Tyrosine kinase syk non-enzymatic inhibitors and potential anti-allergic drug-like compounds discovered by virtual and in vitro screening.
ABSTRACT: 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 IC?? 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.
Project description:Spleen tyrosine kinase (SYK) is an essential mediator of immune cell signaling and has been anticipated as a therapeutic target for autoimmune diseases, notably rheumatoid arthritis, allergic rhinitis, asthma, and cancers. Significant attempts have been undertaken in recent years to develop SYK inhibitors; however, limited success has been achieved due to poor pharmacokinetics and adverse effects of inhibitors. The primary goal of this research was to identify potential inhibitors having high affinity, selectivity based on key molecular interactions, and good drug-like properties than the available inhibitor, fostamatinib. In this study, a 3D-QSAR model was built for SYK based on known inhibitor IC50 values. The best pharmacophore model was then used as a 3D query to screen a drug-like database to retrieve hits with novel chemical scaffolds. The obtained compounds were subjected to binding affinity prediction using the molecular docking approach, and the results were subsequently validated using molecular dynamics (MD) simulations. The simulated compounds were ranked according to binding free energy (ΔG), and the binding affinity was compared with fostamatinib. The binding mode analysis of selected compounds revealed that the hit compounds form hydrogen bond interactions with hinge region residue Ala451, glycine-rich loop residue Lys375, Ser379, and DFG motif Asp512. Identified hits were also observed to form a desirable interaction with Pro455 and Asn457, the rare feature observed in SYK inhibitors. Therefore, we argue that identified hit compounds ZINC98363745, ZINC98365358, ZINC98364133, and ZINC08789982 may help in drug design against SYK.
Project description:The nonreceptor tyrosine kinase Syk, a central regulator of immune cell differentiation and activation, is a promising drug target for treatment of leukemia and allergic and inflammatory diseases. The clinical failure of Syk inhibitors underscores the importance of understanding the regulation of Syk function and activity. A series of previous studies emphasized the importance of three C-terminal tyrosines in Syk for kinase activity regulation, as docking sites for downstream effector molecules, and for Ca2+ mobilization. Here, we investigated the roles of these C-terminal tyrosines in the mouse. Surprisingly, expression of a triple tyrosine-to-phenylalanine human Syk mutant, SYK(Y3F), was not associated with discernible signaling defects either in reconstituted DT40 cells or in B or mast cells from mice expressing SYK(Y3F) instead of wild-type Syk. Remarkably, lymphocyte differentiation, calcium mobilization, and 2,4,6-trinitrophenyl (TNP)-specific immune responses were unperturbed in SYK(Y3F) mice. These results emphasize the capacity of immune cells to compensate for specific molecular defects, likely using redundant intermolecular interactions, and highlight the importance of in vivo analyses for understanding cellular signaling mechanisms.
Project description:Brusatol derivative-34 (Bru-34), a derivative of brusatol, has been shown significantly anti-inflammatory activity in mice in our previously work. However, to our knowledge, there were very limited studies on how Bru-34 affected airway inflammation. Thus, in this present study, the effects and potential mechanisms of Bru-34 on allergic airway inflammation were examined both <i>in vivo</i> and <i>in vitro</i>. The results showed that Bru-34 attenuated the allergic airway inflammation in mice, with significant decreasing of the inflammatory cells and mediators in bronchoalveolar lavage fluids and attenuation of the histopathological alterations in the lung tissues. In addition, Bru-34 significantly inhibited the release of inflammatory cytokines in antigen induced rat basophilic leukemia -2H3 (RBL-2H3) cells. What's more, Bru-34 significantly decreased the expression of spleen tyrosine kinase (Syk), <i>p</i>-Syk, cytoplasmic phospholipase A2 (cPLA2), <i>p</i>-cPLA2, nuclear factor-κB (NF-κB) and p-NF-κB both in allergic mice lung tissue and antigen induced RBL-2H3 cells. Furthermore, the collaborative effects of Bru-34 with inhibitors against Syk, cPLA2, and NF-κB, showed that Syk was an important target of Bru-34, and cPLA2 and NF-κB played important roles in the coordinated inflammatory response. In conclusion, Bru-34 could significantly modulate the allergic airway inflammation, and its potential mechanism was revealed at least partially via down-regulating of Syk-cPLA2 -NF-κB signaling.
Project description:Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1? in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.
Project description:Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor gamma (FcRgamma)-chain-dependent platelet activation through its interaction with Syk.
Project description:Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcepsilonRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.
