Tibor Vántus

Kinase pathways in chemoattractant-induced degranulation of neutrophils: The role of p38 mitogen-activated protein kinase activated by Src family kinases

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck−/−fgr−/−lyn−/− mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.

Dasatinib inhibits proinflammatory functions of mature human neutrophils.

Dasatinib is a tyrosine kinase inhibitor used to treat imatinib-resistant chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. At present, little is known about how dasatinib influences nonmalignant cells. In the present study, we tested the effect of dasatinib on functional responses of normal mature human neutrophils. Dasatinib completely blocked integrin- and Fc-receptor-mediated neutrophil functions, with the lowest IC(50) values below 10nM under serum-free conditions. Dasatinib caused a partial inhibition of neutrophil responses triggered by G-protein-coupled receptors and had a moderate effect on neutrophil responses triggered by microbial compounds. Whereas dasatinib inhibited neutrophil chemotaxis under static conditions in 2 dimensions, it did not affect migration under flow conditions or in 3-dimensional environments. Dasatinib did not have any major effect on phagocytosis or killing of bacteria by neutrophils. Adhesion of human neutrophils in the presence of whole serum was significantly inhibited by 50-100nM dasatinib, which corresponds to the reported serum concentrations in dasatinib-treated patients. Finally, ex vivo adhesion of mouse peripheral blood neutrophils was strongly reduced after oral administration of 5 mg/kg of dasatinib. Those results suggest that dasatinib treatment may affect the proinflammatory functions of mature neutrophils and raise the possibility that dasatinib-related compounds may provide clinical benefit in neutrophil-mediated inflammatory diseases.

Small-Molecule Inhibitors of NADPH Oxidase 4

NOX enzymes are the major contributors in many oxidative damage related diseases. Unfortunately, at present no specific NOX inhibitor is available. Here, we describe the discovery and development of novel NOX4 inhibitors. Compound libraries were tested in a cell-based assay as a primary screen, monitoring H2O2 production. Twenty-four compounds inhibited Nox4 activity with low-micromolar IC(50) values of which three were selected for further drug development.

Nanoparticles based on PLGA: Poloxamer blends for the delivery of proangiogenic growth factors

New blood vessel formation is a critical requirement for treating many vascular and ischemia related diseases, as well as for many tissue engineering applications. Angiogenesis and vasculogenesis, in fact, represent crucial processes for the functional regeneration of complex tissues through tissue engineering strategies. Several growth factors (GFs) and signaling molecules involved in blood vessels formation have been identified, but their application to the clinical setting is still strongly limited by their extremely short half-life in the body. To overcome these limitations, we have developed a new injectable controlled release device based on polymeric nanoparticles for the delivery of two natural proangiogenic GFs: platelet derived growth factor (PDGF-BB) and fibroblast growth factor (FGF-2). The nanoparticle system was prepared by a modified solvent diffusion technique, encapsulating the GF both in presence and in the absence of two stabilizing agents: bovine serum albumin (BSA) and heparin sodium salt (Hp). The developed nanocarriers were characterized for morphology, size, encapsulation efficiency, release kinetics in vitro and GF activity in cell cultures. The results have indicated that the coencapsulation of stabilizing agents can preserve the GF active structure and, in addition, increase their encapsulation efficiency into nanoparticles. Through this optimization process, we were able to raise the encapsulation efficiency of FGF-2 to 63%, and that of PDGF-BB to 87%. These PLGA:poloxamer blend nanoparticles loaded with GFs were able to release PDGF-BB and FGF-2 in a sustained fashion for more than a month. This work also confirms other positive features of PLGA:poloxamer nanoparticles. Namely, they are able to maintain their stability in simulated biological medium, and they are also nontoxic to cell culture models. Incubation of nanoparticles loaded with FGF-2 or PDGF-BB with endothelial cell culture models has confirmed that GFs are released in a bioactive form. Altogether, these results underline the interest of PLGA:poloxamer nanoparticles for the controlled delivery of GFs and substantiate their potential for the treatment of ischemic diseases and for tissue engineering applications.

PLGA:poloxamer blend micro- and nanoparticles as controlled release systems for synthetic proangiogenic factors.

Tissue engineering is one of the most promising research areas in bioregenerative medicine. However, the restoration of biological functionalities by implanting bioartificially engineered tissues is still highly limited because of their lack of vascular networks. The use of proangiogenic molecules delivered from a controlled release device is a promising strategy to induce tissue vascularization. Indeed, the controlled release system can enhance the therapeutic effect in vivo of many short half-life drugs, while circumventing the need for repeated administrations. In this work, PLGA:poloxamer blend based micro- and nanoparticles have been developed for the sustained delivery of a recently developed synthetic proangiogenic compound: SHA-2-22. Drug-loaded PLGA:poloxamer blend microparticles were prepared by an oil-in-oil solvent extraction/evaporation technique. Drug-loaded PLGA:poloxamer nanoparticles were prepared by a modified solvent diffusion technique. These drug carriers were characterized with regard to their physicochemical properties, morphology, drug encapsulation efficiency and release kinetics in vitro. The results show that by adjusting the formulation conditions, it is possible to obtain PLGA:poloxamer micro- and nanoparticles with very high drug loadings, and with the capacity to release the active compound in a controlled way for up to one month. In vitro cell assays performed in an endothelial cell model confirmed the bioactivity of SHA-22-2 encapsulated in PLGA:poloxamer microparticles.

