István Szabadkai

AXL Is a Potential Target for Therapeutic Intervention in Breast Cancer Progression

Protein kinases play important roles in tumor development and progression. A variety of members of this family of signal transduction enzymes serve as targets for therapeutic intervention in cancer. We have identified the receptor tyrosine kinase (RTK) AXL as a potential mediator of motility and invasivity of breast cancer cells. AXL is expressed in most highly invasive breast cancer cells, but not in breast cancer cells of low invasivity. Ectopic expression of AXL was sufficient to confer a highly invasive phenotype to weakly invasive MCF7 breast cancer cells. Experimental inhibition of AXL signaling by a dominant-negative AXL mutant, an antibody against the extracellular domain of AXL, or short hairpin RNA knockdown of AXL decreased motility and invasivity of highly invasive breast cancer cells. To selectively interfere with cancer cell properties defining the rate of disease progression, we identified 3-quinolinecarbonitrile compounds, which displayed potent inhibitory activity against AXL and showed strong interference with motility and invasivity of breast cancer cells. Our findings validated the RTK AXL as a critical element in the signaling network that governs motility and invasivity of breast cancer cells, and allowed the identification of experimental anti-AXL small molecular inhibitors that represent lead substances for the development of antimetastatic breast cancer therapy.

NADPH Oxidase 4 Is Expressed in Pulmonary Artery Adventitia and Contributes to Hypertensive Vascular Remodeling

Objective— Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. Approach and Results— Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. Conclusion— These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

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.

Anticytolytic Screen Identifies Inhibitors of Mycobacterial Virulence Protein Secretion

Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy

The Axl receptor tyrosine kinase (RTK) has been established as a strong candidate for targeted therapy of cancer. However, the benefits of targeted therapies are limited due to acquired resistance and activation of alternative RTKs. Therefore, we asked if cancer cells are able to overcome targeted Axl therapies. Here, we demonstrate that inhibition of Axl by short interfering RNA or the tyrosine kinase inhibitor (TKI) BMS777607 induces the expression of human epidermal growth factor receptor 3 (HER3) and the neuregulin 1(NRG1)-dependent phosphorylation of HER3 in MDA-MB231 and Ovcar8 cells. Moreover, analysis of 20 Axl-expressing cancer cell lines of different tissue origin indicates a low basal phosphorylation of RAC-α serine/threonine-protein kinase (AKT) as a general requirement for HER3 activation on Axl inhibition. Consequently, phosphorylation of AKT arises as an independent biomarker for Axl treatment. Additionally, we introduce phosphorylation of HER3 as an independent pharmacodynamic biomarker for monitoring of anti-Axl therapy response. Inhibition of cell viability by BMS777607 could be rescued by NRG1-dependent activation of HER3, suggesting an escape mechanism by tumor microenvironment. The Axl-TKI MPCD84111 simultaneously blocked Axl and HER2/3 signaling and thereby prohibited HER3 feedback activation. Furthermore, dual inhibition of Axl and HER2/3 using BMS777607 and lapatinib led to a significant inhibition of cell viability in Axl-expressing MDA-MB231 and Ovcar8 cells. Therefore, we conclude that, in patient cohorts with expression of Axl and low basal activity of AKT, a combined inhibition of Axl and HER2/3 kinase would be beneficial to overcome acquired resistance to Axl-targeted therapies.

Developing FGFR4 Inhibitors As Potential Anti-Cancer Agents Via In Silico Design, Supported by In Vitro and Cell-Based Testing

Fibroblast growth factor receptor-4 (FGFR4) is a tyrosine kinase with a range of important physiological functions. However, it is also frequently mutated in various cancers and is now generating significant interest as a potential therapeutic target. Unfortunately, biochemical characterization of its role in disease, and further evaluation as a drug target is hampered by lack of a specific inhibitor. We aimed to discover new inhibitors for FGFR4 ab initio using a strategy combining in silico, in vitro and cell-based assays. We used the homologous FGFR1 to calculate docking scores of a chemically-diverse library of approximately 2000 potential kinase inhibitors. Nineteen potential inhibitors and ten randomly- selected negative controls were taken forward for in vitro FGFR4 kinase assays. All compounds with good docking scores significantly inhibited FGFR4 kinase activity, some with sub-micromolar (most potent being V4-015 with an IC(50) of 0.04 μM). Four of these compounds also demonstrated substantial activity in cellular assays using the FGFR4- overexpressing breast carcinoma cell line, MDA-MB453. Through immunoblot assays, these compounds were shown to block the phosphorylation of the FGFR4 adaptor protein, FGFR substrate protein-2α (FRS2α). The most potent compound to date, V4-015, suppressed proliferation of MDA-MB453 cells at sub-micromolar concentrations, activated the pro-apoptotic caspases 3/7 and inhibited cellular migration. While achieving complete selectivity of this compound for FGFR4 will require further lead optimization, this study has successfully identified new chemical scaffolds with unprecedented FGFR4 inhibition capacities that will support mechanism of action studies and future anti-cancer drug design.

