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1.
Oncotarget ; 10(51): 5255-5266, 2019 Sep 03.
Article in English | MEDLINE | ID: mdl-31523388

ABSTRACT

Targeted therapies against cancer types with more than one driver gene hold bright but elusive promise, since approved drugs are not available for all driver mutations and monotherapies often result in resistance. Targeting multiple driver genes in different pathways at the same time may provide an impact extensive enough to fight resistance. Our goal was to find synergistic drug combinations based on the availability of targeted drugs and their biological activity profiles and created an associated compound library based on driver gene-related protein targets. In this study, we would like to show that driver gene pattern based customized combination therapies are more effective than monotherapies on six cell lines and patient-derived primary cell cultures. We tested 55-102 drug combinations targeting driver genes and driver pathways for each cell line and found 25-85% of these combinations highly synergistic. Blocking 2-5 cancer pathways using only 2-3 targeted drugs was sufficient to reach high rates of tumor cell eradication at remarkably low concentrations. Our results demonstrate that the efficiency of cancer treatment may be significantly improved by combining drugs against multiple tumor specific drivers.

2.
Eur J Med Chem ; 174: 309-329, 2019 Jul 15.
Article in English | MEDLINE | ID: mdl-31055147

ABSTRACT

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.


Subject(s)
Antitubercular Agents/pharmacology , Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Isoquinolines/pharmacology , Mycobacterium tuberculosis/drug effects , Piperazines/pharmacology , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , IMP Dehydrogenase/chemistry , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Structure-Activity Relationship
3.
Tuberculosis (Edinb) ; 103: 52-60, 2017 03.
Article in English | MEDLINE | ID: mdl-28237034

ABSTRACT

There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development.


Subject(s)
Antitubercular Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Drug Design , Machine Learning , Molecular Docking Simulation , Mycobacterium tuberculosis/drug effects , Topoisomerase I Inhibitors/pharmacology , Tuberculosis/drug therapy , Antitubercular Agents/chemistry , Antitubercular Agents/metabolism , Bayes Theorem , Computer-Aided Design , DNA Topoisomerases, Type I/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Targeted Therapy , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/growth & development , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/growth & development , Protein Conformation , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/metabolism , Tuberculosis/microbiology
4.
Mol Microbiol ; 103(1): 13-25, 2017 01.
Article in English | MEDLINE | ID: mdl-27677649

ABSTRACT

There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.


Subject(s)
Antitubercular Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Pyrimidines/pharmacokinetics , Anti-Bacterial Agents/pharmacokinetics , Cell Enlargement , Drug Discovery/methods , Gene Expression Regulation, Bacterial/genetics , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , Sequence Homology, Amino Acid , Sulfonamides/metabolism , Sulfonamides/pharmacokinetics , Time-Lapse Imaging
5.
ACS Infect Dis ; 3(1): 5-17, 2017 01 13.
Article in English | MEDLINE | ID: mdl-27726334

ABSTRACT

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 µM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.


Subject(s)
Antitubercular Agents/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Mycobacterium/drug effects , Sulfones/pharmacology , Tuberculosis/drug therapy , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Discovery , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Genome, Bacterial , IMP Dehydrogenase/genetics , IMP Dehydrogenase/metabolism , Mice , Mice, Inbred C57BL , Mutation , Tuberculosis/microbiology
6.
J Pathol ; 236(4): 407-20, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25810250

ABSTRACT

Inflammation is a main feature of progressive kidney disease. Gremlin binds to bone morphogenetic proteins (BMPs), acting as an antagonist and regulating nephrogenesis and fibrosis among other processes. Gremlin also binds to vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells to induce angiogenesis. In renal cells, gremlin regulates proliferation and fibrosis, but there are no data about inflammatory-related events. We have investigated the direct effects of gremlin in the kidney, evaluating whether VEGFR2 is a functional gremlin receptor. Administration of recombinant gremlin to murine kidneys induced rapid and sustained activation of VEGFR2 signalling, located in proximal tubular epithelial cells. Gremlin bound to VEGFR2 in these cells in vitro, activating this signalling pathway independently of its action as an antagonist of BMPs. In vivo, gremlin caused early renal damage, characterized by activation of the nuclear factor (NF)-κB pathway linked to up-regulation of pro-inflammatory factors and infiltration of immune inflammatory cells. VEGFR2 blockade diminished gremlin-induced renal inflammatory responses. The link between gremlin/VEGFR2 and NF-κB/inflammation was confirmed in vitro. Gremlin overexpression was associated with VEGFR2 activation in human renal disease and in the unilateral ureteral obstruction experimental model, where VEGFR2 kinase inhibition diminished renal inflammation. Our data show that a gremlin/VEGFR2 axis participates in renal inflammation and could be a novel target for kidney disease.


