Daganatos sejtek rezisztenciáját gátló vegyületek fejlesztése = Development of compounds targeting multidrug resistant cancer

A korszerű daganatellenes terápia jelentős sikerei ellenére a kemoterápiával szemben fellépő rezisztencia (multidrog rezisztencia, MDR) továbbra is megoldásra váró klinikai kihívás. Számos rosszindulatú megbetegedés, valamint az áttétet adó daganatok hatékony kezelése a terápia során rendszerint kialakuló MDR hatás miatt a mai napig nem megoldott. A rezisztens fenotípus gyakran társul az ABC-transzporterek családjába tartozó fehérjék emelkedett expressziójával. E család legismertebb képviselője a Pgp (ABCB1) membránfehérje, mely az ATP energiáját felhasználva megakadályozza a citosztatikus vegyületek sejten belüli felhalmozódását. A farmakogenomikai megközelítés révén lehetővé válik a személyre szabott gyógyítás, a daganatos megbetegedések molekuláris profiljához igazított kemoterápiás kezelés. A kutatás fő célja az volt, hogy a korábban kidolgozott farmakogenomikai módszer segítségével olyan ?MDR-inverz? vegyületeket fedezzünk fel, melyek szelektíven elpusztítják az egyébként multidrog rezisztens sejteket. Fontosabb eredményeink a következő pontokban összegezhetők: (i) módszerünk számos további MDR-inverz vegyületet azonosított; (ii) a szerkezetek analízise lehetővé tette QSAR modellek felállítását; (iii) javaslatot tettünk a vegyületek hatásmechanizmusára. Távlati tervünk, hogy a megismert MDR-inverz vegyületekből kiindulva originális gyógyszerkutatást folytassunk a rákos sejteket szelektíven pusztító molekulák preklinikai fejlesztése céljából. | Despite considerable advances in drug discovery, resistance to chemotherapy confounds the effective treatment of cancer patients. Cancer cells can become resistant to a single drug or they may acquire broad cross-resistance to mechanistically and structurally unrelated drugs (multidrug resistance (MDR)). ATP-Binding Cassette (ABC) proteins comprise the largest protein family, many members of which are of immediate medical importance and relevant to human health. The application of pharmacogenetics has the potential to improve the management of patients, particularly by providing the molecular basis for choosing among the increasing number of chemotherapeutic agents available for the treatment. The major aim of this project was to apply a pharmacogenomic approach to discover ?MDR-inverse? compounds that selectively kill multidrug resistant cancer cells. The results can be summarized as follows: (i) we identified a series of MDR-inverse compounds; (ii) we delineated structural features associated with their cytotoxic activity; (iii) we proposed a mechanism of action for the toxicity of newly identified MDR1-inverse compounds. Our future aim is to establish the framework for the preclinical development of the most promising MDR-inverse molecules, setting the stage for a fresh therapeutic approach that may eventually translate into improved patient care

Characterization of Disease-Associated Mutations in Human Transmembrane Proteins.

Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations

Az ABCA1 membránfehérje funkciójának és fehérje-kölcsönhatásainak vizsgálata = Investigation of the function and protein interactions of the ABCA1 membrane protein

