory activity of the 42Me was characterized via the DFT mechanistic calculation on the MAO-B cluster-model. The population analysis affirmed the mechanism of hydride abstraction in the first step of the inhibtion reaction.
Conclusion: Obtained results show that newly designed 42Me is kinetically and thermodynamically more favourable propargylamine inhibitor (MAOI) than known drugs, selegiline and rasagiline. MAO-B inhibition reaction with 42Me is 69x more efficacious than selegiline, or 622x more efficacious than rasagiline. Applied propargylamine MAOI design approach was proved succesful and acquired results will assist in the development of the new inhibitors with better kinetic and thermodynamic parameters
Effect of impaired Arf GEF protein function on cytomegalovirus infection
No full textCitomegalovirus (CMV) je dvolančani DNA virus koji spada u porodicu beta herpes virusa. Svi betaherpesvirusi imaju svojstvo reorganiziranja membranskog sustava stanice sa svrhom da stvore povoljan okoliš za vlastitu replikaciju. Reorganizacija staničnih membrana i odjeljaka za posljedicu ima formiranje citoplazmatskog odjeljka za sklapanje viriona – cVAC. Cilj ovoga rada je ispitati mijenjaju li se izražaj i unutarstanična lokalizacija Arf GEF regulatornih proteina Gbf1, Big1 i Big2 tijekom CMV infekcije te ispitati da li njihova narušena funkcija putem malih inhibitora utječe na odvijanje CMV infekcije. Western blot metodom je pokazano da izražaj Gbf1, Big1 i Big2 proteina raste tijekom CMV infekcije s tim da je izražaj Big1 najveći nakon 6 sati infekcije, dok je izražaj proteina Big2 i Gbf1 najveći nakon 48 sati infekcije. Metodom imunofluorescentne mikroskopije pokazano je da tijekom CMV infekcije dolazi do nakupljanja spomenutih proteina u jukstanuklearnom području inficiranih stanica, s tim da se protein Gbf1 nakuplja i u subplazmalemalnom području. Kolokalizacija proteina Big1, Big2 i Gbf1 s markerima unutrašnjeg i vanjskog dijela VAC-a je pokazala da se proteini Big1, Big2 i Gbf1 nakupljaju uglavnom u unutrašnjem dijelu VAC-a. Nadalje, metodama Western blota, protočne citometrije i imunofluorescentne mikroskopije pokazano je da inhibitori Brefeldin A (BFA), Golgicid A (GCA) i EXO2 narušavaju uspostavu MCMV infekcije i smanjuju izražaj virusnih proteina s tim da se EXO2 pokazao najučinkovitijim inhibitorom iako je učinak BFA imao najveću statističku značajnost. Daljnja istraživanja točne uloge Big1, Big2 i Gbf1 proteina u patogenezi CMV infekcije mogla bi doprinijeti njezinom boljem razumijevanju, a samim time i boljoj terapiji.Cytomegaloviruses (CMV) are a double-stranded DNA virus that belong to the beta herpes virus family. All beta-herpesviruses have the property of reorganizing the cell membrane system to create an environment that is favorable for their own replication. Reorganization of cell compartments and membranes ultimately results in the creation of a cytoplasmic virion assembly compartment - cVAC. The aim of this study was to examine whether the expression and intracellular localization of Arf GEF regulatory proteins Gbf1, Big1 and Big2 change during CMV infection and to examine whether their impaired function by small inhibitors impairs establishment of cytomegalovirus infection. Western blot has shown that expression of Big1, Big2 and Gbf1 increses during CMV infection and Big1 expression is highest after 6 hours of infection, while Big2 and Gbf1 protein expression is highest after 48 hours of infection. Immunofluorescence microscopy has shown that during CMV infection these proteins accumulate in the juxtanuclear region of infected cells, with the Gbf1 protein also accumulating in the subplasmalemal region. Colocalization of Big1, Big2 and Gbf1 proteins with markers of the inner and outer part of the VAC showed that Big1, Big2 and Gbf1 proteins accumulate in the inner part of the VAC. Furthermore, Western blot, flow cytometry and immunofluorescence microscopy methods have shown that small inhibitors Brefeldin A (BFA), Golgicide A (GCA) and EXO2 impare establishment of MCMV infections and reduce viral protein expression. EXO2 was the most effective inhibitor although the effect of BFA showed the greatest statistical significance. Further research of the exact role of Big1, Big2 and Gbf1 proteins in the pathogenesis of CMV infection could contribute to its better understanding and thus better therapy