Subject(s)
Inflammation Mediators/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Kidney/metabolism , Nephritis/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Cell Line, Tumor , Cytokines , Disease Models, Animal , Female , Humans , Kidney/pathology , Male , Mice, Inbred C57BL , NF-kappa B/metabolism , Nephritis/etiology , Nephritis/genetics , Nephritis/pathology , Protein Binding , RNA Interference , Retrospective Studies , Signal Transduction , Time Factors , Transfection , Ureteral Obstruction/complications , Vascular Endothelial Growth Factor Receptor-2/genetics
7.
Tuberculosis (Edinb) ; 95 Suppl 1: S200-6, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25801335

ABSTRACT

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 µM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays.


Subject(s)
Antitubercular Agents/isolation & purification , Drug Design , Small Molecule Libraries , Tuberculosis, Multidrug-Resistant/drug therapy , DNA Gyrase/drug effects , DNA Topoisomerases/drug effects , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Humans , Mannosyltransferases/antagonists & inhibitors , Microbial Sensitivity Tests , Molecular Targeted Therapy/methods , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/isolation & purification , Topoisomerase Inhibitors/chemistry , Topoisomerase Inhibitors/isolation & purification
8.
Antioxid Redox Signal ; 22(1): 29-47, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25065408

ABSTRACT

AIMS: Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions, including cardiovascular diseases, whose product is a matricellular protein that can be degraded to biologically active fragments. Among them, the C-terminal module IV [CCN2(IV)] regulates many cellular functions, but there are no data about redox process. Therefore, we investigated whether CCN2(IV) through redox signaling regulates vascular responses. RESULTS: CCN2(IV) increased superoxide anion (O2(•-)) production in murine aorta (ex vivo and in vivo) and in cultured vascular smooth muscle cells (VSMCs). In isolated murine aorta, CCN2(IV), via O2(•-), increased phenylephrine-induced vascular contraction. CCN2(IV) in vivo regulated several redox-related processes in mice aorta, including increased nonphagocytic NAD(P)H oxidases (Nox)1 activity, protein nitrosylation, endothelial dysfunction, and activation of the nuclear factor-κB (NF-κB) pathway and its related proinflammatory factors. The role of Nox1 in CCN2(IV)-mediated vascular responses in vivo was investigated by gene silencing. The administration of a Nox1 morpholino diminished aortic O2(•-) production, endothelial dysfunction, NF-κB activation, and overexpression of proinflammatory genes in CCN2(IV)-injected mice. The link CCN2(IV)/Nox1/NF-κB/inflammation was confirmed in cultured VSMCs. Epidermal growth factor receptor (EGFR) is a known CCN2 receptor. In VSMCs, CCN2(IV) activates EGFR signaling. Moreover, EGFR kinase inhibition blocked vascular responses in CCN2(IV)-injected mice. INNOVATION AND CONCLUSION: CCN2(IV) is a novel prooxidant factor that in VSMCs induces O2(•-) production via EGFR/Nox1 activation. Our in vivo data demonstrate that CCN2(IV) through EGFR/Nox1 signaling pathway induces endothelial dysfunction and activation of the NF-κB inflammatory pathway. Therefore, CCN2(IV) could be considered a potential therapeutic target for redox-related cardiovascular diseases.


Subject(s)
Connective Tissue Growth Factor/metabolism , ErbB Receptors/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , NADH, NADPH Oxidoreductases/metabolism , NF-kappa B/metabolism , Animals , Immunohistochemistry , Male , Mice , Microscopy, Confocal , NADPH Oxidase 1
9.
Acta Pharm Hung ; 84(3): 91-104, 2014.
Article in Hungarian | MEDLINE | ID: mdl-25470976

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Biochemistry/methods , Cell Line, Tumor/drug effects , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Quinazolines/pharmacology , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Humans , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship
10.
Cell Host Microbe ; 16(4): 538-48, 2014 Oct 08.
Article in English | MEDLINE | ID: mdl-25299337

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Fibroblasts/physiology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Virulence Factors/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Antigens, Bacterial , Cell Survival , Fibroblasts/microbiology , Gene Expression Profiling , Molecular Sequence Data , Sequence Analysis, DNA
11.
Acta Pharm Hung ; 83(3): 88-95, 2013.
Article in Hungarian | MEDLINE | ID: mdl-24369587

ABSTRACT

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.


