CDK9 inhibition as an effective therapy for small cell lung cancer.

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

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.

The chemical class of quinazoline compounds provides a core structure for the design of anticytomegaloviral kinase inhibitors.

HCMV is a member of the family Herpesviridae and represents a worldwide distributed pathogen with seropositivity rates in the adult population ranging between 40% and 90%. Notably, HCMV infection is a serious, sometimes life-threatening medical problem for newborns and immunosuppressed individuals, including transplant recipients and patients under antitumoral chemotherapy. Current standard therapy with valganciclovir has the disadvantage of inducing drug-resistant virus mutants and toxicity-related side effects. Our analysis stresses the earlier finding that kinase inhibitors of the quinazoline class exert an antiviral response by targeting the viral protein kinase pUL97 without inducing resistance. Therefore, quinazolines have been used as a core structure to gain insight in the mode of inhibitor-kinase interaction. Here, we demonstrate that (i) the novel quinazolines Vi7392 and Vi7453 are highly active against HCMV laboratory and clinically relevant strains including maribavir- and ganciclovir-resistant variants, (ii) antiviral activity is not cell-type specific and was also detected in a placental explant tissue model using a genetically intact HCMV strain (iii) the viral kinase pUL97 represents a target of the anticytomegaloviral activity of these compounds, (iv) induction of pUL97-conferring drug resistance was not detectable under single-step selection, thus differed from the induction of ganciclovir resistance, and (v) pUL97 drug docking simulations enabled detailed insights into specific drug-target binding properties providing a promising basis for the design of optimized kinase inhibitors. These novel findings may open new prospects for the future medical use of quinazoline drug candidates and the use of drug-target dynamic simulations for rational design of antivirals.

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.

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.

Characterization of binding mode of imatinib to human α1-acid glycoprotein.

Imatinib (IMT) is a selective tyrosine kinase inhibitor, used in the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Its strong plasma protein binding was found to belong to the F1*S genetic variant of α(1)-acid glycoprotein (AGP). In this work, comparative AGP binding studies were performed with IMT fragment molecules to reveal which parts of the molecule are important in the high-affinity interaction provoking specific spectral changes. Molecular modeling calculations indicated that IMT docked into the X-ray structure of AGP/F1 adopts a bent, compact conformation. This binding mode is similar to those found in its complexes with some low-affinity kinases and a quinone reductase, being strikingly different from the extended conformation of IMT in its high-affinity kinase targets.

Novel, selective CDK9 inhibitors for the treatment of HIV infection.

Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties.

BACKGROUND AND PURPOSE: ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity. EXPERIMENTAL APPROACH: MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity. KEY RESULTS: Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters. CONCLUSIONS AND IMPLICATIONS: A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter-TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs.

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.

Structure -activity relationships of PDE5 inhibitors.

cGMP has a short-term effect on smooth muscle tone and a longer-term effect on responses to chronic drug treatment or proliferative signals. cGMP-Phosphodiesterase type 5 (PDE5) hydrolizes cGMP, and the result is smooth muscle contraction. PDE5 is a relatively novel therapeutic target of various diseases, such as erectile dysfunction and pulmonary hypertension. The most intensively examined and marketed PDE5 inhibitor was sildenafil (Viagra) but recently vardenafil (Levitra) and tadalafil (Cialis) were launched with beneficial ADME parameters and PDE5 selectivity. The increasing interest in PDE5 inhibition made it reasonable to collect the available inhibitory data from the scientific literature and set up a structure-activity relationship study. Chemical structures of 438 compounds and their cGMP-PDE5 inhibitory data (IC50) were collected from recently published articles. In this paper physiology, regulation and inhibition of PDE5 (and briefly other PDE-s) are discussed and inhibitors are tabulated by the core structures. Finally, a general QSAR model built from these data is presented. All data used in the QSAR study were summarized in a Supplement (for description please see the online version of the article).

Prediction oriented QSAR modelling of EGFR inhibition.

Epidermal Growth Factor Receptor (EGFR) is a high priority target in anticancer drug research. Thousands of very effective EGFR inhibitors have been developed in the last decade. The known inhibitors are originated from a very diverse chemical space but--without exception--all of them act at the Adenosine TriPhosphate (ATP) binding site of the enzyme. We have collected all of the diverse inhibitor structures and the relevant biological data obtained from comparable assays and built prediction oriented Quantitative Structure-Activity Relationship (QSAR) which models the ATP binding pocket's interactive surface from the ligand side. We describe a QSAR method with automatic Variable Subset Selection (VSS) by Genetic Algorithm (GA) and goodness-of-prediction driven QSAR model building, resulting an externally validated EGFR inhibitory model built from pIC50 values of a diverse structural set of 623 EGFR inhibitors. Repeated Trainings/Evaluations (RTE) were used to obtain model fitness values and the effectiveness of VSS is amplified by using predictive ability scores of descriptors. Numerous models were generated by different methods and viable models were collected. Then, intensive RTE were applied to identify ultimate models for external validations. Finally, suitable models were validated by statistical tests. Since we use calculated molecular descriptors in the modeling, these models are suitable for virtual screening for obtaining novel potential EGFR inhibitors.

Comparison of measured and calculated lipophilicity of substituted aurones and related compounds.

A molecule library containing 55 aurone- and thioaurone-type structures has been designed and synthesised. Reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed to separate these compounds and to characterise their lipophilicity by experimental method (k'). The experimental lipophilicity data have been compared with the computer calculated lipophilicity parameters (CLOGPs) of the same molecules. In general, good correlations between the measured and calculated lipophilicities have been found with the exception of structure isomers and compounds capable for hydrogen bonding. The chromatographic method was suitable to separate the structure (ortho and para) isomers of aurone and thioaurones and was sensitive enough to differentiate their lipophilicities. Our findings suggest the usefulness of the chromatographic method in fast characterisation of the lipophilicity of structurally closely related molecules.

