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Additive-controlled crystallization is a promising method to improve crystal morphology and produce solid drug particles with the desired technological and pharmacological properties. However, its adaptation to continuous operation is a hardly researched area. Accordingly, in this work, we aimed to come up with a methodology that provides the systematic and fast development of a continuous three-stage MSMPR cascade crystallizer. For that, a cooling crystallization of famotidine (FMT) from water, in the presence of a formulation additive, poly(vinylpyrrolidone) (PVP-K12), was developed. Process parameters with a significant impact on product quality and quantity were examined in batch mode through a 24-1 fractional factorial design for the implementation of additive-controlled continuous crystallization. These batch experiments represented one residence time of the continuous system. Based on the statistical analysis, the residence time (RT) had the highest effect on yield, while the polymer amount was critical from the product polymorphism, crystal size, and flowability points of view. The values of critical process parameters in continuous operation were fixed according to the batch results. Two continuous cooling crystallization experiments were carried out, one with 1.25 w/wFMT% PVP-K12 and one with no additive. A mixture of FMT polymorphs (Form A and Form B) crystallized without the additive through five residence times (>6.5 h) with 70.8% overall yield. On the other hand, the additive-controlled continuous experiment resulted pure and homogeneous Form A product with excellent flowability. The system could be operated for >6.5 h without clogging with a 71.1% overall yield and a 4-fold improvement in productivity compared to its batch equivalent.
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Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system, was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures.
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Alzheimer's disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aß) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (ß-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC50 between 4-7 µM) and BACE-1 (IC50 between 50-65 µM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aß42.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Ratones , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Acetilcolinesterasa , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Encéfalo/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismoRESUMEN
Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.
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COVID-19 , Fluorocarburos , Antibacterianos/química , Antivirales/química , Catepsinas/farmacología , Fluorocarburos/farmacología , Glicopéptidos/química , Bacterias Grampositivas , Humanos , SARS-CoV-2 , Teicoplanina/farmacologíaRESUMEN
Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.
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Insectos/genética , Insectos/metabolismo , Hormonas Placentarias/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Estabilidad de Enzimas , Humanos , Hormonas Placentarias/aislamiento & purificación , Hormonas Placentarias/metabolismo , Plásmidos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , TemperaturaRESUMEN
The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and ß-apo-8'carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.
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Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach in early drug discovery. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compounds' databases. This approach can be combined with physico-chemical parameter and diversity filtering, bioisosteric replacements, and fragment-based approaches for performing a first round biological screening. Our objectives were to investigate the combination of 2D similarity search with various 3D ligand and structure-based methods for hit expansion and validation, in order to increase the hit rate and novelty. In the present account, six case studies are described and the efficiency of mixing is evaluated. While sequentially combined 2D/3D similarity approach increases the hit rate significantly, sequential combination of 2D similarity with pharmacophore model or 3D docking enriched the resulting focused library with novel chemotypes. Parallel integrated approaches allowed the comparison of the various 2D and 3D methods and revealed that 2D similarity-based and 3D ligand and structure-based techniques are often complementary, and their combinations represent a powerful synergy. Finally, the lessons we learnt including the advantages and pitfalls of the described approaches are discussed.
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Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular/métodos , Bibliotecas de Moléculas Pequeñas/química , Bases de Datos de Compuestos Químicos , Humanos , Relación Estructura-Actividad Cuantitativa , Análisis de Secuencia de Proteína/métodos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The complement system is associated with various diseases such as inflammation or auto-immune diseases. Complement-targeted drugs could provide novel therapeutic intervention against the above diseases. C1s, a serine protease, plays an important role in the CS and could be an attractive target since it blocks the system at an early stage of the complement cascade. Designing C1 inhibitors is particularly challenging since known inhibitors are restricted to a narrow bioactive chemical space in addition selectivity over other serine proteases is an important requirement. The typical architecture of a small molecule inhibitor of C1s contains an amidine (or guanidine) residue, however, the discovery of non-amidine inhibitors might have high value, particularly if novel chemotypes and/or compounds displaying improved selectivity are identified. We applied various virtual screening approaches to identify C1s focused libraries that lack the amidine/guanidine functionalities, then the in silico generated libraries were evaluated by in vitro biological assays. While 3D structure-based methods were not suitable for virtual screening of C1s inhibitors, and a 2D similarity search did not lead to novel chemotypes, pharmacophore model generation allowed us to identify two novel chemotypes with submicromolar activities. In three screening rounds we tested altogether 89 compounds and identified 20 hit compounds (<10 µM activities; overall hit rate: 22.5%). The highest activity determined was 12 nM (1,2,4-triazole), while for the newly identified chemotypes (1,3-benzoxazin-4-one and thieno[2,3-d][1,3]oxazin-4-one) it was 241 nM and 549 nM, respectively.