Subject(s)
Amide Synthases/antagonists & inhibitors , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Computer Simulation , Models, Chemical , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapy , Amides/chemistry , Amides/pharmacology , Coinfection/epidemiology , HIV Infections/epidemiology , Humans , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Protein Serine-Threonine Kinases/metabolism , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Tuberculosis/epidemiology , Tuberculosis, Multidrug-Resistant/drug therapy
12.
Bioorg Med Chem Lett ; 23(22): 6152-5, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24095095

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Quinazolines/pharmacology , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Erlotinib Hydrochloride , Humans , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistry , Signal Transduction
13.
J Pathol ; 231(4): 480-94, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24037740

ABSTRACT

TWEAK, a member of the TNF superfamily, binds to the Fn14 receptor, eliciting biological responses. EGFR signalling is involved in experimental renal injury. Our aim was to investigate the relationship between TWEAK and EGFR in the kidney. Systemic TWEAK administration into C57BL/6 mice increased renal EGFR phosphorylation, mainly in tubular epithelial cells. In vitro, in these cells TWEAK phosphorylated EGFR via Fn14 binding, ADAM17 activation and subsequent release of the EGFR ligands HB-EGF and TGFα. In vivo the EGFR kinase inhibitor Erlotinib inhibited TWEAK-induced renal EGFR activation and downstream signalling, including ERK activation, up-regulation of proinflammatory factors and inflammatory cell infiltration. Moreover, the ADAM17 inhibitor WTACE-2 also prevented those TWEAK-induced renal effects. In vitro TWEAK induction of proinflammatory factors was prevented by EGFR, ERK or ADAM17 inhibition. In contrast, EGFR transactivation did not modify TWEAK-mediated NF-κB activation. Our data suggest that TWEAK transactivates EGFR in the kidney, leading to modulation of downstream effects, including ERK activation and inflammation, and suggest that inhibition of EGFR signalling could be a novel therapeutic tool for renal inflammation.


Subject(s)
ErbB Receptors/metabolism , Nephritis/metabolism , Tumor Necrosis Factors/pharmacology , ADAM Proteins/metabolism , ADAM Proteins/physiology , ADAM17 Protein , Animals , Cells, Cultured , Cytokine TWEAK , Epithelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Erlotinib Hydrochloride , Female , Gene Silencing , Heparin-binding EGF-like Growth Factor , Humans , Inflammation Mediators/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Kidney Tubules/metabolism , Ligands , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Nephritis/chemically induced , Nephritis/pathology , Nephritis/prevention & control , Phosphorylation/drug effects , Quinazolines/pharmacology , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , TWEAK Receptor , Transcriptional Activation/drug effects , Transcriptional Activation/physiology , Transforming Growth Factor alpha/metabolism , Tumor Necrosis Factors/metabolism
14.
J Mol Cell Biol ; 5(5): 323-35, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23929714

ABSTRACT

Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-ß-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.


Subject(s)
Connective Tissue Growth Factor/metabolism , ErbB Receptors/metabolism , ADAM Proteins/metabolism , Animals , Cells, Cultured , Connective Tissue Growth Factor/chemistry , Epithelial Cells/drug effects , Epithelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Integrins/metabolism , Kidney Tubules/cytology , Ligands , MAP Kinase Signaling System/drug effects , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , Phosphorylation/drug effects , Protein Binding/drug effects , Receptor Cross-Talk/drug effects , Receptor, trkA/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology
15.
Bioconjug Chem ; 22(4): 540-5, 2011 Apr 20.
Article in English | MEDLINE | ID: mdl-21443263

ABSTRACT

Multikinase inhibitors are potent anticancer drugs that simultaneously intervene in multiple related signaling cascades, thus being capable of blocking salvage pathways that may play a role in the development of drug resistance. Multikinase inhibitors are increasingly evaluated for indications other than cancer, but long-term safety risks dictated by off-organ toxicities of these agents may prevent their safe and effective use. Here, we describe a new approach in which platinum coordination chemistry is applied for the development of a cell-selective multikinase inhibitor bioconjugate. The platinum(II) kinase inhibitor bioconjugate was designed to be active with the linker attached to the inhibitor and displayed improved activity by enhanced cell specificity as well as enhanced intracellular retention, thereby prolonging its pharmacological activity. In addition, the utilized platinum-based linkage technology potentiated the inhibitory activity of the multikinase inhibitor. These features in combination with carrier-mediated uptake in the target cells may revolutionize dosing regimens and safety profiles of (multi)kinase inhibitors.