A kutatás célja az ABCA1 membránfehérje működésének és fehérje-kölcsönhatásainak jellemzése volt. Új modellrendszereket alakítottunk ki Sf9 rovarsejt-bakulovírus és retrovirális expressziós rendszerek segítségével, részletesen vizsgáltuk a vad-típusú és mutáns ABCA1 fehérjék sejten belüli lokalizációját, működését és PDZ fehérjékkel való kölcsönhatását. Bizonyítottuk, hogy az ABCA1 fehérje mind az ApoA1-függő koleszterin kiáramlás, mind a Ca2+-aktivált sejtfelszíni foszfatidilszerin expozíció folyamatában fontos szerepet játszik. Elsőként mutattunk ki összefüggést egy vérzékenységi betegség és az ABCA1 működése között. Elemeztük az ABCA1 mutációnak hatását a betegségre jellemző hibás foszfatidilszerin expozícióban. Vizsgáltuk a lipidanyagcserére ható vegyületek hatását az ABCA1-hez köthető funkciókra, azonosítottunk két új gátló vegyületet. Megállapítottuk, hogy egy speciális PDZ fehérje a vizsgált ABC fehérjék közül egyedül az ABCA1 fehérjével lép kölcsönhatásba, más ABC transzporterekhez kötő egyéb PDZ fehérjék nem kötődtek az ABCA1-hez. Kimutattuk polarizált sejtekben az ABCA1, a b2-syntrophin és az utrophin bazolaterális ko-lokalizációját. A kidolgozott mérési módszereket más ABC transzporterek működésének vizsgálatára is eredményesen alkalmaztuk. Megkezdtük a foszfolipid-transzportért felelős ABC fehérjék azonosítását trombocitákban. | The aims of this project were the functional characterization of the ABCA1 protein and identification of its potential interactions with intracellular proteins. We installed new assay systems to analyse the function, subcellular localization and protein interactions of the wild-type and mutant ABCA1 versions, by using two expression systems: the baculovirus-Sf9 insect cell system and retrovirus based expression system for mammalian cells. We proved that ABCA1 plays a key role both in cellular ApoAI-mediated cholesterol removal pathway, and in the exofacial translocation of phosphatidylserine. Our results provided the first link between a defect in a transbilayer phospholipid transport pathway, that of ABCA1, and the bleeding phenotype. We analysed the effects of various mutations of ABCA1 on the Ca2+-stimulated PS exposition. We screened the influence of potential inhibitors on the ABCA1-dependent processes and identified new inhibitors of the PS exposition. We demonstrated that among the examined ABC transporters only ABCA1 binds b2-syntrophin. A diverse group of PDZ proteins that interacts with other ABC proteins does not bind to ABCA1. We showed basolateral colocalization of ABCA1 protein with b2-syntrophin and utrophin. The assays for ABCA1 characterization were applied for studying other ABC proteins successfully. We started the identification of ABC proteins involved in phospholipid translocation in platelets

A novel mathematical model describing adaptive cellular drug metabolism and toxicity in the chemoimmune system

Cells cope with the threat of xenobiotic stress by activating a complex molecular network that recognizes and eliminates chemically diverse toxic compounds. This "chemoimmune system" consists of cellular Phase I and Phase II metabolic enzymes, Phase 0 and Phase III ATP Binding Cassette (ABC) membrane transporters, and nuclear receptors regulating these components. In order to provide a systems biology characterization of the chemoimmune network, we designed a reaction kinetic model based on differential equations describing Phase 0-III participants and regulatory elements, and characterized cellular fitness to evaluate toxicity. In spite of the simplifications, the model recapitulates changes associated with acquired drug resistance and allows toxicity predictions under variable protein expression and xenobiotic exposure conditions. Our simulations suggest that multidrug ABC transporters at Phase 0 significantly facilitate the defense function of successive network members by lowering intracellular drug concentrations. The model was extended with a novel toxicity framework which opened the possibility of performing in silico cytotoxicity assays. The alterations of the in silico cytotoxicity curves show good agreement with in vitro cell killing experiments. The behavior of the simplified kinetic model suggests that it can serve as a basis for more complex models to efficiently predict xenobiotic and drug metabolism for human medical applications

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter

Impact of copper and iron binding properties on the anticancer activity of 8-hydroxyquinoline derived Mannich bases.

The anticancer activity of 8-hydroxyquinolines relies on complex formation with redox active copper and iron ions. Here we employ UV-visible spectrophotometry and EPR spectroscopy to compare proton dissociation and complex formation processes of the reference compound 8-hydroxyquinoline (Q-1) and three related Mannich bases to reveal possible correlations with biological activity. The studied derivatives harbor a CH2-N moiety at position 7 linked to morpholine (Q-2), piperidine (Q-3), and chlorine and fluorobenzylamino (Q-4) substituents. Solid phase structures of Q-3, Q-4·HCl·H2O, [(Cu(HQ-2)2)2]·(CH3OH)2·Cl4·(H2O)2, [Cu(Q-3)2]·Cl2 and [Cu(HQ-4)2(CH3OH)]·ZnCl4·CH3OH were characterized by single-crystal X-ray diffraction analysis. In addition, the redox properties of the copper and iron complexes were studied by cyclic voltammetry, and the direct reaction with physiologically relevant reductants (glutathione and ascorbic acid) was monitored. In vitro cytotoxicity studies conducted with the human uterine sarcoma MES-SA/Dx5 cell line reveal the significant cytotoxicity of Q-2, Q-3, and Q-4 in the sub- to low micromolar range (IC50 values 0.2-3.3 μM). Correlation analysis of the anticancer activity and the metal binding properties of the compound series indicates that, at physiological pH, weaker copper(ii) and iron(iii) binding results in elevated toxicity (e.g.Q4: pCu = 13.0, pFe = 6.8, IC50 = 0.2 μM vs.Q1: pCu = 15.1, pFe = 13.0 IC50 = 2.5 μM). Although the studied 8-hydroxyquinolines preferentially bind copper(ii) over iron(iii), the cyclic voltammetry data revealed that the more cytotoxic ligands preferentially stabilize the lower oxidation state of the metal ions. A linear relationship between the pKa (OH) and IC50 values of the studied 8-hydroxyquinolines was found. In summary, we identify Q-4 as a potent and selective anticancer candidate with significant toxicity in drug resistant cells

