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Na(v)1.5 sodium channels contribute to the generation of the rapid upstroke of the myocardial action potential and thereby play a central role in the excitability of myocardial cells. At present, the patch clamp method is the gold standard for ion channel inhibitor screening. However, this method has disadvantages such as high technical difficulty, high cost and low speed. In this study, novel machine learning models to screen chemical blockers were developed to overcome the above shortage. The data from the ChEMBL Database were employed to establish the machine learning models. Firstly, six molecular fingerprints together with five machine learning algorithms were used to develop 30 classification models to predict effective inhibitors. A validation and a test set were used to evaluate the performance of the models. Subsequently, the privileged substructures tightly associated with the inhibition of the Na(v)1.5 ion channel were extracted using the bioalerts Python package. In the validation set, the RF-Graph model performed best. Similarly, RF-Graph produced the best result in the test set in which the Prediction Accuracy (Q) was 0.9309 and Matthew's correlation coefficient was 0.8627, further indicating the model had high classification ability. The results of the privileged substructures indicated Sulfa structures and fragments with large Steric hindrance tend to block Na(v)1.5. In the unsupervised learning task of identifying sulfa drugs, MACCS and Graph fingerprints had good results. In summary, effective machine learning models have been constructed which help to screen potential inhibitors of the Na(v)1.5 ion channel and key privileged substructures with high affinity were also extracted.

BReast tumor Kinase (BRK, also known as PTK6) is a non-receptor tyrosine kinase that is highly expressed in breast carcinomas while having low expression in the normal mammary gland, which hints at the oncogenic nature of this kinase in breast cancer. In the past twenty-six years since the discovery of BRK, an increasing number of studies have strived to understand the cellular roles of BRK in breast cancer. Since then, BRK has been found both in vitro and in vivo to activate a multitude of oncoproteins to promote cell proliferation, metastasis, and cancer development. The compelling evidence concerning the oncogenic roles of BRK has also led, since then, to the rapid and exponential development of inhibitors against BRK. This review highlights recent advances in BRK biology in contributing to the “hallmarks of cancer”, as well as BRK's therapeutic significance. Importantly, this review consolidates all known inhibitors of BRK activity and highlights the connection between drug action and BRK-mediated effects. Despite the volume of inhibitors designed against BRK, none have progressed into clinical phase. Understanding the successes and challenges of these inhibitor developments are crucial for the future improvements of new inhibitors that can be clinically relevant.

Given the broad utility of humanized yeast to model and study human biology, a reference set of human genes that can replace cognate yeast genes and operate in yeast is needed. Hamza et al. present..., Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.

Natural gas hydrates represent a valid opportunity in terms of energy supplying, carbon dioxide permanent storage and climate change contrast. Research is more and more involved in performing CO2 replacement competitive strategies. In this context, the inhibitor effect of sodium chloride on hydrate formation and stability needs to be investigated in depth. The present work analyses how NaCl intervenes on CO2 hydrate formation, comparing results with the same typology of tests carried out with methane, in order to highlight the influence that salt produced on hydrate equilibrium conditions and possibilities which arise from here for improving the replacement process efficiency. Sodium chloride influence was then tested on five CO2/CH4 replacement tests, carried out via depressurization. In relation with the same typology of tests, realised in pure demineralised water and available elsewhere in literature, three main differences were found. Before the replacement phase, CH4 hydrate formation was particularly contained; moles of methane involved were in the range 0.059–0.103 mol. On the contrary, carbon dioxide moles entrapped into water cages were 0.085–0.206 mol or a significantly higher quantity. That may be justified by the greater presence of space and free water due to the lower CH4 hydrate formation, which led to a more massive new hydrate structure formation. Moreover, only a small part of methane moles remained entrapped into hydrates after the replacement phase (in the range of 0.023–0.042 mol), proving that, in presence of sodium chloride, CO2/CH4 exchange interested the greater part of hydrates. Thus, the possibility to conclude that sodium chloride presence during the CO2 replacement process provided positive and encouraging results in terms of methane recovery, carbon dioxide permanent storage and, consequently, replacement process efficiency.