Subject(s)
Coordination Complexes/chemical synthesis , Muramidase/chemistry , Organoplatinum Compounds/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Coordination Complexes/chemistry , Humans , Models, Molecular , Molecular Structure , Muramidase/metabolism , Organoplatinum Compounds/chemistry , Protein Kinase Inhibitors/chemistry
16.
Tuberculosis (Edinb) ; 90(6): 354-60, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20934382

ABSTRACT

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find antimycobacterial scaffolds, we screened a kinase inhibitor library of more than 12,000 compounds using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and a target-based assay with the protein kinase PknA. Seventeen "hits" came from the whole cell-based screening approach, from which three displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10µM and were non-mutagenic and non-cytotoxic. Two of these hits were specific for M. tuberculosis versus C. glutamicum and none of them was found to inhibit the essential serine/threonine protein kinases, PknA and PknB present in both bacteria. One of the most active hits, VI-18469, had a benzoquinoxaline pharmacophore while another, VI-9376, is structurally related to a new class of antimycobacterial agents, the benzothiazinones (BTZ). Like the BTZ, VI-9376 was shown to act on the essential enzyme decaprenylphosphoryl-ß-D-ribose 2'-epimerase, DprE1, required for arabinan synthesis.


Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/drug effects , Tuberculosis/drug therapy , Gene Library , Humans , Mycobacterium tuberculosis/metabolism , Protein Serine-Threonine Kinases/genetics , Tuberculosis/genetics , Tuberculosis/metabolism
17.
J Control Release ; 132(3): 200-7, 2008 Dec 18.
Article in English | MEDLINE | ID: mdl-18793687

ABSTRACT

Cell-specific targeting to renal tubular cells is an interesting approach to enhance the accumulation of drugs in the kidney. Low molecular weight proteins are rapidly filtered and extensively accumulate in proximal tubular cells. We therefore have used lysozyme (LZM, 14 kDa) as a tubular cell-specific carrier for the delivery of kinase inhibitors. Two different kinase inhibitors (LY364947 and erlotinib, directed to either the TGF-beta receptor kinase or the EGF receptor) were individually conjugated to LZM via a novel platinum-based linker (Universal Linkage System; ULS). The cellular handling and pharmacological efficacy of the conjugates were evaluated in cultured proximal tubular cells (HK-2 cells). Both conjugates were efficiently internalized via endocytosis. TGF-beta or EGF activated HK-2 cells showed a strong activation of the studied kinases and the conjugates inhibited these events, as was demonstrated by Western blotting of phosphorylated downstream mediators and quantitative gene expression analysis. In conclusion, we have developed tubular cell-specific kinase inhibitor-LZM conjugates via a novel linker strategy, which both showed to be effective in vitro. Future in vivo studies should show their potential for the treatment of renal diseases.


Subject(s)
Drug Carriers , Intercellular Signaling Peptides and Proteins/metabolism , Kidney Tubules, Proximal/drug effects , Muramidase/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrroles/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Cells, Cultured , Chemistry, Pharmaceutical , Endocytosis , Epidermal Growth Factor/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Fibrosis , Gene Expression Regulation/drug effects , Humans , Kidney Tubules, Proximal/enzymology , Kinetics , Muramidase/chemistry , Phosphorylation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrroles/chemistry , Pyrroles/metabolism , Quinazolines/chemistry , Quinazolines/metabolism , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/metabolism
18.
J Recept Signal Transduct Res ; 28(4): 361-73, 2008.
Article in English | MEDLINE | ID: mdl-18702009

ABSTRACT

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.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Viral Proteins/metabolism , Cytomegalovirus , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Mutant Proteins/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Quinazolines/chemical synthesis , Quinazolines/chemistry , Quinazolines/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Viral Proteins/antagonists & inhibitors , Viral Proteins/drug effects
19.
Immunol Lett ; 116(2): 225-31, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18258308

ABSTRACT

The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.


Subject(s)
Drug Design , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Tuberculosis/drug therapy , Tuberculosis/microbiology , Amides/chemistry , Amides/pharmacology , Animals , Bacterial Proteins/metabolism , Cells, Cultured , Inhibitory Concentration 50 , Macrophages/drug effects , Mice , Microbial Sensitivity Tests , Mycobacterium tuberculosis/enzymology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/metabolism , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Tuberculosis/enzymology
20.
Assay Drug Dev Technol ; 3(5): 543-51, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16305311

ABSTRACT

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.


Subject(s)
Chemistry, Pharmaceutical/methods , Combinatorial Chemistry Techniques/methods , Databases, Factual , Drug Design , Pharmaceutical Preparations/chemistry , Protein Kinase Inhibitors/chemistry , Technology, Pharmaceutical/methods , Algorithms , Pharmaceutical Preparations/analysis , Structure-Activity Relationship
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