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Complemento C1s/antagonistas & inhibidores , Complemento C1s/química , Diseño de Fármacos , Descubrimiento de Drogas , Modelos Moleculares , Desarrollo de Medicamentos , Descubrimiento de Drogas/métodos , Estructura Molecular , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas PequeñasRESUMEN
Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.
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Inhibidores Enzimáticos/farmacología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Aminopropionitrilo/química , Aminopropionitrilo/farmacología , Disulfiram/química , Disulfiram/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ligandos , Estructura Molecular , Proteína-Lisina 6-Oxidasa/metabolismo , Piridinas/química , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Tionas/química , Tionas/farmacología , Tiram/química , Tiram/farmacologíaRESUMEN
A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.
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Aminoaciltransferasas/antagonistas & inhibidores , Simulación por Computador , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Aminoaciltransferasas/química , Aminoaciltransferasas/metabolismo , Dominio Catalítico , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Simulación del Acoplamiento Molecular , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Aminergic G-protein coupled receptors (GPRCs) represent well-known targets of central nervous-system related diseases. In this study a structure-based consensus virtual screening scheme was developed for designing targeted fragment libraries against class A aminergic GPCRs. Nine representative aminergic GPCR structures were selected by first clustering available X-ray structures and then choosing the one in each cluster that performs best in self-docking calculations. A consensus scoring protocol was developed using known promiscuous aminergic ligands and decoys as a training set. The consensus score (FrACS-fragment aminergic consensus score) calculated for the optimized protein ensemble showed improved enrichments in most cases as compared to stand-alone structures. Retrospective validation was carried out on public screening data for aminergic targets (5-HT1 serotonin receptor, TA1 trace-amine receptor) showing 8-17-fold enrichments using an ensemble of aminergic receptor structures. The performance of the structure based FrACS in combination with our ligand-based prefilter (FrAGS) was investigated both in a retrospective validation on the ChEMBL database and in a prospective validation on an in-house fragment library. In prospective validation virtual fragment hits were tested on 5-HT6 serotonin receptors not involved in the development of FrACS. Six out of the 36 experimentally tested fragments exhibited remarkable antagonist efficacies, and 4 showed IC50 values in the low micromolar or submicromolar range in a cell-based assay. Both retrospective and prospective validations revealed that the methodology is suitable for designing focused class A GPCR fragment libraries from large screening decks, commercial compound collections, or virtual databases.
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Aminas/química , Receptores Acoplados a Proteínas G/química , Animales , Células CHO , Cricetinae , Cricetulus , Modelos Químicos , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
Rapid in silico selection of target-focused libraries from commercial repositories is an attractive and cost-effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5-HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second-round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first-round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed.
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Descubrimiento de Drogas , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/química , Antagonistas de la Serotonina/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bases de Datos Farmacéuticas , Descubrimiento de Drogas/métodos , Humanos , Ligandos , Modelos MolecularesRESUMEN
Increasing number of evidences support the role of glycosylation in the evolution of autoimmunity. We examined carbohydrate-reactive natural autoantibodies systematically for the first time in patients with autoimmune myasthenia gravis. Antibodies reactive to glycosaminoglycans were measured with CovaLink ELISA in the sera of 59 myasthenia patients as well as in 54 healthy controls. We used the GlycoChip carbohydrate array to characterize individual carbohydrate recognition patterns. Chondroitin-sulphate C and anti-α-mannose-specific IgG levels were significantly elevated in myasthenia patients. Unexpectedly, we found that immunosuppressants increased the levels of the protective IgM glycosaminoglycan-reactive natural antibodies demonstrating a new role for these agents in immunoregulation.