WOS: 000394539800022, Carotenoids are vital for most photosynthetic organisms because of their crucial role in prevention of the damage caused by excess light or stress conditions like heat or nutrient deprivation. Some of them are also valuable for biotechnology with respect to their colorful feature and antioxidant properties. In this study, a natural beta-carotene producer, green microalga Dunaliella salina, was evaluated for its potential to produce different valuable carotenoids through the inhibition of cyclization reactions in the carotenoid pathway by the chemical inhibitors 2-methylimidazole (2MI) and 3-amino-1,2,4-triazole (Amitrol). 2MI was shown to be effective on lutein accumulation in D. salina cultures grown at high temperature, without affecting cell viability. Addition of 2MI to the culture at 1 mM concentration resulted a 1.7-fold increase in lutein content in the cell with the highest amount of 3.45 pg/cell, and an associated decrease in beta-carotene content suggesting that this inhibitor is more effective on lycopene beta-cyclase activity, thus shifting the pathway from beta-carotene arm to the alpha-carotene direction. The results of this study may give the opportunity to use D. salina for the production of valuable carotenoids other than beta-carotene., Turkish General Directorate of Agricultural Research and Policy [TAGEM/10/ARGE/06], This work was supported by the projects from the Turkish General Directorate of Agricultural Research and Policy (TAGEM/10/ARGE/06).

Funding acknowledgement The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome.

International audience; An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of "second generation", chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark.

The essential phosphobase methylation pathway for synthesis of phosphocholine is unique to nematodes, protozoa and plants, and thus an attractive antiparasitic molecular target. Herein, we screened compounds from the National Cancer Institute (Developmental Therapeutics Program Open Chemical Repository) for specific inhibitory activity against Haemonchus contortus phosphoethanolamine methyltransferases (HcPMT1 and HcPMT2), and tested candidate compounds for anthelmintic activity against adult and third-stage larvae of H. contortus. We identified compound NSC-641296 with IC50 values of 8.3 ± 1.1 μM and 5.1 ± 1.8 μM for inhibition of the catalytic activity of HcPMT1 alone and HcPMT1/HcPMT2 combination, respectively. Additionally we identified compound NSC-668394 with inhibitory IC50 values of 5.9 ± 0.9 μM and 2.8 ± 0.6 μM for HcPMT1 alone and HcPMT1/HcPMT2 combination, respectively. Of the two compounds, NSC-641296 depicted significant anthelmintic activity against third-stage larvae (IC50 = 15 ± 2.9 μM) and adult stages (IC50 = 7 ± 2.9 μM) of H. contortus, with optimal effective in vitro concentrations being 2-fold and 4-fold, respectively, lower than its cytotoxic IC50 (29 ± 2.1 μM) in a mammalian cell line. Additionally, we identified two compounds, NSC-158011 and NSC-323241, with low inhibitory activity against the combined activity of HcPMT1 and HcPMT2, but both compounds did not show any anthelmintic activity against H. contortus. The identification of NSC-641296 that specifically inhibits a unique biosynthetic pathway in H. contortus and has anthelmintic activity against both larval and adult stages of H. contortus, provides impetus for the development of urgently needed new efficacious anthelmintics to address the prevailing problem of anthelmintic-resistant H. contortus., Graphical abstract, Highlights • NSC-641296 and NSC-668394 inhibit HcPMT1 and HcPMT2 enzymes in Haemonchus contortus. • NSC-641296 has in vitro anthelmintic activity against larvae and adult H. contortus. • H. contortus HcPMT1 and HcPMT2 are two unique targets for anthelmintic development.

In this work p38 protein kinases are studied. These proteins are involved in human diseases such as cancer, rheumatoid arthritis, or psoriasis. Among the group of p38 proteins, one can find 4 different proteins, where p38α is the most studied one and will be the reference of this work. This protein is relevant from the pharmacological point of view and the design of novel inhibitors is required. The structure consists of two large domains that are connected by the ATP binding region that forms the active site of the protein. The active site presents conformational changes depending on the inhibitor that is interaction. There are three main types of inhibitors of protein kinases: Type I, Type II, and Type I/II. The objective of this work is to establish a computational protocol to study the interaction between inhibitors of different types and p38α protein in order to be able to design new, more potent and selective inhibitors for this protein. In the first place, an assessment of which molecular docking software properly describes the interactions between the different proteins and inhibitors is performed. In order to answer this question, the SwissDock software will be compared to AutoDock. The interactions established by these inhibitors with the amino acids in the active center are studied in detail to determine the molecular basis of the inhibition process. Based on these interactions, computational tools are used to determine the pharmacophore of the drug and to use this information to design a more potent inhibitor for this protein. We have tried to design a more potent inhibitor for Type I, Type II, as for Type I / II. Based on the improved inhibitors, the interaction and selectivity between p38α protein inhibitors and the other three types of p38 protein (γ, β and Δ) are studied. Finally, computational results are compared with results based on the IC50 inhibitor Type I / II of the p38α protein, if there is a relationship between the study model and the experimental results, it will be used to see the limitations of the model. It is expected that the proposed computational protocol is sufficiently robust to identify new p38α protein inhibitors

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.

