Styrylquinazoline derivatives as ABL inhibitors selective for different DFG orientations.

Among tyrosine kinase inhibitors, quinazoline-based compounds represent a large and well-known group of multi-target agents. Our previous studies have shown interesting kinases inhibition activity for a series of 4-aminostyrylquinazolines based on the CP-31398 scaffold. Here, we synthesised a new series of styrylquinazolines with a thioaryl moiety in the C4 position and evaluated in detail their biological activity. Our results showed high inhibition potential against non-receptor tyrosine kinases for several compounds. Molecular docking studies showed differential binding to the DFG conformational states of ABL kinase for two derivatives. The compounds showed sub-micromolar activity against leukaemia. Finally, in-depth cellular studies revealed the full landscape of the mechanism of action of the most active compounds. We conclude that S4-substituted styrylquinazolines can be considered as a promising scaffold for the development of multi-kinase inhibitors targeting a desired binding mode to kinases as effective anticancer drugs.

Increased EGFRvIII Epitope Accessibility after Tyrosine Kinase Inhibitor Treatment of Glioblastoma Cells Creates More Opportunities for Immunotherapy.

The number of glioblastoma (GB) cases is increasing every year, and the currently available therapies remain ineffective. A prospective antigen for GB therapy is EGFRvIII, an EGFR deletion mutant containing a unique epitope that is recognized by the L8A4 antibody used in CAR-T (chimeric antigen receptor T cell) therapy. In this study, we observed that the concomitant use of L8A4 with particular tyrosine kinase inhibitors (TKIs) does not impede the interaction between L8A4 and EGFRvIII; moreover, in this case, the stabilization of formed dimers results in increased epitope display. Unlike in wild-type EGFR, a free cysteine at position 16 (C16) is exposed in the extracellular structure of EGFRvIII monomers, leading to covalent dimer formation in the region of L8A4-EGFRvIII mutual interaction. Following in silico analysis of cysteines possibly involved in covalent homodimerization, we prepared constructs containing cysteine-serine substitutions of EGFRvIII in adjacent regions. We found that the extracellular part of EGFRvIII possesses plasticity in the formation of disulfide bridges within EGFRvIII monomers and dimers due to the engagement of cysteines other than C16. Our results suggest that the EGFRvIII-specific L8A4 antibody recognizes both EGFRvIII monomers and covalent dimers, regardless of the cysteine bridging structure. To summarize, immunotherapy based on the L8A4 antibody, including CAR-T combined with TKIs, can potentially increase the chances of success in anti-GB therapy.

Mathematical modeling of regulatory networks of intracellular processes - Aims and selected methods.

Regulatory networks structure and signaling pathways dynamics are uncovered in time- and resource consuming experimental work. However, it is increasingly supported by modeling, analytical and computational techniques as well as discrete mathematics and artificial intelligence applied to to extract knowledge from existing databases. This review is focused on mathematical modeling used to analyze dynamics and robustness of these networks. This paper presents a review of selected modeling methods that facilitate advances in molecular biology.

Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

Terpyridines as promising antitumor agents: an overview of their discovery and development.

INTRODUCTION: The fused aromatic system of terpyridines makes them good, innocent ligands for various metals. The resulting complexes have been extensively studied for both their biological activity and physico-chemical properties. However, although free ligands also have an interesting biological activity, their share in recent research is considerably limited. AREAS COVERED: This review covers the literature on the anticancer activity of terpyridines with special attention being paid to their use as free ligands. Whenever possible, the mechanism of action has been discussed, thereby providing evidence of the substantial differences between sole ligands or less stable complexes and those that have heavier elements. EXPERT OPINION: The existing literature indicates that there is a specific attitude for investigating terpyridines and their transition metal complexes. While the latter have been well explored and recognized in the scientific community, the free terpyridines are considered to be useful solely due to their complexing ability. At the same time, terpyridines could have similar or even higher anticancer potency than their complexes. Moreover, a mechanistic analysis of the stability and intracellular activity would provide information that would be useful for designing new drugs.

Neuromedin U induces an invasive phenotype in CRC cells expressing the NMUR2 receptor.