Project description:Spleen tyrosine kinase (Syk) is a critical target protein for treating immunoreceptor signalling-mediated allergies. In this study, a virtual screening of an in-house Chinese medicine database followed by biological assays was carried out to identify novel Syk inhibitors. A molecular docking method was employed to screen for compounds with potential Syk inhibitory activity. Then, an in vitro kinase inhibition assay was performed to verify the Syk inhibitory activity of the virtual screening hits. Subsequently, a ?-hexosaminidase release assay was conducted to evaluate the anti-mast cell degranulation activity of the active compounds. Finally, tanshinone I was confirmed as a Syk inhibitor (IC50 = 1.64 ?M) and exhibited anti-mast cell degranulation activity in vitro (IC50 = 2.76 ?M). Docking studies showed that Pro455, Gln462, Leu377, and Lys458 were key amino acid residues for Syk inhibitory activity. This study demonstrated that tanshinone I is a Syk inhibitor with mast cell degranulation inhibitory activity. Tanshinone I may be a potential lead compound for developing effective and safe Syk-inhibiting drugs.
Project description:The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.
Project description:Resistance to antimalarial drugs has spread rapidly over the past few decades. The WHO recommends artemisinin-based combination therapies for the treatment of uncomplicated malaria, but unfortunately these approaches are losing their efficacy in large areas of Southeast Asia. In 2016, artemisinin resistance was confirmed in 5 countries of the Greater Mekong subregion. We focused our study on Syk inhibitors as antimalarial drugs. The Syk protein is present in human erythrocytes, and the membrane of protein band 3 is its major target following activation by oxidant stress. Tyr phosphorylation of band 3 occurs during P. falciparum growth, leading to the release of microparticles containing hemicromes and structural weakening of the host cell membrane, simplifying merozoite reinfection. Syk inhibitors block these events by interacting with the Syk protein's catalytic site. We performed in vitro proteomics and in silico studies and compared the results. In vitro studies were based on treatment of the parasite's cellular cultures with different concentrations of Syk inhibitors, while proteomics studies were focused on the Tyr phosphorylation of band 3 by Syk protein with the same concentrations of drugs. In silico studies were based on different molecular modeling approaches in order to analyze and optimize the ligand-protein interactions and obtain the highest efficacy in vitro. In the presence of Syk inhibitors, we observed a marked decrease of band 3 Tyr phosphorylation according to the increase of the drug's concentration. Our studies could be useful for the structural optimization of these compounds and for the design of novel Syk inhibitors in the future.
Project description:<h4>Background</h4>SYK gene regulates the expression of SYK kinase (Spleen tyrosine kinase), an important non-receptor protein-tyrosine kinase for immunological receptor-mediated signaling, which is also considered a tumor growth metastasis initiator. An onco-informatics analysis was adopted to evaluate the expression and prognostic value of the SYK gene in colorectal cancer (CRC), the third most fatal cancer type; of late, it may be a biomarker as another targeted site for CRC. In addition, identify the potential phytochemicals that may inhibit the overexpression of the SYK kinase protein and minimize the human CRC.<h4>Materials & methods</h4>The differential expression of the SYK gene was analyzed using several transcriptomic databases, including Oncomine, UALCAN, GENT2, and GEPIA2. The server cBioPortal was used to analyze the mutations and copy number alterations, whereas GENT2, Gene Expression Profiling Interactive Analysis (GEPIA), Onco-Lnc, and PrognoScan were used to examine the survival rate. The protein-protein interaction network of SYK kinase and its co-expressed genes was conducted via Gene-MANIA. Considering the SYK kinase may be the targeted site, the selected phytochemicals were assessed by molecular docking using PyRx 0.8 packages. Molecular interactions were also observed by following the Ligplot+ version 2.2. YASARA molecular dynamics simulator was applied for the post-validation of the selected phytochemicals.<h4>Results</h4>Our result reveals an increased level of mRNA expression of the SYK gene in colorectal adenocarcinoma (COAD) samples compared to those in normal tissues. A significant methylation level and various genetic alterations recurrence of the SYK gene were analyzed where the fluctuation of the SYK alteration frequency was detected across different CRC studies. As a result, a lower level of SYK expression was related to higher chances of survival. This was evidenced by multiple bioinformatics platforms and web resources, which demonstrated that the SYK gene can be a potential biomarker for CRC. In this study, aromatic phytochemicals, such as kaempferol and glabridin that target the macromolecule (SYK kinase), showed higher stability than the controls, and we have estimated that these bioactive potential phytochemicals might be a useful option for CRC patients after the clinical trial.<h4>Conclusions</h4>Our onco-informatics investigation suggests that the SYK gene can be a potential prognostic biomarker of CRC. On the contrary, SYK kinase would be a major target, and all selected compounds were validated against the protein using in-silico drug design approaches. Here, more in vitro and in vivo analysis is required for targeting SYK protein in CRC.