57 research outputs found
The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling
The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction
Tyrosine Kinase Syk Non-Enzymatic Inhibitors and Potential Anti-Allergic Drug-Like Compounds Discovered by Virtual and In Vitro Screening
In the past decade, the spleen tyrosine kinase (Syk) has shown a high potential for the discovery of new treatments for inflammatory and autoimmune disorders. Pharmacological inhibitors of Syk catalytic site bearing therapeutic potential have been developed, with however limited specificity towards Syk. To address this topic, we opted for the design of drug-like compounds that could impede the interaction of Syk with its cellular partners while maintaining an active kinase protein. To achieve this challenging task, we used the powerful potential of intracellular antibodies for the modulation of cellular functions in vivo, combined to structure-based in silico screening. In our previous studies, we reported the anti-allergic properties of the intracellular antibody G4G11. With the aim of finding functional mimics of G4G11, we developed an Antibody Displacement Assay and we isolated the drug-like compound C-13, with promising in vivo anti-allergic activity. The likely binding cavity of this compound is located at the close vicinity of G4G11 epitope, far away from the catalytic site of Syk. Here we report the virtual screen of a collection of 500,000 molecules against this new cavity, which led to the isolation of 1000 compounds subsequently evaluated for their in vitro inhibitory effects using the Antibody Displacement Assay. Eighty five compounds were selected and evaluated for their ability to inhibit the liberation of allergic mediators from mast cells. Among them, 10 compounds inhibited degranulation with IC50 values ≤10 µM. The most bioactive compounds combine biological activity, significant inhibition of antibody binding and strong affinity for Syk. Moreover, these molecules show a good potential for oral bioavailability and are not kinase catalytic site inhibitors. These bioactive compounds could be used as starting points for the development of new classes of non-enzymatic inhibitors of Syk and for drug discovery endeavour in the field of inflammation related disorders
Designing Focused Chemical Libraries Enriched in Protein-Protein Interaction Inhibitors using Machine-Learning Methods
Protein-protein interactions (PPIs) may represent one of the next major classes of therapeutic targets. So far, only a minute fraction of the estimated 650,000 PPIs that comprise the human interactome are known with a tiny number of complexes being drugged. Such intricate biological systems cannot be cost-efficiently tackled using conventional high-throughput screening methods. Rather, time has come for designing new strategies that will maximize the chance for hit identification through a rationalization of the PPI inhibitor chemical space and the design of PPI-focused compound libraries (global or target-specific). Here, we train machine-learning-based models, mainly decision trees, using a dataset of known PPI inhibitors and of regular drugs in order to determine a global physico-chemical profile for putative PPI inhibitors. This statistical analysis unravels two important molecular descriptors for PPI inhibitors characterizing specific molecular shapes and the presence of a privileged number of aromatic bonds. The best model has been transposed into a computer program, PPI-HitProfiler, that can output from any drug-like compound collection a focused chemical library enriched in putative PPI inhibitors. Our PPI inhibitor profiler is challenged on the experimental screening results of 11 different PPIs among which the p53/MDM2 interaction screened within our own CDithem platform, that in addition to the validation of our concept led to the identification of 4 novel p53/MDM2 inhibitors. Collectively, our tool shows a robust behavior on the 11 experimental datasets by correctly profiling 70% of the experimentally identified hits while removing 52% of the inactive compounds from the initial compound collections. We strongly believe that this new tool can be used as a global PPI inhibitor profiler prior to screening assays to reduce the size of the compound collections to be experimentally screened while keeping most of the true PPI inhibitors. PPI-HitProfiler is freely available on request from our CDithem platform website, www.CDithem.com
Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl–like molecules binding
A novel IgE antibody targeting the prostate-specific antigen as a potential prostate cancer therapy
Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The
prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 andCD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa.Fil: Daniels-Wells, Tracy R. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Helguera, Gustavo Fernando. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Tecnologia Farmaceutica; Argentina; University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Leuchter, Richard K. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Quintero, Rafael. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Kozman, Maggie. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Rodríguez, José A.. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; University of California. The Molecular Biology Institute; Estados Unidos de América;Fil: Ortiz-Sánchez, E. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; Biomedical Research in Cancer. Basic Research Division. National Institute of Cancerology; Mexico.;Fil: Martínez-Maza, Otonel. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América;Fil: Schultes, Brigit C.. Advanced Immune Therapeutics; Estados Unidos de América;Fil: Nicodemus Christopher. Advanced Immune Therapeutics; Estados Unidos de América;Fil: Penichet, Manuel. University of California. David Geffen School of Medicine. Department of Surgery. Division of Surgical Oncology; Estados Unidos de América; University of California. The Molecular Biology Institute; Estados Unidos de América
Yersinia enterocolitica Serum Resistance Proteins YadA and Ail Bind the Complement Regulator C4b-Binding Protein
Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host
Major prospects for exploring canine vector borne diseases and novel intervention methods using 'omic technologies
Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, 'omic technologies. The aim of the present article is to take a prospective view of the impact that next-generation, 'omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a 'systems biology' context and could lead to radically new intervention and management strategies against CVBDs
Structure-Based Virtual Screening for Drug Discovery: a Problem-Centric Review
Structure-based virtual screening (SBVS) has been widely applied in early-stage drug discovery. From a problem-centric perspective, we reviewed the recent advances and applications in SBVS with a special focus on docking-based virtual screening. We emphasized the researchers’ practical efforts in real projects by understanding the ligand-target binding interactions as a premise. We also highlighted the recent progress in developing target-biased scoring functions by optimizing current generic scoring functions toward certain target classes, as well as in developing novel ones by means of machine learning techniques
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