Spontaneous heating and fire in coal mines is a major problem worldwide and has been a great concern both for the industry and researchers in this field. Majority of fires existing today in different coalfields are mainly due to spontaneous combustion of coal. The auto oxidation of coal ultimately leads to spontaneous combustion which is the major root cause for the disastrous of coal mine in leading and coal producing countries like USA, China, Australia, India and Germany. It is a slow process and the heat evolved is carried away by air. This process of self-heating of coal or other carbonaceous material resulting eventually in its ignition is termed as “spontaneous heating” or “auto oxidation”. Coal can interact with oxygen in the air at ambient temperature liberating heat. If the heat is allowed to accumulate the interaction rate increases and may ultimately lead to fires – known as spontaneous fires. The exact mechanism of the reaction of oxygen with coal is not completely understood as the chemical nature of coal is not yet fully established. But most of the workers agree that the reaction of oxygen with coal is a surface phenomenon and proceeds through a loosely bound coal-oxygen-water complex with subsequent steps being breakdown of the complex to simpler molecules such as CO, CO2 and H2O etc. Most popular hypothesis is that the overall reaction proceeds through a chain mechanism with moisture facilitating the formation of free radicals that act as chain carrier. Due to fire in coal mines, hues quantity of noxious gases liberated in the atmosphere which damage the ecological balance of nature. There are various technologies available in different parts of world to prevent and control spontaneous heating. Out of these technologies, chemical inhibitors play a vital role to control and combat fires. The main objective of this paper is to elaborate the causes, mechanism of spontaneous heating and technological advancement mainly development of chemical inhibitors for controlling and combating fire in coal mines.

Summary All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins’ physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin’s role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly., Graphical Abstract, Highlights • Ribozinoindoles are potent chemical inhibitors of eukaryotic ribosome assembly • Activity of four of Mdn1’s six ATPase sites is likely needed for cell growth • Ribozinoindoles inhibit recombinant full-length Mdn1’s ATPase activity in vitro • Assembly of Nsa1 particles, precursors of the 60S subunit, depends on Mdn1, Selective potent inhibitors of eukaryotic ribosome biogenesis are found through a chemical synthetic lethal screen.

Heat shock protein 90 (Hsp90) is a conserved and constitutively expressed molecular chaperone and it has been shown to stabilize oncoproteins and facilitate cancer development. Hsp90 has been considered as a therapeutic target for cancers and three classes of Hsp90 inhibitors have been developed: (1) benzoquinone ansamycin and its derivatives, (2) radicicol and its derivates, and (3) small synthetic inhibitors. The roles of these inhibitors in cancer treatment have been studied in laboratories and clinical trials, and some encouraging results have been obtained. Interestingly, targeting of Hsp90 has been shown to be effective in inhibition of cancer stem cells responsible for leukemia initiation and progression, providing a strategy for finding a cure. Because cancer stem cells are well defined in some human leukemias, we will focus on hematologic malignancies in this review.

Memoria presentada por Marta Vázquez Cedeira para optar al grado de Doctor por la Universidad de Salamanca y realizada en el Instituto de Biología Molecular y Celular del Cancer de Salamanca., Esta memoria ha sido realizada siendo Marta Vázquez Cedeira beneficiaria de una beca de Iniciación a la Investigación y una beca predoctoral JAE del Consejo Superior de Investigaciones Científicas-Fondo Social Europeo para el desarrollo de la tesis doctoral (2006-2010). La investigación en el laboratorio ha sido financiada por los siguientes proyectos: • Ministerio de Educación y Ciencia (SAF2004-02900; SAF2007-60242; CDS-2007-0017 y SAF2010-14935) • Junta de Castilla y León, Consejería de Sanidad (SAN/673/SA05/08; BOCyL Nº 170) • Junta de Castilla y León, Consejería de Educación (CSI14A08; CSI006A11-2 y grupo de excelencia GR-15) • Kutxa-Fundación INBIOMED

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

NAD + biosynthesis through nicotinamide phosphoribosyltransferase (NAMPT) holds potential as a target for the treatment of inflammatory disorders due to NAD + ’s role in immune cell signaling and metabolism. In addition to its activity as an enzyme, NAMPT is also secreted in the extracellular space where it acts as a pro-inflammatory and proangiogenic cytokine. NAMPT inhibition with FK866 has anti-inflammatory activity in different models of immune disorders and it prevents ischemia-reperfusion-induced heart damage by dampening the production of neutrophil chemoattractants. NAMPT blockade with a neutralizing antibody has beneficial effects in an acute lung injury model. Last, but not least, the anticancer activity of NAMPT inhibitors may also reflect, at least in part, their ability to modify the cancer microenvironment through their anti-inflammatory properties. Overall, NAMPT inhibition holds potential for the treatment of inflammation-related disorders and the development of effective and safe NAMPT inhibitors remains an area of strong interest in pharmaceutical research.