BACKGROUND: Successful colorectal cancer (CRC) therapy often depends on the accurate identification of primary tumours with invasive potential. There is still a lack of identified pathological factors associated with disease recurrence that could help in making treatment decisions. Neuromedin U (NMU) is a secretory neuropeptide that was first isolated from the porcine spinal cord, and it has emerged as a novel factor involved in the tumorigenesis and/or metastasis of many types of cancers. Previously associated with processes leading to CRC cell invasiveness, NMU has the potential to be a marker of poor outcome, but it has not been extensively studied in CRC. METHODS: Data from The Cancer Genome Atlas (TCGA) were used to analyse NMU and NMU receptor (NMUR1 and NMUR2) expression in CRC tissues vs. normal tissues, and real-time PCR was used for NMU and NMU receptor expression analysis. NMU protein detection was performed by immunoblotting. Secreted NMU was immunoprecipitated from cell culture-conditioned media and analysed by immunoblotting and protein sequencing. DNA demethylation by 5-aza-CdR was used to analyse the regulation of NMUR1 and NMUR2 expression. NMU receptor activity was monitored by detecting calcium mobilisation in cells loaded with fluo-4, and ERK1/2 kinase activation was detected after treatment with NMU or receptor agonist. Cell migration and invasion were investigated using membrane filters. Integrin expression was evaluated by flow cytometry. RESULTS: The obtained data revealed elevated expression of NMU and NMUR2 in CRC tissue samples and variable expression in the analysed CRC cell lines. We have shown, for the first time, that NMUR2 activation induces signalling in CRC cells and that NMU increases the motility and invasiveness of NMUR2-positive CRC cells and increases prometastatic integrin receptor subunit expression. CONCLUSIONS: Our results show the ability of CRC cells to respond to NMU via activation of the NMUR2 receptor, which ultimately leads to a shift in the CRC phenotype towards a more invasive phenotype.

Interaction of new tri-, tetra-, and pentacyclic azaphenothiazine derivatives with calf thymus DNA: Spectroscopic and molecular docking studies.

Azaphenothiazines (AZA), modified phenothiazine derivatives, have been reported to exhibit a wide spectrum of biological activities, including anticancer activities, but the mechanisms of their interactions with biomolecules are not fully recognized. In this work, the mode of interaction of selected AZA with calf thymus DNA was investigated using UV-Vis absorption, fluorescence spectroscopy (competition experiment with ethidium bromide, quenching of fluorescence) and molecular docking. The investigated AZA represent dipyrido[3,4-b;3'4'-e][1,4]thiazine, quino[3,2-b]benzo[1,4]thiazine and diquino[3,2-b;2',3'-e][1,4]thiazine possessing tricyclic, tetracyclic and pentacyclic ring system with the additional N,N-dimethylaminopropyl group at the nitrogen atom in the 1,4 thiazine ring. The results obtained from spectroscopic studies showed that AZA bind to DNA by insertion of a fragment of the fused rings system between the base pair stack in the double helix of DNA. In addition, the number of rings in the AZA structures seemed to be related to the strength of the interaction, because pentacyclic AZA (binding constant Kb = 6.31 × 106 L/mol) demonstrated 10-fold higher affinity towards DNA than the tetracyclic AZA and about 100-fold higher affinity than that of tricyclic AZA. The molecular docking results showed that the binding mode of AZA to DNA helix was an intercalation mode with the partial insertion of one planar part of the AZA structure (the pyridine or quinoline ring) into the neighboring bases of one of the DNA chains with additional hydrogen bonding with the minor groove through the positively charged N,N-dimethylaminopropyl group. Chemical potential (µ), chemical hardness (ƞ), electronegativity (χ) and the value of electrons transferred from one system to another (ΔN) calculated from the HOMO and LUMO energies by the density functional theory method indicated that AZA acted as the electron acceptors to the DNA bases.

A Possible Role for Arylsulfatase G in Dermatan Sulfate Metabolism.

Perturbations of glycosaminoglycan metabolism lead to mucopolysaccharidoses (MPS)-lysosomal storage diseases. One type of MPS (type VI) is associated with a deficiency of arylsulfatase B (ARSB), for which we previously established a cellular model using pulmonary artery endothelial cells with a silenced ARSB gene. Here, we explored the effects of silencing the ARSB gene on the growth of human pulmonary artery smooth muscle cells in the presence of different concentrations of dermatan sulfate (DS). The viability of pulmonary artery smooth muscle cells with a silenced ARSB gene was stimulated by the dermatan sulfate. In contrast, the growth of pulmonary artery endothelial cells was not affected. As shown by microarray analysis, the expression of the arylsulfatase G (ARSG) in pulmonary artery smooth muscle cells increased after silencing the arylsulfatase B gene, but the expression of genes encoding other enzymes involved in the degradation of dermatan sulfate did not. The active site of arylsulfatase G closely resembles that of arylsulfatase B, as shown by molecular modeling. Together, these results lead us to propose that arylsulfatase G can take part in DS degradation; therefore, it can affect the functioning of the cells with a silenced arylsulfatase B gene.