Az ABC-fehérjék Tudományos Iskolája: a gének regulációjától a transzport-mechanizmusig = The School of ABC-proteins: From Gene Regulation to Transport Mechanism

Megállapítottuk, hogy az ABCC6 gén duplikációi a low-copy repeat 16a elemekhez kötődnek, és ilyen duplikációk több alkalommal is bekövetkeztek különböző főemlős fajokban. Populációgenetikai vizsgálatunkban kimutattuk, hogy egy inaktív ABCC6 allél növeli a koronáriás artéria betegség (CAD) kialakulásának esélyét. Az ABCC6 expressziójánk szabályozását is vizsgáltuk, megállapítottuk, hogy az ERK1/2 szignál-útvonal és a HNF4 transzkripciós faktor felelős az ABCC6 szövet-specifikus szabályozásáért. Megalkottuk az ABCC6 transzporter homológia modelljét, és tanulmányoztuk az ismert 119 misszensz PXE-t okozó mutáció eloszlását. A komplex domén-domén határfelületeken a misszensz mutációk jelentős feldúsúlását figyeltük meg, ami ezen kapcsolatok fontosságának genetikai bizonyítéka. Az ABCC6 viszgálatára alkalmas új állat modellt fejlesztettünk ki: a zebrahal (Danio rerio) modell-rendszert. Bemutattuk, hogy az ecetmuslica MRP az ortológ humán ABCC fehérjékhez hasonló biokémiai tulajdonságokkal rendelkezik, így azok magas turnover-rel rendelkező modellje. Megfigyeltük, hogy a koleszterin szelektíven módosítja az ABCG2 aktivitását. Tanulmányoztuk az ABCG2 katalitikus ciklusa során fellépő intramolekuláris átrendeződéseket. Kifejlesztettünk egy sejtes modell-rendszert az ABCA1, egy fontos koleszterin transzporter tanulmányozására. Új módszert fejlesztettünk ki a kvantitatív PCR reprodukálhatóságának javítására. 15 közleményt publikáltunk szakmailag lektorált nemzetközi folyóiratokban. | We established that duplications of ABCC6 are associated to low-copy repeat 16a and such duplications have occurred several times in different primates. Our population genetic study revealed that one inactive allele of ABCC6 increases the risk of coronary artery disease (CAD) significantly. Th signal transduction pathways leading to the modulation of ABCC6 expression have also been deciphered: the ERK1/2 pathway and HNF4 are responsible for the tissue-specific regulation of ABCC6. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the structure. Significant clustering of the missense mutations has been found at domain-domain interfaces providing a genetic proof of the importance of these domain-domain interactions. A novel animal model to investigate ABCC6 has been developed: the zebrafish (Danio rerio) model system. We have demonstrated that the Drosophila MRP shares the biochemical features of its human ABCC orthologues and serves as a high turn-over model protein of human ABCC-type transporters. We have found that cholesterol selectively modulates the activity of ABCG2. We have studied the intramolecular rearrangments during the catalytic cycle of ABCG2. We have developed a cellular model system to study ABCA1, an important cholesterol transporter. We have developed a novel method to improve the quantitative PCR technique. We have published 15 papers in peer-reviewed international journals

Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance

There is a constant need for new therapies against multidrug resistant (MDR) cancer. An attractive strategy is to develop chelators that display significant antitumor activity in multidrug resistant cancer cell lines overexpressing the drug efflux pump P-glycoprotein. In this study we used a panel of sensitive and MDR cancer cell lines to evaluate the toxicity of picolinylidene and salicylidene thiosemicarbazone, arylhydrazone, as well as picolinylidene and salicylidene hydrazino-benzothiazole derivatives. Our results confirm the collateral sensitivity of MDR cells to isatin-β-thiosemicarbazones, and identify several chelator scaffolds with a potential to overcome multidrug resistance. Analysis of structure-activity-relationships within the investigated compound library indicates that NNS and NNN donor chelators show superior toxicity as compared to ONS derivatives regardless of the resistance status of the cells. © 2016 Elsevier Masson SAS