Statin and rottlerin small-molecule inhibitors restrict colon cancer progression and metastasis via MACC1

MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is—to the best of our knowledge—the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients

Glycolytic flux control by drugging phosphoglycolate phosphatase

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates

Article Translocation Biosensors – Cellular System Integrators to Dissect CRM1-Dependent Nuclear Export by Chemicogenomics

sensor

Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential

BACKGROUND: Deregulation of the protein kinase MARK has been linked to Alzheimer disease. - RESULTS: Mark-specific inhibitors and a biosensor are identified. - CONCLUSION: The inhibitors and the biosensor are tools to provide new insights into the role of MARK during polarity establishment and maintenance of neurons. - SIGNIFICANCE: The inhibitors might possess therapeutic potential by interfering with abnormal Tau phosphorylation in Alzheimer disease.Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific single-substrate enzymes (HisA and TrpF), sharing a similar (β/α)8-barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept

Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy

Software quality assurance

Dissertação de Mestrado em Desenvolvimento de Software e Sistemas Interactivos apresentada à Escola Superior de Tecnologia do Instituto Politécnico de Castelo Branco.O desenvolvimento de software é caracterizado por um conjunto de actividades, as quais estão em grande parte relacionadas com habilidades humanas. Assim, como em qualquer outra actividade humana, o desenvolvimento de software pode estar sujeito a erros. Em função disto, a aplicação de práticas de garantia da qualidade ao longo do processo de desenvolvimento, torna-se um aspecto fundamental para a redução e prevenção desses erros inevitáveis. Considerando ainda a importância que a inovação de produtos tem sobre o sucesso das organizações, é imprescindível que a qualidade seja agregada ao processo de desenvolvimento de software, garantindo assim a qualidade do produto através da definição e normalização deste processo. Testar o software é uma das formas de verificação que mais tem sido utilizada na prática, particularmente o teste funcional, por estar baseado na especificação do software. Esta técnica reduz os custos inerentes ao processo de teste, uma vez que é praticada paralelamente ao desenvolvimento do software. Em função disto, estão a ser feitas diversas pesquisas com o objectivo de produzir técnicas efectivas para a construção de testes a partir da especificação dos sistemas. Existe assim uma convergência, entre diversos autores e especialistas da área, na necessidade de automação destas técnicas, de forma a permitir que todo o processo de teste possa ser executado e re-executado com a menor intervenção humana possível. Dada a importância da qualidade de software para o sucesso das organizações, este trabalho incide em avaliar conceitos, estudos e teorias de especialistas da área de Software Quality Assurance quer no planeamento quer no desenvolvimento, bem como na validação do software. Serão apresentadas duas das normas mais utilizadas para a certificação dos processos de desenvolvimento de software, boas práticas que podem ser aplicadas ao mesmo e um caso de estudo exemplificativo, de uma empresa deste ramo certificada, onde são exemplificados os processos utilizados e aplicadas as boas práticas sugeridas.Abstract: Software development is characterized by a set of activities, which are largely related to human skills. So, like any other human activity, software development may be subject to errors. Because of this, the application of quality assurance practices during the development process becomes a fundamental aspect for the reduction and prevention of such inevitable errors. Considering also the importance of product innovation on the success of organizations, is essential that quality is tied to the process of software development, thus ensuring product quality through the development and standardization of this process. Testing software is one way of checking that it has been used in practice, particularly the functional test, because it is based on the software specification. This technique reduces the cost of the testing process as it is done in addition to software development. Because of this, are being made various studies in order to produce effective techniques for the construction of tests from the specification of systems. There is thus a convergence, between different authors and experts, on the need for automation of these techniques, to allow the entire testing process can be executed and re-executed with the least human intervention possible. Given the importance of software quality for the success of the organizations, this work focuses on evaluating concepts, studies and theories of experts in the field of Software Quality Assurance in planning, development and validation of software. Will be presented two of the most widely used standards for the certification of the software development process, good practices that can be applied to it and a case study, of a company that is certified, exemplifying the used processes and the suggested best practices

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling

<div><p>Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20.</p><p>Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.</p></div

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway.

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs