Kinase Syk Inhibitor Research Articles

Millingtonia hortensis L.f. ethanol extract exerts in vivo and in vitro anti-inflammatory activities through inhibition of Syk kinase in NF-κB pathway

Ethnopharmacological relevanceMillingtonia hortensis L.f., commonly known as tree jasmine or Indian cork tree, is native to South Asia and Southeast Asia. Traditionally, its stem bark, leaves, and roots are employed for pulmonary, gastrointestinal, and antimicrobial purposes, while the flowers are used in treating asthma and sinusitis.Aim of the study: The underlying anti-inflammatory mechanisms of M. hortensis remain relatively unexplored. Therefore, we studied the anti-inflammatory effects of M. hortensis and the molecular mechanisms of its ethanol extracts (Mh-EE) both in vitro and in vivo. Materials and methodsNitric oxide (NO) production was assessed using Griess reagent, while cell viability of RAW264.7 cell and HEK293T cells were determined via the MTT assay. Constituent analysis of Mh-EE using GC/MS-MS and HPLC, and mRNA expression of inflammatory cytokines was measured through PCR and RT-PCR. Protein levels were analyzed using western blotting. The thermal stability of Mh-EE was evaluated by CESTA. Lastly, a gastritis in vivo model was induced by HCl/EtOH, and protein expression levels were measured using western blotting. ResultsMh-EE significantly reduced NO production in LPS-induced RAW264.7 cells without substantially affecting cell viability. Additionally, Mh-EE decreased the expression of proinflammatory factors, such as iNOS, IL-1β and COX2. Furthermore, Mh-EE downregulated TLR4 expression, altered MyD88 recruitment, and suppressed phosphorylation of Syk, IKKα, IκBα and AKT. Simultaneously, Mh-EE also attenuated NF-κB signaling in HCl/EtOH-induced mice. ConclusionsMh-EE exerts anti-inflammatory effects by suppressing p-Syk in the NF-κB pathway, and it has potential as a novel treatment agent for inflammatory diseases.

Chronic spontaneous urticaria—status quo and future

AbstractChronic spontaneous urticaria (CsU) is a chronic inflammatory dermatosis whose etiology is not yet fully understood. In affected patients, it is often associated with a high limitation of health-related quality of life, which necessitates effective therapeutic management. Different immune cell populations such as mast cells, eosinophilic and basophilic granulocytes, and T cells are involved in the pathogenesis of CsU, whereby mast cells playing a key role. In addition, type I autoallergic reactions with auto IgE antibodies or type IIb autoimmune reactions with auto IgG antibodies have been identified in a proportion of patients. The current international guideline initially recommends the use of second-generation H1 antihistamines, first in standard, then in off-label quadruple dosing. Subsequently, the anti-IgE antibody omalizumab should be added. However, this therapy algorithm does not lead to freedom from manifestations in all patients. Therefore, various targeted therapies are currently being evaluated for their efficacy in CsU, such as off-label use of the anti-interleukin receptor alpha (IL4Rα) antibody dupilumab, the anti-IL-17A antibody secukinumab, or interleukin‑5 blockade using mepolizumab, reslizumab, or benralizumab. In addition, new promising compounds such as the Bruton tyrosine kinase (BTK) inhibitors remibrutinib and fenebrutinib, the anti-cKIT antibody barzolvolimab, the anti-SIGLEC8 antibody lirentelimab, the anti-TSLP antibody tezepelumab, the anti-C5aR1 antibody advoralimab, or the topical application of Syk kinase inhibitors are being tested, which were developed according to new insights into the pathogenesis of CsU. The BTK inhibitor fenebrutinib is currently not being pursued due to a less favorable side effect profile compared to remibrutinib, as well as the anti-IgE antibody ligelizumab, which was inferior to omalizumab therapy in a phase 3 study. Overall, there is a high need for new therapeutic strategies to better treat CsU both symptomatically and curatively. This requires a more comprehensive understanding of pathogenesis of the disease in order to develop new targeted therapies.

The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm

IntroductionAbdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. MethodsIn the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, RAW 264.7 macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. ResultsMacrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNFα and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by IC in macrophages. ConclusionsActivation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Developing a Robust and High Throughput Assay for Screening Innate Immune Training Molecules

Abstract Innate immune training molecules have several potential applications in the biomedical field. We developed an assay to screen for new training molecules by generating HiBiT tagged TNF cells in the THP1 monocytic cell line through CRISPR knock-in. The HiBiT tag, an 11 amino acid long peptide, was fused to the TNF C-terminus to produce luminescence upon addition of the LgBiT substrate. To optimize the assay, we first titrated different concentrations of multiple TLR ligands including pIC, p(dAdT), PGN, FLGN, LPS, R848, and P3C. LPS, PGN, and P3C induced the strongest signal to noise (S/N) ratio in the pool of TNF-HiBiT cells. From the TNF-HiBiT pool, we then single cell cloned and characterized them for optimum TNF response. The clones D3 and D6 had significantly higher S/N ratio compared to the pools, especially in response to P3C and PGN. To optimize the TNF-HiBiT cells for a training screen, we used previously known training molecules such as Syk kinase inhibitor, beta-Glucan and Rutaecarpine, to train multiple TNF-HiBiT cell clones. We found that the clones that induced a lower TNF signal upon acute TLR activation exhibited a more robust training phenotype compared to the clones that induced a more robust acute TNF signal. We found therefore that the optimal protocol to assess trained immunity is to use lower concentrations of activating TLR ligands (0.1, 0.1, and 0.0001 ng/mL of P3C, LPS and PGN) in the trained cells. This may be due to saturation of the TNF-HiBit reporter signal when cells are challenged with higher TLR ligand concentrations (10, 100, LPS, and 10 ng/mL of P3C, LPS and PGN). In brief, we have developed and optimized a robust assay for screening novel training molecules. This work was supported by the Intramural Research Program of NIAID, NIH Supported by grants from Intramural Research Program of NIAID, NIH and the Office of AIDS Research at NIH.

Restoration of aberrant gene expression of monocytes in systemic lupus erythematosus via a combined transcriptome-reversal and network-based drug repurposing strategy

BackgroundMonocytes -key regulators of the innate immune response- are actively involved in the pathogenesis of systemic lupus erythematosus (SLE). We sought to identify novel compounds that might serve as monocyte-directed targeted therapies in SLE.ResultsWe performed mRNA sequencing in monocytes from 15 patients with active SLE and 10 healthy individuals. Disease activity was assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K). Leveraging the drug repurposing platforms iLINCS, CLUE and L1000CDS2, we identified perturbagens capable of reversing the SLE monocyte signature. We identified transcription factors and microRNAs (miRNAs) that regulate the transcriptome of SLE monocytes, using the TRRUST and miRWalk databases, respectively. A gene regulatory network, integrating implicated transcription factors and miRNAs was constructed, and drugs targeting central components of the network were retrieved from the DGIDb database. Inhibitors of the NF-κB pathway, compounds targeting the heat shock protein 90 (HSP90), as well as a small molecule disrupting the Pim-1/NFATc1/NLRP3 signaling axis were predicted to efficiently counteract the aberrant monocyte gene signature in SLE. An additional analysis was conducted, to enhance the specificity of our drug repurposing approach on monocytes, using the iLINCS, CLUE and L1000CDS2 platforms on publicly available datasets from circulating B-lymphocytes, CD4+ and CD8+ T-cells, derived from SLE patients. Through this approach we identified, small molecule compounds, that could potentially affect more selectively the transcriptome of SLE monocytes, such as, certain NF-κB pathway inhibitors, Pim-1 and SYK kinase inhibitors. Furthermore, according to our network-based drug repurposing approach, an IL-12/23 inhibitor and an EGFR inhibitor may represent potential drug candidates in SLE.ConclusionsApplication of two independent - a transcriptome-reversal and a network-based -drug repurposing strategies uncovered novel agents that might remedy transcriptional disturbances of monocytes in SLE.

