Alternatív sejtelhalási mechanizmusok, mint a tumor ellenes terápia célpontjai = Alternative mechanisms of the cell death as targets for anti-tumor therapy

A daganatellenes kemoterápiában alakalmazott vegyületek egyik fontos hatása az, hogy daganatsejtekben aktív sejtelhalást váltanak ki. Az alternatív sejtelhalási típusok közül legismeretebb az apoptotikus és a nekrotikus forma, amelyeknek eltérő immunológiai következménye is vélelmezhető. Az apoptotikus jelpálya centrális elemei a kaszpáz proteázok. Azonban kaszpázaktivitás hiányában is (génexpressziós eltérések, oxidatív környezet) indukálódhat aktív sejtelhalás és fordítva: a kaszpázaktivitás nem jár együtt mindig sejtelhalással. Kutatásaink célja az volt, hogy olyan, elsősorban proteolitikus aktivitásokat azonosítsunk daganatsejtekben, amelyek helyettesíthetik a kaszpázok hiányát, illetve kaszpáz aktiválódás mellett is befolyásolják a sejtelhalást. Távlati célunk az, hogy ezeket a proteolitikus aktivitásokat megcélozva hatékonyabb daganatellenes terápiák lehetőségét alapozzunk meg. Eredményeink azt mutatják, hogy a cisztein katepszinek nem helyettesítik a kaszpázok apoptótikus funkcióját az általunk kidolgozott leukémia modellekben, de befolyásolhatják a kialakuló sejtelhalás típusát (apoptózis-nekrózis váltás). A proteaszóma aktivitás pro- és antiapoptotikus hatású is lehet kaszpáz-mediálta sejtelhaláskor függően az indukáló szerektől és sejttípustól. Kidolgoztunk egy új eljárást nekrotikus sejtek detektálására áramlásos citométerrel. | Induction of active cell death is the main effect of therapeutic anticancer drugs. The most known alternative cell death forms are apoptosis and necrosis that have also alternative immunological consequences. Caspase proteases are the central elements of the apoptotic pathway. However, in the absence of caspase activity (change in gene expression, oxidative environment) cell death can proceed and vica versa: caspase activation is not always accompanied with cell death. The purpose of our project was to identify proteolytic activities that may replace caspases in apoptosis or modulate the outcome of apoptotic process in the presence of caspase activation in cancer cells. Taking a long view, our aim is to target these proteolytic activities to improve the anticancer activities of therapeutic drugs. Our resuls demonstrated that cystein cathepsins do not substitute caspase activity in our leukemic models, but influence the emerging mode of cell death (apoptosis-necrosis switch). Proteasome activity can promote both pro- and anti-apoptotic effects in caspase-mediated cell death depending on inducer drugs and cell types. We have developed a new method to detect necrotic cells by flow cytometry

Identification of protein kinase inhibitors with a selective negative effect on the viability of Epstein-Barr virus infected B cell lines

Epstein-Barr virus (EBV) is a human herpesvirus, which is causally associated with the development of several B lymphocytic malignancies that include Burkitt's lymphomas, Hodgkin's disease, AIDS and posttransplant associated lymphomas. The transforming activity of EBV is orchestrated by several latent viral proteins that mimic and modulate cellular growth promoting and antiapoptotic signaling pathways, which involve among others the activity of protein kinases. In an effort to identify small molecule inhibitors of the growth of EBV-transformed B lymphocytes a library of 254 kinase inhibitors was screened. This effort identified two tyrosine kinase inhibitors and two MEK inhibitors that compromised preferentially the viability of EBV-infected human B lymphocytes. Our findings highlight the possible dependence of EBV-infected B lymphocytes on specific kinase-regulated pathways underlining the potential for the development of small molecule-based therapeutics that could target selectively EBV-associated human B lymphocyte malignancies. © 2014 Mavromatidis et al

