Interakcija kompleksa platine i rutenijuma sa pankreasnom fosfolipazom A2 i fosfolipidima

Abstract

Kompleksi platine se koriste u tretmanima razlicitih tipova kancera od kasnih sedamdesetih godina prošlog veka, kada je zvanicno odobrena upotreba neorganskog kompleksa cis-diamino-dihloroplatine (II). Princip terapije zasnovane na kompleksima platine je interakcija sa molekulom DNK, pri cemu se onemogucava dalja deoba celija i podstice programirana smrt celija, odnosno apoptoza. Glavni nedostaci ovog vida terapije su sporedni toksicni efekti, koji su rezultat interakcije kompleksa sa zdravim celijama i razlicitim biomolekulima na njihovom putu do celijskog jedra, kao i razvijanje celijske rezistencije na lek. Osim sinteze novih kompleksa, kombinacijom drugih liganada i metalnih centara i razvoja metoda za ciljanu dostavu lekova, jedan od nacina poboljšanja hemoterapije jeste izucavanje novih celijskih meta, kao što su enzimi neophodni za rast i razvoj tkiva kancera i stvaranje metastaza. Jedan od enzima oznacenih kao „meta“ ili ciljani molekul u terapiji kancera i zapaljenskih procesa je fosfolipaza A2. Ovaj enzim katalizuje hidrolizu membranskih fosfolipida, pri cemu se oslobadaju odgovarajuci lizolipidi i masne kiseline, odnosno signalni molekuli koji ucestvuju u procesima apoptoze, karcinogeneze i zapaljenskih procesa. Nivo fosfolipaze A2 i njena aktivnost su povišeni u kanceroznim tkvima i celijama, dok je pokazano da specificni inhibitori ovog enzima umanjuju metastatski potencijal kancera. U ovom radu ispitan je uticaj kompleksa platine i rutenijuma: [PtCl2(dach)], [PtCl4(bipy)], [PtCl4(en)], [PtCl2(en)], [RuCl2(bipy)2]Cl, [RuCl2(en)2]Cl i cis- [PtCl2(NH3)2] na strukturu i aktivnost pankreasne fosfolipaze A2. Imajuci u vidu da enzimska aktivnost fosfolipaze A2 zavisi od sastava i organizacije supstrata, odnosno membranskih lipida, istovremeno je ispitan i efekat koji isti kompleksi metala imaju na fosfolipidni dvosloj (lipozome i eritrocite) i strukturu fosfolipida. viii Eksperimentalni rezultati interakcije kompleksa sa fosfolipazom A2 i sa fosfolipidima prikupljeni su korišcenjem metoda: MALDI-TOF masene spektrometrije, fluorescentne spektrometrije, tankoslojne hromatografije, nuklearne magnetne rezonancije i UV/VIS spektrofotometrije. Svi ispitani kompleksi uticu na organizaciju i propustljivost fosfolipidnih membrana i posredno uticu na aktivnost fosfolipaze A2. Primenom navedenih metoda nije detektovano stvaranje adukata izmedu kompleksa metala i fosfolipida. Dva kompleksa, [PtCl4(bipy)] i [RuCl2(bipy)2]Cl, stvaraju adukte sa fosfolipazom A2 i direktno inhibiraju njenu enzimatsku aktivnost. Tip inhibicije je akompetetivan, što znaci da se oba kompleksa intenzivnije vezuju i inhibiraju enzim u prisustvu supstrata, što je potvrdeno odgovarajucim kinetickih parametarima i razlikom u masenim spektrima kompleksa i enzima sa i bez dodatka supstrata.

Platinum complexes have been used for treatments of various types of cancer since the late seventies, when the use of inorganic complex cisdiamminedichloroplatinum( II) was officially approved. The principle of therapy based on platinum complexes is their interaction with DNA molecules, which prevents further cell division and promotes programmed cell death or apoptosis. The main disadvantages of this therapy are toxic side effects, as a consequence of interaction of platinum complexes with a variety of biomolecules and healthy cells on their way to the cell nucleus, as well as the development of cellular resistance to the drug. In addition to the synthesis of new metal complexes with different ligands or/and metal centers and to the development of targeted drug delivery, the improvement of chemotherapy is possible to achieve by revealing new cellular targets, such as enzymes necessary for the growth and spread of cancer tissues and formation of metastasis. One of the enzymes marked as a target of cancer and inflammation therapeutics is phospholipase A2. Phospholipase A2 catalyzes the hydrolysis of membrane phospholipids followed by the release of appropriate lysophospholipids and fatty acids, signaling molecules involved in apoptosis, inflammation and carcinogenesis. Cancer tissues are associated to elevated concentration and activity of phospholipase A2 and it is well known that specific inhibitors of this enzyme reduce the metastatic potential of cancer. In this dissertation the effect of platinum and ruthenium complexes: [PtCl2(dach)], [PtCl4(bipy)], [PtCl4(en)], [PtCl2(en)], [RuCl2(bipy)2]Cl, [RuCl2(en)2]Cl and cis-[PtCl2(NH3)2] on the structure and activity of pancreatic phospholipase A2 is investigated. Bearing in mind that the enzymatic activity of phospholipase A2 depends on the composition and organization of the substrate, the effect of same metal complexes on the phospholipid bilayers (liposomes and erythrocytes) is examined. x Experimental results were obtained by following methods: MALDI-TOF mass spectrometry, fluorescence spectrometry, thin-layer chromatography, nuclear magnetic resonance and UV/VIS spectrophotometry. All investigated complexes affect the organization and permeability of phospholipid membranes and indirectly modify the activity of phospholipase A2. Adducts between metal complexes and phospholipids were not detected by chosen methods. Complexes, [PtCl4(bipy)] and [RuCl2(bipy)2]Cl, create adducts with phospholipase A2 and directly inhibit its enzymatic activity. The mechanism of inhibition is mainly uncompetetive, which means that both complexes bind and inhibit the enzyme intensively in the presence of substrate. Type of inhibition is determined due to calculation of kinetic parameters and confirmed by the differences in mass spectra of the enzyme and metal complexes with and without the presence of substrate.