The Landscape of the Anti-Kinase Activity of the IDH1 Inhibitors.

Isocitrate dehydrogenases constitute a class of enzymes that are crucial for cellular metabolism. The overexpression or mutation of isocitrate dehydrogenases are often found in leukemias, glioblastomas, lung cancers, and ductal pancreatic cancer among others. Mutation R132H, which changes the functionality of an enzyme to produce mutagenic 2-hydroxyglutarate instead of a normal product, is particularly important in this field. A series of inhibitors were described for these enzymes of which ivosidenib was the first to be approved for treating leukemia and bile duct cancers in 2018. Here, we investigated the polypharmacological landscape of the activity for known sulfamoyl derivatives that are inhibitors, which are selective towards IDH1 R132H. These compounds appeared to be effective inhibitors of several non-receptor kinases at a similar level as imatinib and axitinib. The antiproliferative activity of these compounds against a panel of cancer cells was tested and is explained based on the relative expression levels of the investigated proteins. The multitargeted activity of these compounds makes them valuable agents against a wide range of cancers, regardless of the status of IDH1.

The p53 stabilizing agent CP-31398 and multi-kinase inhibitors. Designing, synthesizing and screening of styrylquinazoline series.

Quinazoline derivatives constitute a large family of small-molecule inhibitors of tyrosine kinases. In the current study, the p53 protein reactivator CP-31398 was tested against a panel of kinases on the assumption that it was structurally similar to other active inhibitors. Although it was found to be active in the enzyme-based assay, this compound did not block the proliferation of cancer cells at a feasible concentration level. The styrylquinazoline was used to design new structures that might be potential multitarget inhibitors. Subsequently, a series of compounds was obtained and characterized. Their inhibitory activity in a panel of tyrosine kinases had an antiproliferative effect against several cancer cell lines that have different expression levels of those proteins. The mode of protein interaction was tested for the most active compound in docking experiments.

Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation.

Despite intensive research no therapies targeted against the oncogenic EGFRvIII are present in the clinic. One of the reasons is the elusive nature of the molecular structure and activity of the truncated receptor. The recent publications indicate the EGF-bound wild-type EGFR to trans-phosphorylate the EGFRvIII initiating aberrant signaling cascade. The elevated stability of the mutant receptor contributes towards oncogenic potential, preventing termination of signaling by receptor degradation. Here, we show that inhibition of phosphatases leads to a marked increase in phosphorylation of wild-type EGFR and EGFRvIII, indicating that both undergo cyclic rounds of phosphorylation and dephosphorylation on all investigated tyrosine residues, including Tyr1045. Still, we observe elevated stability of the mutant receptor, suggesting phosphorylation as insufficient to cause degradation. Hyperphosphorylation of EGFRvIII was hindered only by EGFR tyrosine kinase inhibitors. Co-immunoprecipitation as well as semi-native Western blotting structural analyses together with functional investigation of EGFRvIII's phosphorylation following depletion of wild-type EGFR by shRNA or EGF-mediated degradation indicated homodimerization as the predominant quaternary structure of the mutant receptor. Dimers were observed only under non-reducing conditions, suggesting that homodimerization is mediated by covalent bonds. Previous reports indicated cysteine at position 16 to mediate covalent homodimerization. Upon its substitution to serine, we have observed impaired formation of dimers and lower phosphorylation levels of the mutated oncogene. Based on the obtained results we propose that EGFRvIII is predominantly regulated dynamically by phosphatases that counteract the process of trans-phosphorylation occurring within the homodimers.

EMQIT: a machine learning approach for energy based PWM matrix quality improvement.

BACKGROUND: Transcription factor binding affinities to DNA play a key role for the gene regulation. Learning the specificity of the mechanisms of binding TFs to DNA is important both to experimentalists and theoreticians. With the development of high-throughput methods such as, e.g., ChiP-seq the need to provide unbiased models of binding events has been made apparent. We present EMQIT a modification to the approach introduced by Alamanova et al. and later implemented as 3DTF server. We observed that tuning of Boltzmann factor weights, used for conversion of calculated energies to nucleotide probabilities, has a significant impact on the quality of the associated PWM matrix. RESULTS: Consequently, we proposed to use receiver operator characteristics curves and the 10-fold cross-validation to learn best weights using experimentally verified data from TRANSFAC database. We applied our method to data available for various TFs. We verified the efficiency of detecting TF binding sites by the 3DTF matrices improved with our technique using experimental data from the TRANSFAC database. The comparison showed a significant similarity and comparable performance between the improved and the experimental matrices (TRANSFAC). Improved 3DTF matrices achieved significantly higher AUC values than the original 3DTF matrices (at least by 0.1) and, at the same time, detected notably more experimentally verified TFBSs. CONCLUSIONS: The resulting new improved PWM matrices for analyzed factors show similarity to TRANSFAC matrices. Matrices had comparable predictive capabilities. Moreover, improved PWMs achieve better results than matrices downloaded from 3DTF server. Presented approach is general and applicable to any energy-based matrices. EMQIT is available online at http://biosolvers.polsl.pl:3838/emqit . REVIEWERS: This article was reviewed by Oliviero Carugo, Marek Kimmel and István Simon.

