Proučavanje interakcija biološki aktivnog hinona avarona i njegovih derivata sa lizozimom, linearnom i cirkularnom dezoksiribonukleinskom kiselinom

Abstract

Cilj našeg rada bio je ispitivanje biološke aktivnosti metilamino- i metoksiderivataavarona i razjašnjavanje mehanizma njihovog biološkog dejstva.Za sintezu su izabrani derivati za koje se očekivalo da će imati negativnijipolutalasni potencijal od avarona i samim tim pokazivati povećanu aktivnost. Sintetisanisu 4'-(metilamino)-avaron, 3'-(metilamino)-avaron i 3'-metoksi-avaron.Antibakterijska aktivnost dobijenih derivata je ispitivana prema gram-pozitivnimbakterijama: Bacillus subtilis, Clostridium sporogenes, Streptosporangiumlongisporum, Micrococcus flavus, Sarcina lutea i Staphylococcus aureus, prema gramnegativnimbakterijama: Klebsiella pneumoniae, Proteus vulgaris, Pseudomonasaeruginosa, Salmonella enteritidis, Escherichia coli i prema kulturama gljivica:Aspergillus niger, Candida albicans i Saccharomyces cerevisiae disk-difuzionommetodom. Takođe, ispitivana je i toksičnost derivata na račiće Artemia salina.Potencijalna antioksidativna aktivnost avarona i dobijenih derivata ispitivana jetestom 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 naestrogeni receptor, rak dojke negativan na estrogeni receptor, rak pluća, leukemijaT-ćelija i promijelocitna leukemija) pri čemu je ispitivana i njihova citotoksičnost nalimfocite.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 imasenom spektrometrijom. Mesto vezivanja hinonskih derivata za molekul lizozima određeno je tehnikom MALDI TOF posle tripsinske digestije modifikata. S obzirom nauočeno 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 aktivnostilizozim-hinonskog adukta...

aim of our work has been investigation of biological activity ofmethylamino- and metoxy- derivatives of avarone, their bioconjugates of lysozyme andstudy of the mechanism of their biological action.For synthesis were chosen derivatives for which it was expected to have morenegative half-wave potential than avarone and therefore a higher activity. The selectedcompounds are 3’-methylamino, 4’-methylamino- and 3’-methoxyavarone.Antimicrobial activity of the synthesized derivatives was investigated towardsGram positive bacteria: Bacilus subtilis, Clostridium sporogenes, Sreptosporangiumlongisporum., Micrococcus flavus, Sarcina lutea and Staphylococcus aureus, Gramnegative 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. Toxicityagainst Artemia salina nauplii was surveyed as well.Antioxidant activity of avarone and its derivatives was assessed by DPPH assay.Antitumor activity was determined for all derivatives towards eight lines oftumor cells (myelogenous leukemia (K562), cervix carcinoma (HeLa), humanmalignant melanoma cells (Fem-X), Jurkat T cell leukemia, estrogen receptor negativebreast carcinoma (MDA-MB-231), estrogen receptor positive breast carcinoma (MCF7),human fetal lung fibroblast (MRC-5) and human promyelocytic leukemia (HL-60)). Forall derivatives toxicity towards lymphocytes was determined.Model enzyme lysozyme was modified with the synthesized quinones and for allthe obtained bioconjugates MIC value towards Gram positive and Gram negativebacteria were determined. Modification reaction was monitored by UV/VIS spectrophotometry, SDS electrophoresis and mass spectrometry. Binding position ofquinone derivatives on lysozyme was determined by MALDI TOF spectrometry aftertrypsin digestion. Since the avarone derivatives were found to bind to lysine (Lys-97) inlysozyme, 4’-((5-((tert-butoxycarbonyl)amino)-5-carboxypentyl)-amino)avarone hasbeen synthesized as a model compound for further investigation of the biologicalactivity of lysozyme―quinone aduct..,