SSTR4 illetve capsaicin receptor mediált neurogéngyulladás gátló gyógyszerhatóanyagok optimalizációja, karakterizációja és farmakofor model generálása = Optimization, characterization and pharmacophore modeling of SSTR4 or capsaicin receptor mediated neurogenic inflammation inhibiting drug candidates

A Klinika II fázisban lévő SSTR4 receptoron ható TT-232 heptapeptid olyan új, eltérő szerkezetű származékait állítottuk elő, ahol a biológiai hatás szempontjából fontos aminosavakat egyrészt egy pirazinon gyűrűvel ciklizáltuk, másrészt egy flexibilis magra építettünk fel szimmetrikusan három aminosavat, végül dipeptidamidokig egyszerűsítettük a szerkezetet. Az új követő-molekulákra erős sejtproliferáció- és hatékony neurogén gyulladás-gátlás a jellemző orális adagolás mellett is. A TT-232 védett N-terminális tripeptidje egerekben, az angiogenezis indukálás vizsgálatára szolgáló 'matrigél modellben' tízszer annyi vérér képződését eredményezte, mint a kontroll állatokban. A vegyületek és biológiai hatásuk védelmére szabadalmi bejelentéseket tettünk. A TT-232 molekula bizonyos szerkezeti elemeit felhasználva célzott molekulakönyvtárakat állítottunk elő, amelyekben a neurogén gyulladást gátló, de a kiindulási molekulánál kedvezőbb ADME paraméterekkel rendelkező tagokat találtunk. HPLC módszert dolgoztunk ki, amelyben a mért és számított lipofilitási adatok alapján meghatározhatjuk azt a lipofilitási tartományt (egy adott könyvtár körében) ahol nagy valószínűséggel jó antiproliferatív hatást mutató molekulák találhatók. A szomatosztatin és capsaicin receptor mediálásában fontos szerepet játzószó kináz molekulák tesztelésére beállítottuk az IMAP fluoreszcens mérési módszert, és az ezáltal kiválasztott hitmolekulákat karakterizáltuk szabadalmaztatási eljáráshoz. | Novel structurally different follow-up molecules of the heptapeptide acting on SSTR4 receptor TT-232, presently in Clinical phase II trial, have been synthesized. The biologically important amino acids were cyclized by a pyrazinon ring or the three amino acids were coupled symmetrically to a flexible core. The structure of the parent peptide was finally simplified into dipepitde amides. The new molecules effectively inhibit tumorous cell proliferation and neurogenic inflammation even during oral administration. The protected N-terminus tripeptide of TT-232 proved to be a strong angiogenesis promoting material in the so called matrigel test. The number of the newly formed vessels in the matrigel was ten times as much as in the control animals. New focused molecular libraries were generated using certain elements of the TT-232 molecule. Chemical and biological evaluation of the libraries resulted in active molecules with improved ADME parameters. An effective HPLC method has been developed for the determination of the lipophylicity range for good antiproliferative activity based on measured and calculated lipofilicity parameters of a given library. For screening kinase enzymes mediating the effect of somatostatin and capsaicin receptors a fluorescence assay (IMAP) has been developed and the selected hit molecules have been characterized for patenting

Characterization of new, efficient Mycobacterium tuberculosis topoisomerase-I inhibitors and their interaction with human ABC multidrug transporters

Drug resistant tuberculosis (TB) is a major worldwide health problem. In addition to the bacterial mechanisms, human drug transporters limiting the cellular accumulation and the pharmacological disposition of drugs also influence the efficacy of treatment. Mycobacterium tuberculosis topoisomerase-I (MtTopo-I) is a promising target for antimicrobial treatment. In our previous work we have identified several hit compounds targeting the MtTopo-I by in silico docking. Here we expand the scope of the compounds around three scaffolds associated with potent MtTopo-I inhibition. In addition to measuring the effect of newly generated compounds on MtTopo-I activity, we characterized the compounds' antimicrobial activity, toxicity in human cells, and interactions with human multidrug transporters. Some of the newly developed MtTopo-I inhibitors have strong antimicrobial activity and do not harm mammalian cells. Moreover, our studies revealed significant human ABC drug transporter interactions for several MtTopo-I compounds that may modify their ADME-Tox parameters and cellular effects. Promising new drug candidates may be selected based on these studies for further anti-TB drug development