Pro-apoptotic and anti-apoptotic molecules affecting pathways of signal transduction.

Selective inhibition of the "false" proliferative signals via targeting tyrosine kinases resulting in the induction of apoptosis by depletion of the "survival factors" is one of the most studied and widely accepted concepts of modern chemotherapy. We have synthesized a series of potent tyrosine kinase inhibitors and tested these compounds for apoptosis induction. Some of the tyrosine kinase inhibitors caused either apoptotic or cytoplasmic vacuolar cell death in various tumor cell cultures. The somatostatin analogue oligopeptide TT-232, which indirectly inhibits tyrosine kinases, exerted a dose-dependent apoptosis-inducing effect. The tumor growth-inhibitory effect of TT-232 and some tyrosine kinase inhibitors has also been proven by in vivo experiments, using human tumor xenografts. On the other hand, a dose-dependent pro- or anti-apoptotic activity of (-)-deprenyl has been shown in melanoma cell cultures, the lower doses inhibiting and the higher doses inducing apoptosis. Various metabolites of (-)-deprenyl are responsible for these actions. The effect of (-)-deprenyl is connected with depolarization of mitochondrial membranes. The kinase inhibitors act on the growth factor receptor signaling pathways (survival factor pathways) and initiate the caspase cascade. The key enzyme for the action of both pro-apoptotic and anti-apoptotic compounds is caspase 3.

Reliability of logP predictions based on calculated molecular descriptors: a critical review.

Correct QSAR analysis requires reliable measured or calculated logP values, being logP the most frequently utilized and most important physico-chemical parameter in such studies. Since the publication of theoretical fundamentals of logP prediction, many commercial software solutions are available. These programs are all based on experimental data of huge databases therefore the predicted logP values are mostly acceptable - especially for known structures and their derivatives. In this study we critically reviewed the published methods and compared the predictive power of commercial softwares (CLOGP, KOWWIN, SciLogP/ULTRA) to each other and to our recently developed automatic QS(P)AR program. We have selected a very diverse set of 625 known drugs (98%) and drug-like molecules with experimentally validated logP values. We have collected 78 reported "outliers" as well, which could not be predicted by the "traditional" methods. We used these data in the model building and validation. Finally, we used an external validation set of compounds missing from public databases. We emphasized the importance of data quality, descriptor calculation and selection, and presented a general, reliable descriptor selection and validation technique for such kind of studies. Our method is based on the strictest mathematical and statistical rules, fully automatic and after the initial settings there is no option for user intervention. Three approaches were applied: multiple linear regression, partial least squares analysis and artificial neural network. LogP predictions with a multiple linear regression model showed acceptable accuracy for new compounds therefore it can be used for "in-silico-screening" and/or planning virtual/combinatorial libraries.

A novel normalized retention factor in micellar electrokinetic chromatography.

Characteristic properties of the expression k'' = (t(m)-t(o))/(t(mc)-t(o)) and its applicability in micellar electrokinetic capillary chromatography (MEKC) were compared to the previous expression, k' = (t(m)-t(o))/t(o)(1-t(m)/t(mc)), introduced by Terabe. It was proved with theoretical calculations (curve shape analysis) that the properties of function k'' are in full accordance with the properties of the MEKC system and k'' could be applied advantageously to characterize hydrophobicity of the analytes. This conclusion is now supported by experimental data obtained with homolog series of alkylbenzenes and alkylphenones as well as with hydrophobic protected peptides. Migration times, k', k'' values, and software-calculated hydrophobicity data are summarized and analyzed in the present study. Since k'' is a normalized parameter, good relationships between the migration time, the software-calculated hydrophobicity, and the k'' values were obtained. Differences in hydrophobicity of the analytes could be estimated in a more realistic way with the aid of function k'' than by using function k'. Hydrophobicity data estimated on the basis of the k'' values proved to be in good accordance with the expectations based on the migration times and on the chemical structures of the compounds investigated.

[Preparation and characterization of potential antineoplastic agents]. / Potenciális daganatellenes hatású vegyületek elóállítása és vizsgálata.

A parallel combinatorial library of over sixteen hundred compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads that is aimed for intervening with the substrate binding site of the pp60c-src enzyme. The new structures were based on known PTK inhibitors having at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl type inhibitory compounds were found in the range of 18-100 micromolar IC50 concentrations from combinations of twelve different substituents. Molecular modeling of the active compounds showed a characteristic distance of 13-14 A between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c-src PTK [5,6,7] showed that the energy minimized conformers had the same distance between two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis inducing effect on HT-29 human colon tumor cells.

Application of neural networks in structure-activity relationships.

Methodology and application of artificial neural networks in structure-activity relationships are reviewed focusing on the most frequently used three-layer feedforward back-propagation procedure. Two applications of neural networks are presented and a comparison of the performance with those of CoMFA and a classical QSAR analysis is also discussed.

[Biological activity and structure of antitumor compounds from Plantago media L]. / Plantago media L antitumor hatóanyagainak szerkezete és biológiai aktivitása.

Tyrosine kinase inhibition and tumor growth inhibition activity of verbascoside and homoplantaginin are described. Both molecules proved to be equally significant inhibitors of isolated EGF-R tyrosine kinases, nevertheless their in vitro antiproliferative activity was variable in cellular assays. Their different inhibitory efficacies could be interpreted on the basis of conformational analysis and lipophilicity evaluation.