Sampling of conformational ensemble for virtual screening using molecular dynamics simulations and normal mode analysis

Aim: Molecular dynamics simulations and normal mode analysis are well-established approaches to generate receptor conformational ensembles (RCEs) for ligand docking and virtual screening. Here, we report new fast molecular dynamics-based and normal mode analysis-based protocols combined with conformational pocket classifications to efficiently generate RCEs. Materials \& methods: We assessed our protocols on two well-characterized protein targets showing local active site flexibility, dihydrofolate reductase and large collective movements, CDK2. The performance of the RCEs was validated by distinguishing known ligands of dihydrofolate reductase and CDK2 among a dataset of diverse chemical decoys. Results \& discussion: Our results show that different simulation protocols can be efficient for generation of RCEs depending on different kind of protein flexibility

FAF-Drugs2: Free ADME/tox filtering tool to assist drug discovery and chemical biology projects

<p>Abstract</p> <p>Background</p> <p>Drug discovery and chemical biology are exceedingly complex and demanding enterprises. In recent years there are been increasing awareness about the importance of predicting/optimizing the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of small chemical compounds along the search process rather than at the final stages. Fast methods for evaluating ADMET properties of small molecules often involve applying a set of simple empirical rules (educated guesses) and as such, compound collections' property profiling can be performed <it>in silico</it>. Clearly, these rules cannot assess the full complexity of the human body but can provide valuable information and assist decision-making.</p> <p>Results</p> <p>This paper presents FAF-Drugs2, a free adaptable tool for ADMET filtering of electronic compound collections. FAF-Drugs2 is a command line utility program (e.g., written in Python) based on the open source chemistry toolkit OpenBabel, which performs various physicochemical calculations, identifies key functional groups, some toxic and unstable molecules/functional groups. In addition to filtered collections, FAF-Drugs2 can provide, via Gnuplot, several distribution diagrams of major physicochemical properties of the screened compound libraries.</p> <p>Conclusion</p> <p>We have developed FAF-Drugs2 to facilitate compound collection preparation, prior to (or after) experimental screening or virtual screening computations. Users can select to apply various filtering thresholds and add rules as needed for a given project. As it stands, FAF-Drugs2 implements numerous filtering rules (23 physicochemical rules and 204 substructure searching rules) that can be easily tuned.</p

Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

Background: During inflammation, beta(2)-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. Results: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk(-/-) hematopoietic system. In unstimulated venules, Syk(-/-) leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk(-/-) and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk(-/-) leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk(-/-) leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk(-/-) chimeric mice was due to compromised beta(2)-integrin-mediated outside-in signaling. Conclusion: We provide substantial evidence for an important role of Syk in mediating beta(2)-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo

Cell-crossing functional network driven by microRNA-125a regulates endothelial permeability and monocyte trafficking in acute inflammation

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications

Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-β-dependent mechanisms

ACKNOWLEDGMENTS. We thank Phillip B. Murray for help with the shRNA mapping pipeline and Francesc Lopez-Giraldez for help with RNAseq mapping software.Peer reviewedPostprintPostprin

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo

RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner

<p>Abstract</p> <p>Background</p> <p>The receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the β₂-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated β₂-integrin-dependent leukocyte adhesion in <it>RAGE^-/-</it>and <it>Icam1^-/-</it>mice in different cremaster muscle models of inflammation using intravital microscopy.</p> <p>Results</p> <p>We demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo.</p> <p>Conclusion</p> <p>Our results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.</p

Critical role of phospholipase Cγ2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis

β2 integrins and Fcγ receptors are critically involved in neutrophil activation at the site of inflammation. Both receptor types trigger a receptor-proximal tyrosine phosphorylation cascade through Src family kinases and Syk, but further downstream signaling events are poorly understood. We show that phospholipase C (PLC) γ2 is phosphorylated downstream of Src family kinases and Syk during integrin or Fc receptor-mediated activation of neutrophils. PLCγ2−/− neutrophils are completely defective in β2 integrin or Fcγ receptor-mediated functional responses such as respiratory burst, degranulation, or cell spreading in vitro and show reduced adhesion/spreading in inflamed capillary venules in vivo. However, PLCγ2−/− neutrophils respond normally to various other agonists, including chemokines, bacterial formyl peptides, Toll-like receptor ligands, or proinflammatory cytokines, and migrate normally both in vitro and in vivo. To confirm the in vivo relevance of these observations, the effect of the PLCγ2−/− mutation was tested in the K/B×N serum transfer arthritis model, which is known to require β2 integrins, Fcγ receptors, and neutrophils. PLCγ2 deficiency completely protected mice from clinical signs and histological features of arthritis as well as from arthritis-induced loss of articular function. These results identify PLCγ2 as a critical player of integrin and Fc receptor-mediated neutrophil functions and the neutrophil-mediated effector phase of autoimmune arthritis

Gravitational Waves From Known Pulsars: Results From The Initial Detector Era

We present the results of searches for gravitational waves from a large selection of pulsars using data from the most recent science runs (S6, VSR2 and VSR4) of the initial generation of interferometric gravitational wave detectors LIGO (Laser Interferometric Gravitational-wave Observatory) and Virgo. We do not see evidence for gravitational wave emission from any of the targeted sources but produce upper limits on the emission amplitude. We highlight the results from seven young pulsars with large spin-down luminosities. We reach within a factor of five of the canonical spin-down limit for all seven of these, whilst for the Crab and Vela pulsars we further surpass their spin-down limits. We present new or updated limits for 172 other pulsars (including both young and millisecond pulsars). Now that the detectors are undergoing major upgrades, and, for completeness, we bring together all of the most up-to-date results from all pulsars searched for during the operations of the first-generation LIGO, Virgo and GEO600 detectors. This gives a total of 195 pulsars including the most recent results described in this paper.United States National Science FoundationScience and Technology Facilities Council of the United KingdomMax-Planck-SocietyState of Niedersachsen/GermanyAustralian Research CouncilInternational Science Linkages program of the Commonwealth of AustraliaCouncil of Scientific and Industrial Research of IndiaIstituto Nazionale di Fisica Nucleare of ItalySpanish Ministerio de Economia y CompetitividadConselleria d'Economia Hisenda i Innovacio of the Govern de les Illes BalearsNetherlands Organisation for Scientific ResearchPolish Ministry of Science and Higher EducationFOCUS Programme of Foundation for Polish ScienceRoyal SocietyScottish Funding CouncilScottish Universities Physics AllianceNational Aeronautics and Space AdministrationOTKA of HungaryLyon Institute of Origins (LIO)National Research Foundation of KoreaIndustry CanadaProvince of Ontario through the Ministry of Economic Development and InnovationNational Science and Engineering Research Council CanadaCarnegie TrustLeverhulme TrustDavid and Lucile Packard FoundationResearch CorporationAlfred P. Sloan FoundationAstronom

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