Combined inhibition of AXL, Lyn and p130Cas kinases block migration of triple negative breast cancer cells

Blocking the migration of metastatic cancer cells is a major goal in the therapy of cancer. The receptor tyrosine kinase AXL is one of the main triggers for cancer cell migration in neoplasia of breast, colon, skin, thyroid and prostate. In our study we analyzed the effect of AXL inhibition on cell motility and viability in triple negative breast cancer cell lines overexpressing AXL. Thereby we reveal that the compound BMS777607, exhibiting the lowest IC50 values for inhibition of AXL kinase activity in the studied cell lines, attenuates cell motility to a lower extent than the kinase inhibitors MPCD84111 and SKI606. By analyzing the target kinases of MPCD84111 and SKI606 with kinase profiling assays we identified Lyn, a Src family kinase, as a target of both compounds. Knockdown of Lyn and the migration-related CRK-associated substrate (p130Cas), had a significant inhibitory effect on cell migration. Taken together, our findings highlight the importance of combinatorial or multikinase inhibition of non-receptor tyrosine kinases and AXL receptor tyrosine kinase in the therapy of triple negative breast cancer.

Pharmacophore and binding analysis of known and novel B-RAF kinase inhibitors.

The extensively investigated serine/threonine kinase, B-RAF, is a member of the RAS/RAF/MEK/ERK pathway. It plays important role in the regulation of cell growth, differentiation and survival. The mutation of B-RAF occurs frequently in melanomas and colon tumors; therefore, it is considered as an outstanding therapeutic target. In recent years a great number of B-RAF inhibitors have been reported and this number is expected to increase. The aim of our work was to compare the structures and binding mode of the published B-RAF inhibitors, and then to apply the correlations found for the explanation of our experimental results. In the first part of this paper we describe the main pharmacophore features of the co-crysallized B-RAF inhibitors published in the literature, focusing on the binding modes and common structural elements. In the second part we present and characterize our recently developed B-RAF inhibitor family by application of in silico methods and in vitro kinetic profiling. The inhibitory activity of these compounds was determined in biochemical kinase- and cell-based assays. The docking and assay results support our conclusion that the presented compound family belongs to the type I 1/2 subgroup, they inhibit B-RAF and B-RAF(V600E) mutant in a sub-micromolar range and most of them show selectivity towards B-RAF(V600E) mutant expressing cell lines with equal or even better IC50 values than sorafenib.

Novel members of quinoline compound family enhance insulin secretion in RIN-5AH beta cells and in rat pancreatic islet microtissue

According to clinical data, some tyrosine kinase inhibitors (TKIs) possess antidiabetic effects. Several proposed mechanisms were assigned to them, however their mode of action is not clear. Our hypothesis was that they directly stimulate insulin release in beta cells. In our screening approach we demonstrated that some commercially available TKIs and many novel synthesized analogues were able to induce insulin secretion in RIN-5AH beta cells. Our aim was to find efficient, more selective and less toxic compounds. Out of several hits, we chose members from a compound family with quinoline core structure for further investigation. Here we present the studies done with these novel compounds and reveal structure activity relationships and mechanism of action. One of the most potent compounds (compound 9) lost its affinity to kinases, but efficiently increased calcium influx. In the presence of calcium channel inhibitors, the insulinotropic effect was attenuated or completely abrogated. While the quinoline TKI, bosutinib substantially inhibited tyrosine phosphorylation, compound 9 had no such effect. Molecular docking studies further supported our data. We confirmed that some TKIs possess antidiabetic effects, moreover, we present a novel compound family developed from the TKI, bosutinib and optimized for the modulation of insulin secretion.

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.