Current Trends in Applications of Circulatory Microchimerism Detection in Transplantation

Primarily due to recent advances of detection techniques, microchimerism (the proportion of minor variant population is below 1%) has recently gained increasing attention in the field of transplantation. Availability of polymorphic markers, such as deletion insertion or single nucleotide polymorphisms along with a vast array of high sensitivity detection techniques, allow the accurate detection of small quantities of donor- or recipient-related materials. This diagnostic information can improve monitoring of allograft injuries in solid organ transplantations (SOT) as well as facilitate early detection of relapse in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the present review, genetic marker and detection platform options applicable for microchimerism detection are discussed. Furthermore, current results of relevant clinical studies in the context of microchimerism and SOT or allo-HSCT respectively are also summarized

Tetralone derivatives are MIF tautomerase inhibitors and attenuate macrophage activation and amplify the hypothermic response in endotoxemic mice

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine playing crucial role in immunity. MIF exerts a unique tautomerase enzymatic activity that has relevance concerning its multiple functions and its small molecule inhibitors have been proven to block its pro-inflammatory effects. Here we demonstrate that some of the E-2-arylmethylene-1-tetralones and their heteroanalogues efficiently bind to MIF’s active site and inhibit MIF tautomeric (enolase, ketolase activity) functions. A small set of the synthesised derivatives, namely compounds (4), (23), (24), (26) and (32), reduced inflammatory macrophage activation. Two of the selected compounds (24) and (26), however, markedly inhibited ROS and nitrite production, NF-κB activation, TNF-α, IL-6 and CCL-2 cytokine expression. Pre-treatment of mice with compound (24) exaggerated the hypothermic response to high dose of bacterial endotoxin. Our experiments suggest that tetralones and their derivatives inhibit MIF’s tautomeric functions and regulate macrophage activation and thermal changes in severe forms of systemic inflammation

Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes

Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in beta-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes. (C) 2015 Elsevier Ltd. All rights reserved

Inclusion complexation of the anticancer drug pomalidomide with cyclodextrins: fast dissolution and improved solubility

Pomalidomide (POM), a potent anticancer thalidomide analogue was characterized in terms of cyclodextrin complexation to improve its aqueous solubility and maintain its anti-angiogenic activity. The most promising cyclodextrin derivatives were selected by phase-solubility studies. From the investigated nine cyclodextrins – differing in cavity size, nature of substituents, degree of substitution and charge – the highest solubility increase was observed with sulfobutylether-β-cyclodextrin (SBE-β-CD). The inclusion complexation between POM and SBE-β-CD was further characterized with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), circular dichroism spectroscopy, fluorescence spectroscopy as well as X-ray powder diffraction method (XRD). Job plot titration by NMR and the AL-type phase-solubility diagram indicated 1:1 stoichiometry in a liquid state. Complementary analytical methods were employed for the determination of the stability constant of the complex; the advantages and disadvantages of the different approaches are also discussed. Inclusion complex formation was also assessed by molecular modelling study. Solid state complexation in a 1:1 M ratio was carried out by lyophilization and investigated by IR and XRD. The complex exhibited fast-dissolution with immediate release of POM, when compared to the pure drug at acidic and neutral pH. Kinetic analysis of POM release from lyophilized complex shows that Korsmeyer-Peppas and Weibull model described the best the dissolution kinetics. The cytotoxicity of the complex was tested against the LP-1 human myeloma cell line which revealed that supramolecular interactions did not significantly affect the anti-cancer activity of the drug. Overall, our results suggest that the inclusion complexation of POM with SBE-β-CD could be a promising approach for developing more effective POM formulations with increased solubility