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Autoanticuerpos/sangre , Glicosaminoglicanos/inmunología , Inmunosupresores/inmunología , Miastenia Gravis/sangre , Adulto , Análisis de Varianza , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Receptores Colinérgicos/inmunologíaRESUMEN
Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.
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Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa 5 , Evaluación Preclínica de Medicamentos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.
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Anticuerpos Monoclonales/química , Epítopos/química , Inmunoglobulinas/sangre , Biblioteca de Péptidos , Péptidos/química , Proteómica/métodos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Epítopos/genética , Epítopos/inmunología , Expresión Génica , Humanos , Inmunoensayo , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Cinética , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/inmunología , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.
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Bases de Datos de Compuestos Químicos , Bases de Datos Farmacéuticas , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Moleculares , Estructura Molecular , Receptores de Somatostatina/antagonistas & inhibidores , Aequorina/análisis , Aequorina/química , Química Farmacéutica , Ciclohexilaminas/química , Descubrimiento de Drogas , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Luz , Piperidinas/química , Quinazolinas/química , Interfaz Usuario-ComputadorRESUMEN
A novel chemocentric approach to identifying cancer-relevant targets is introduced. Starting with a large chemical collection, the strategy uses the list of small molecule hits arising from a differential cytotoxicity screening on tumor HCT116 and normal MRC-5 cell lines to identify proteins associated with cancer emerging from a differential virtual target profiling of the most selective compounds detected in both cell lines. It is shown that this smart combination of differential in vitro and in silico screenings (DIVISS) is capable of detecting a list of proteins that are already well accepted cancer drug targets, while complementing it with additional proteins that, targeted selectively or in combination with others, could lead to synergistic benefits for cancer therapeutics. The complete list of 115 proteins identified as being hit uniquely by compounds showing selective antiproliferative effects for tumor cell lines is provided.
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Antineoplásicos/farmacología , Modelos Biológicos , Proteínas de Neoplasias/genética , Oncogenes , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Proteínas de Neoplasias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach when starting new drug discovery projects. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compounds' databases. This in silico approach can be combined with physico-chemical parameter filtering based on the property space of the active compounds and 3D virtual screening if the structure of the target protein is available. A multi-step virtual screening procedure was developed and applied to select potential phosphodiesterase 5 (PDE5) inhibitors in real time. The combined 2D/3D in silico method resulted in the identification of 14 novel PDE5 inhibitors with <1 µMIC(50) values and the hit rate in the second in silico selection and in vitro screening round exceeded the 20%.
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Biología Computacional/métodos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Fosfodiesterasa 5/análisis , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Sulfonas/análisis , Animales , Línea Celular , Modelos Moleculares , Inhibidores de Fosfodiesterasa 5/química , Piperazinas/química , Piperazinas/farmacología , Mapas de Interacción de Proteínas , Purinas/análisis , Purinas/química , Purinas/farmacología , Estándares de Referencia , Citrato de Sildenafil , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
INTRODUCTION: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC). METHODS: A total of 94 patients with stage I-IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. RESULTS: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples compared with normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival. CONCLUSIONS: This study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.
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Adenocarcinoma/metabolismo , Carcinoma de Células Grandes/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias de Células Escamosas/metabolismo , Neovascularización Patológica/metabolismo , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apelina , Western Blotting , Carcinoma de Células Grandes/irrigación sanguínea , Carcinoma de Células Grandes/patología , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Neoplasias de Células Escamosas/irrigación sanguínea , Neoplasias de Células Escamosas/patología , Neovascularización Patológica/patología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
C1, the first component of the complement system, is a Ca(2+)-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Noncatalytic modules in C1r have been proposed to provide the flexibility required for function. Using a recombinant CUB2-CCP1 domain pair and the individual CCP1 module, we showed that binding of Ca(2+) induces the folding of the CUB2 domain and stabilizes its structure. In the presence of Ca(2+), CUB2 shows a compact, folded structure, whereas in the absence of Ca(2+), it has a flexible, disordered conformation. CCP1 module is Ca(2+)-insensitive. Isothermal titration calorimetry revealed that CUB2 binds a single Ca(2+) with a relatively high K(D) (430 mum). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca(2+), therefore the disordered, Ca(2+)-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca(2+) on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca(2+) concentration decreases, this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain.