Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

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 ß-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.

Targeting vascular endothelial growth factor receptor 2 and protein kinase D1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways.

[Pyrido[2,3-b]pyrazines inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines, and their preparation via regioselective condensation reaction]. / Erlotinib-érzékeny és erlotinib-rezisztens sejtvonalakat gátló pirido[2,3-b] pirazinok, és eloállításuk régiószelektív kondenzációs reakcióval.

The EGFR inhibitor erlotinib possesses high anti-tumor effect but despite the good clinical responses in most of the cases recrudescence occures. This can be attributed to a secondary, acquired mutation causing resistance to tyrosine kinase inhibitors. In our work we were looking for small-molecule inhibitors, which simultaneously affect on the proliferation of erlotinib-sensitive PC9 cells and PC9-ER erlotinib-resistant cells. A set of molecules were selected from Vichem Chemie Research Ltd.'s kinase inhibitor compound library (Nested Chemical Library™). According to the results of medium throughput screening (MTS) of this set of compounds, novel structures with pyrido[2,3-b]pyrazine core were designed. These compounds were proved to be effective inhibitors of resistant cells in phenotypic screening. Based on these results structure-activity relationships were set up. The pyrido[2,3-b]pyrazine core was synthesized by a condensation reaction, which resulting two asymmetric products. In the reaction two regioisomer intermediates formed, and one of the products is the intermediate of the effective compounds. This condensation reaction was optimized, the regioisomers were identified by NMR analysis and X-ray crystallography. As a result of optimization we found that lower reaction temperature and replacement of dimethylformamide solvent with trifluoroacetic acid provided the undesired isomer in less than 2 % ratio.

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.

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.

Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors.

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

[Development and study of structure-activity relationship of drugs against Mycobacterium tuberculosis]. / Mycobacterium tuberculosis ellenes hatóanyagok fejlesztése és szerkezet-hatás összefüggéseinek vizsgálata.

Tuberculosis is considered to be one of the major health problem not only in the less developed countries but in the economically developed countries as well. Roughly one third of the world's population are infected with Mycobacterium tuberculosis and a significant part of them are carriers of latent tuberculosis. From ten percent of these latent infections are developing the active TB disease and fifty percent of them die from the illness without appropriate treatment. The drug-resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) and TB-HIV co-infection attracted attention to the most serious infectious disease. Inhibition of alternative signaling pathways were an important part of the research strategies for cancer and inflammatory diseases in recent years. In case of Mycobacterium tuberculosis such pathways were also identified, for example, three serine-threonine kinases (PknA, PknB, PknG) which are necessary and essential for bacterial growth. In this paper we summarize our best anti-TB active compounds, their biological effects and structure-activity relationships using in silico modeling, biochemical measurements and tests on active bacteria.

[Developing FGFR inhibitors as potential anti-cancer agents]. / Potenciális antitumor hatású FGFR inhibitorok fejlesztése.

Fibroblast Growth Factor Receptor (FGFR) family is a sequentially highly related subgroup of membrane proteins consisting of four tyrosine kinase type enzyme: FGFR1, FGFR2, FGFR3 and FGFR4. These are kinases of great interest in a wide spectrum of physiological processes such as tissue repair via controlling cell proliferation. As initiatiors of cell proliferation, in some cases they have leading roles in several types of cancer, eg. breast cancer, pancreas cancer, gastric tumors and multiple myeloma via overexpression and/or mutation. This phenomenon makes them promising targets for drug development in order to develop signal transduction therapies based on small molecule FGFR inhibitors. We have developed two main groups of lead molecules: compounds with benzotiophene and oxindole cores utilizing numerous methods from in silico modelling via in vitro biochemichal assays and testing on relevant cell lines to cytotoxicity assays.

[Development and biochemical characterization of EGFR/c-Met dual inhibitors]. / EGFR/c-Met kettosgátlók fejlesztése és biokémiai vizsgálata.

The epidermal growth factor receptor (EGFR) family has been well-known for more than ten years as the target of non-small lung carcinoma (NSCLC) which is one of the leading cause of mortality among the cancer types. The receptor tyrosine kinase inhibitors (gefitinib, erlotinib, lapatinib) which have been applied in the therapy, are not able to inhibit the progression of this disease perfectly because of resistance. It has been demonstrated that the amplification of mesenchymal-epithelial transition factor (c-Met) or secondary mutation of EGFR kinase causes the resistance against EGFR inhibitors in 18-20 percent of the cases. Clinical candidates inhibiting both of EGFR and c-Met kinases are unknown in the literature. We have developed quinoline-based inhibitors in our research project, which inhibit both kinases in submicromolar range in enzymatic assays, moreover we have demonstrated by western blot analysis that these compounds inhibit the autophosphorylation in vivo. The binding of the effective compounds was examined by in silico and docking simulations.

[Synthesis of 4-Chloro-6-(substituted-phenyl)-pyrimidines]. / Eljárás 4-klór-6-(szubsztituált-fenil)-pirimidinek eloállítására.

4-Phenylamino-6-phenyl-pyrimidine is a common core structure of several, important enzyme inhibitors. The common key intermediate of these compounds, 4-chloro-6-substituted phenyl pyrimidine, was synthesised by conventional methods without the application of special catalysts in contrary to the published methods. Here we present two different synthetic procedures for the preparation of the title compound. The results of these processes depend on the substituent of the 6-phenyl ring as it is demonstrated on the presented examples. The reactions can be performed via traditional heating either with microwave assistance.

Proteomics strategy for quantitative protein interaction profiling in cell extracts.

We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.

Large-scale proteomics analysis of the human kinome.

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4-11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.

Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.

2,3-benzodiazepine-type AMPA receptor antagonists and their neuroprotective effects.

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1-4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure-activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.

Drug discovery in the kinase inhibitory field using the Nested Chemical Library technology.

Kinase inhibitors are at the forefront of modern drug research, where mostly three technologies are used for hit-and-lead finding: high throughput screening of random libraries, three-dimensional structure-based drug design based on X-ray data, and focused libraries around limited number of new cores. Our novel Nested Chemical Library (NCL) (Vichem Chemie Research Ltd., Budapest, Hungary) technology is based on a knowledge base approach, where focused libraries around selected cores are used to generate pharmacophore models. NCL was designed on the platform of a diverse kinase inhibitory library organized around 97 core structures. We have established a unique, proprietary kinase inhibitory chemistry around these core structures with small focused sublibraries around each core. All the compounds in our NCL library are stored in a big unified Structured Query Language database along with their measured and calculated physicochemical and ADME/toxicity (ADMET) properties, together with thousands of molecular descriptors calculated for each compound. Biochemical kinase inhibitory assays on selected, cloned kinase enzymes for a few hundred NCL compound sets can provide sufficient biological data for rational computerized design of new analogues, based on our pharmacophore model-generating 3DNET4W QSPAR (quantitative structure-property/activity relationships) approach. Using this pharmacophore modeling approach and the ADMET filters, we can preselect synthesizable compounds for hit-and-lead optimization. Starting from this point and integrating the information from QSPAR, high-quality leads can be generated within a small number of optimization cycles. Applying NCL technology we have developed lead compounds for several validated kinase targets.

Cellular targets of gefitinib.

Targeted inhibition of protein kinases with small molecule drugs has evolved into a viable approach for anticancer therapy. However, the true selectivity of these therapeutic agents has remained unclear. Here, we used a proteomic method to profile the cellular targets of the clinical epidermal growth factor receptor kinase inhibitor gefitinib. Our data suggest alternative cellular modes of action for gefitinib and provide rationales for the development of related drugs.

Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors.

Some protein kinases are known to acquire resistance to selective small molecule inhibitors upon mutation of a conserved threonine at the ATP binding site to a larger residue. Here, we performed a comprehensive mutational analysis of this structural element and determined the cellular sensitivities of several disease-relevant tyrosine kinases against various inhibitors. Mutant kinases possessing a larger side chain at the critical site showed resistance to most compounds tested, such as ZD1839, PP1, AG1296, STI571, and a pyrido[2,3-d]pyrimidine inhibitor. In contrast, indolinones affected both wild-type and mutant kinases with similar potencies. Resistant mutants were established for pharmacological analysis of betaPDGF receptor-mediated signaling and allowed the generation of a drug-inducible system of cellular Src kinase activity. Our data establish a conserved structural determinant of protein kinase sensitivity relevant for both signal transduction research and drug development.