Nóra Breza

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.

Signalling Inhibitors Against Mycobacterium tuberculosis – Early Days of a New Therapeutic Concept in Tuberculosis

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains.

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.

Biochemical Assay-Based Selectivity Profiling of Clinically Relevant Kinase Inhibitors on Mutant Forms of EGF Receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.

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.

Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.

Synthesis and biological evaluation of novel pyrido[2,3-b]pyrazines inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines

A series of novel pyrido[2,3-b]pyrazines were synthesized as potential antitumor agents for erlotinib-resistant tumors. Known signal inhibitor compounds from our Nested Chemical Library were tested in phenotypic assays on erlotinib-sensitive PC9 and erlotinib-resistant PC9-ER cell lines to find a compound class to be active on erlotinib resistant cell lines. Based on the screening data, novel pyrido[2,3-b]pyrazines were designed and synthesized. The effect of the substituent position of the heteroaromatic moiety in position 7 and the importance of unsubstituted position 2 of the pyridopyrazine core were explored. Compound 7n had an IC50 value of 0.09 μM for the inhibition of PC9 and 0.15 μM for the inhibition of PC9-ER. We found that some lead compounds of these structures overcome erlotinib-resistance which might become promising drug candidates to fight against NSCLC with EGFR T790M mutation. The signaling network(s) involved in the mechanism(s) of action of these novel compounds in overcoming erlotinib resistance remain to be elucidated.

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.