The tumor-selective somatostatin analog, TT2-32 induces a biphasic activation of phosphotyrosine phosphatase activity in human colon tumor cell line, SW620

Somatostatin has been demonstrated to activate phosphotyrosine phosphatases (PTPases) in pancreatic cells. In this work we studied the effect of a tumor-selective somatostatin structural derivative, TT2-32, on the PTPase activity in the SW620 human colon tumor cell line. TT2-32 caused a strong inhibition of cell proliferation. In response to TT2-32 we found a rapid and sustained increase (5-30 min) in PTPase activity showing two maxima at 0.1 and 30 microM concentrations, respectively. During short-term incubation tyrosine kinase activity was much less affected by TT2-32. TT2-32-induced activation of PTPases may be an important early step in the signaling cascade in the inhibition of cell proliferation in colon carcinomas.

Synthesis of Somatostatin Analogues Containing C-Terminal Adamantane and Their Antiproliferative Properties

On the basis of the structure of somatostatin analogue TT-232 (1), which exhibited a highly potent antitumor activity, we synthesized small linear peptide derivatives and evaluated their antitumor and apoptotic activity. Of them, Boc-Tyr-D-Trp-1-adamantylamide (5) had the most potent cell antiproliferative activity in SW480 and A431 cell lines, which was supported in A431 cell lines by FACS analysis that demonstrated a major increase in DNA fragmentation in the subG1 fraction.

Discovery and Biological Evaluation of Novel Dual EGFR/c-Met Inhibitors.

Activating mutations in the epidermal growth factor receptor (EGFR) have been identified in a subset of non-small cell lung cancer (NSCLC), which is one of the leading cancer types worldwide. Application of EGFR tyrosine kinase inhibitors leads to acquired resistance by secondary EGFR mutations or by amplification of the hepatocyte growth factor receptor (c-Met) gene. Although several EGFR and c-Met inhibitors have been reported, potent dual EGFR/c-Met inhibitors, which can overcome this latter resistance mechanism, have hitherto not been published and have not reached clinical trials. In the present study we have identified dual EGFR/c-Met inhibitors and designed novel N-[4-(quinolin-4-yloxy)-phenyl]-biarylsulfonamide derivatives, which inhibit the c-Met receptor and both the wild-type and the activating mutant EGFR kinases in nanomolar range. We have demonstrated by Western blot analysis that compound 10 inhibits EGFR and c-Met phosphorylation at cellular level and effectively inhibits viability of the NSCLC cell lines.

Comparative Characterization of Experimental and Calculated Lipophilicity and Anti-Tumour Activity of Isochromanone Derivatives

Compound lipophilicity connected to ADME(T)(a) has great importance in drug development and it has to be evaluated by the generally used drug developmental process. In addition to the importance of lipophilicity in ADMET, recently it has been reported that lipophilicity of small molecules correlates with their antiproliferative activity because of certain specific hydrophobic and lipophilic interactions. Due to the complexity of ADME(T) parameters an efficient and fast method is needed to characterize the many promising candidate lead molecules as a preselection in order not to be rejected from the latter phase of drug development. In the present paper we provide an overview of the importance of lipophilicity of drug candidates for biological action and for ADME(T) and describe a novel approach for drug-likeness characterization of a molecular library using correlation study between lipophilicity and biological activity. Lipophilicity and molecular characteristics have been measured, predicted and optimized for a diverse library from which the best members have been selected to describe their biological, chemical and drug-likeness properties. Molecules were selected from the family of alpha,beta-unsaturated ketones and thorough HPLC characterization for lipophilicity and morphological, antiproliferative and flow cytometric studies were carried out on them. Based on the results 17 member isochromanone library including E and Z geometric isomers were selected for further characterization. In this focused library linear correlation has been found between the calculated and measured lipophilicity and significant parabolic correlation was found between the antiproliferative effect and lipophilicity. Using our efficient and fast method, from a diverse library, we identified an outstandingly effective inhibitor of A431 tumour cell growth via a PARP(a) cleavage dependent apoptosis. In summary the optimized HPLC analyses of lipophilicity combined with the cell-culture assay, introduced above, resulted in the determination of an optimal lipophilicity range. This optimized lipophilicity range should be used in designing novel antiproliferative compounds.

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 β-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.