Proučavanje interakcija biološki aktivnog hinona avarona i njegovih derivata sa lizozimom, linearnom i cirkularnom dezoksiribonukleinskom kiselinom
Investigation of interactions of biologically active quinone avarone and its derivates with lysozyme, linear and circular deoxyribonucleic acid
Author
Novaković, Irena T.Mentor
Sladić, DušanCommittee members
Kostić-Rajačić, SlađanaVujčić, Zoran
Roglić, Goran
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Show full item recordAbstract
Cilj našeg rada bio je ispitivanje biološke aktivnosti metilamino- i metoksiderivata
avarona i razjašnjavanje mehanizma njihovog biološkog dejstva.
Za sintezu su izabrani derivati za koje se očekivalo da će imati negativniji
polutalasni potencijal od avarona i samim tim pokazivati povećanu aktivnost. Sintetisani
su 4'-(metilamino)-avaron, 3'-(metilamino)-avaron i 3'-metoksi-avaron.
Antibakterijska aktivnost dobijenih derivata je ispitivana prema gram-pozitivnim
bakterijama: Bacillus subtilis, Clostridium sporogenes, Streptosporangium
longisporum, Micrococcus flavus, Sarcina lutea i Staphylococcus aureus, prema gramnegativnim
bakterijama: Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas
aeruginosa, Salmonella enteritidis, Escherichia coli i prema kulturama gljivica:
Aspergillus niger, Candida albicans i Saccharomyces cerevisiae disk-difuzionom
metodom. Takođe, ispitivana je i toksičnost derivata na račiće Artemia salina.
Potencijalna antioksidativna aktivnost avarona i dobijenih der...ivata ispitivana je
testom sa DPPH.
Antitumorska aktivnost ispitivana je za sve derivate na osam vrsta ćelija raka
(ćelijske linije raka grlića materice, melanoma i leukemije, rak dojke pozitivan na
estrogeni receptor, rak dojke negativan na estrogeni receptor, rak pluća, leukemija
T-ćelija i promijelocitna leukemija) pri čemu je ispitivana i njihova citotoksičnost na
limfocite.
Svim hinonskim derivatima hemijski je modifikovan model-enzim lizozim.
Dobijenim modifikatima lizozima je nakon modifikacije određena enzimska aktivnost.
Sama modifikacija je praćena UV/Vis spektrofotometrijom, SDS elektroforezom i
masenom spektrometrijom. Mesto vezivanja hinonskih derivata za molekul lizozima određeno je tehnikom MALDI TOF posle tripsinske digestije modifikata. S obzirom na
uočeno vezivanje avaronskih derivata za ε-amino grupu lizina (Lys-97) u lizozimu,
sintetisano je jedinjenje 4'-((5-tert-butoksikarbonil)amino)-5-karboksipentil)amino)-
avarona, koje je poslužilo kao model jedinjenje za dalja ispitivanja biološke aktivnosti
lizozim-hinonskog adukta...
aim of our work has been investigation of biological activity of
methylamino- and metoxy- derivatives of avarone, their bioconjugates of lysozyme and
study of the mechanism of their biological action.
For synthesis were chosen derivatives for which it was expected to have more
negative half-wave potential than avarone and therefore a higher activity. The selected
compounds are 3’-methylamino, 4’-methylamino- and 3’-methoxyavarone.
Antimicrobial activity of the synthesized derivatives was investigated towards
Gram positive bacteria: Bacilus subtilis, Clostridium sporogenes, Sreptosporangium
longisporum., Micrococcus flavus, Sarcina lutea and Staphylococcus aureus, Gram
negative bacteria: Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa,
Salmonella enteritidis and Escherichia coli and fungi cultures: Aspergilus niger,
Candida albicans and Sacharomyces cerevisiae, all by disc diffusion method. Toxicity
against Artemia salina nauplii was surveyed as well.
Antioxidant activit...y of avarone and its derivatives was assessed by DPPH assay.
Antitumor activity was determined for all derivatives towards eight lines of
tumor cells (myelogenous leukemia (K562), cervix carcinoma (HeLa), human
malignant melanoma cells (Fem-X), Jurkat T cell leukemia, estrogen receptor negative
breast carcinoma (MDA-MB-231), estrogen receptor positive breast carcinoma (MCF7),
human fetal lung fibroblast (MRC-5) and human promyelocytic leukemia (HL-60)). For
all derivatives toxicity towards lymphocytes was determined.
Model enzyme lysozyme was modified with the synthesized quinones and for all
the obtained bioconjugates MIC value towards Gram positive and Gram negative
bacteria were determined. Modification reaction was monitored by UV/VIS spectrophotometry, SDS electrophoresis and mass spectrometry. Binding position of
quinone derivatives on lysozyme was determined by MALDI TOF spectrometry after
trypsin digestion. Since the avarone derivatives were found to bind to lysine (Lys-97) in
lysozyme, 4’-((5-((tert-butoxycarbonyl)amino)-5-carboxypentyl)-amino)avarone has
been synthesized as a model compound for further investigation of the biological
activity of lysozyme―quinone aduct..,