PIN3 duplication may be partially responsible for TP53 haploinsufficiency.

BACKGROUND: Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon. METHODS: 250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed. RESULTS: Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes. CONCLUSION: TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.

Different mutational characteristics of TSG in cell lines and surgical specimens.

One of the most crucial concerns of cancer research pertains to the differences between the neoplastic cells in tumor specimens in vivo and their counterparts in cell lines. The huge amount of results deposited in cancer genetic databases allows to address this issue from a wider perspective. Our analysis of the Sanger Institute Catalog Of Somatic Mutations In Cancer (COSMIC) database v61 showed a lower percentage of homozygous mutations in a group of tumor suppressor genes in surgical samples (in vivo) in comparison to their frequency in cell lines (in vitro). Similarly, the mutations resulting in the lack of protein (e.g., nonsense mutations or whole gene deletions) of several tumor suppressor genes (TSGs) were more frequently observed in vitro than in vivo. In this article, we suggest two potential explanations of these data. Firstly, TSG heterozygous mutations resulting in the modified protein (e.g., missense mutations) may be gradually (when the specific molecular context is achieved) changed to homozygous mutations resulting in the lack of protein during carcinogenesis. Secondly, among different independent pathways of tumorigenesis, those leading to homozygous nonsense mutations are characteristic for cells which are more efficiently stabilized in vitro. To conclude, these observations may be interesting for researchers working with cell line in vitro models illustrating the extent to which they reflect the tumors in vivo.

Exploring the landscape of protein-ligand interaction energy using probabilistic approach.

Analysis of protein/small molecule interactions is crucial in the discovery of new drug candidates and lead structure optimization. Small biomolecules (ligands) are highly flexible and may adopt numerous conformations upon binding to the protein. Using computer simulations instead of sophisticated laboratory procedures may significantly reduce cost of some stages of drug development. Inspired by probabilistic path planning in robotics, stochastic roadmap methodology can be regarded as a very interesting approach to effective sampling of ligand conformational space around a protein molecule. Protein-ligand interactions are divided into two parts: electrostatics, modeled by the Poisson-Boltzmann equation, and van der Waals interactions, represented by the Lennard-Jones potential. The results are promising; it can be shown that locations of binding sites predicted by the simulation are in agreement with those revealed by experimental x-ray crystallography of protein-ligand complexes. We wanted to extend our knowledge beyond the current molecular modeling tools to arrive at a better understanding of the ligand-binding process. To this end, we investigated a two-level model of protein-ligand interaction and sampling of ligand conformational space covering the entire surface of protein target.

Analysis of differences in amino acid substitution patterns, using multilevel G-tests.

In this paper, a new algorithm is presented, which makes possible multilevel comparison of BLOSUM protein substitution matrices based on data from different groups of organisms. As an example, a comparison between substitution matrices based on data from two groups of bacterial genomes with different GC content is presented. Our approach includes evaluating the number of amino acid pairs in BLOCKS databases created separately for the two groups of bacteria using protein sequences deposited in the COG database. Differences of distributions of amino acid pair counts are tested using the chi-squared based G-test. Different analysis levels make it possible to distinguish different patterns of amino acid substitution. Application of the algorithm reveals statistically significant differences in amino acid substitution patterns between AT-rich and GC-rich groups of bacterial organisms. The differences are particularly visible in the overall substitution pattern, amino acid conservation pattern and in comparison of substitution patterns for single amino acids. The algorithm presented in this paper can be considered a novel method for multi-level comparison of amino acid substitution patterns. The presented approach is not limited to bacterial organisms and BLOSUM substitution matrices. Statistically significant differences between substitution patterns in the two groups of bacterial organisms with respect to amino acid conservation pattern can be the evidence of different rate of evolutionary change between AT-rich and GC-rich bacterial organisms.