La presencia de receptores Fc de IgG activadores en macrófagos agrava el desarrollo de aneurisma aórtico abdominal experimental

IntroductionAbdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. MethodsIn the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, mouse macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. ResultsMacrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNF-α and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by immune complexes in macrophages. ConclusionsActivation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Older Adults and Immune Thrombocytopenia: Considerations for the Clinician

Many epidemiological studies have shown that the incidence of immune thrombocytopenia (ITP) increases after age 60 years and peaks in patients over age 80 years. Therefore, ITP is a concern for physicians taking care of older patients, especially regarding its diagnosis and management. The diagnostic work-up should exclude other causes of thrombocytopenia and secondary ITP, including myelodysplastic syndrome and drug-induced ITP. The treatment decision is influenced by an increased risk of bleeding, infectious diseases and thrombosis in this population and should take into account comorbidities and concomitant medications such as anticoagulant drugs. First-line treatment is based on short corticosteroids courses and intravenous immunoglobulin, which should be reserved for patients with more severe bleeding complications, with their higher risk of toxic effects as compared with younger patients. Second-line treatment should be tailored to the patient's history, comorbidities and preferences. Preferred second-line treatments are thrombopoietin receptor agonists for most groups and guidelines given their good efficacy/tolerance ratio, but the thrombotic risk is increased in older people. Other second-line options that can be good alternatives depending on the clinical context include rituximab, dapsone, fostamatinib or immunosuppressive drugs. Splenectomy is less often performed but remains an option for fit patients with chronic refractory disease. Emerging treatments such as Syk or Bruton tyrosine kinase inhibitors and FcRn antagonists are becoming available for ITP and may modify the treatment algorithm in the near future. The aim of this review is to describe the particularities of the diagnosis and treatment of ITP in older people, including the response and tolerance to the currently available drugs. We also discuss some situations related to co-morbidities that can frequently lead to adapt the management strategy in older patients.

Loss of NF1 in Melanoma Confers Sensitivity to SYK Kinase Inhibition.

A kinase inhibitor screen identifies SYK as a targetable vulnerability in melanoma cells with NF1 loss of function.

Treatment of congenital thrombocytopenia and decreased collagen reactivity in G6b-B–deficient mice

AbstractMice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2β1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2β1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2β1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2β1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases, providing extended long-term memory. In a screen of biologically active small molecules for regulators of TNF induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing LPS-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits β-glucan-induced H3K4Me3 marks at the promoters of several proinflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for β-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of SARS CoV-2 and OC43 coronavirus infection, highlighting a potential application of this molecule and other Syk kinase inhibitors as prophylactic treatments for viral susceptibility.

Search for putative heme binding sites in the integrated membrane protein of human erythrocytes SLC4A1 and protein kinases SYK and LYN

SLC4A1 protein or band 3 protein (band 3) is one of the most common erythrocyte membrane proteins. Along with the function of the anion exchanger, it contributes to the control of cell shape and lifespan through the formation of various complexes with cytoskeletal components and enzymes. Under oxidative stress, the protein oxidative modifications occur, in particular, due to the binding of hemoglobin aggregates, but the direct effect of heme as the major product of hemoglobin degradation on the band 3 protein activity has not been described in the literature. It is known that hemolytic conditions are accompanied by phosphorylation of the SLC4A1 protein, non-receptor tyrosine protein kinases LYN (by residue Y359), and SYK (by residues Y8 and Y21), while SYK kinase inhibitors have a stabilizing effect on erythrocytes. The regulatory effect of heme on Src kinases, which include SYK and LIN kinases, has been clarified, but the sites of their interaction with heme have not been investigated. Therefore, the aim of this study was to predict putative heme binding sites in the integral protein of the erythrocyte membrane SLC4A1, as well as in the protein kinases SYK and LYN and their complexes with SLC4A1, modeled in silico. Sequence analysis of proteins with HeMoQuest revealed several nonapeptides with potential heme binding sites in the SLC4A1 protein, including the cytosolic His98 and Tyr553 and Tyr555 residues in the region between the TM5 and TM6 transmembrane segments. These residues, as well as the amino acids Tyr216, His303, and His192, were also predicted as heme-binding sites by HemeBind tool. The largest number of putative heme binding sites was found for SYK protein kinase, including the two cytosolic residues Tyr216 and His303. Molecular docking of the SLC4A1 protein revealed a heme binding site in the cavity between His192 and region 173–176 in the cytosolic domain structure (PDB ID 4KY9 and 1HYN), also as part of a simulated complex with SYK or LYN kinases. It should be noted that site 175–185 is known as the ankyrin binding site. Docking heme to the membrane domain (PDB ID 4YZF) revealed a putative heme binding site near Lys539 in TM5, which, according to the literature, belongs to one of the reactive centers sensitive to the anionic transport inhibitor DIDS. Molecular docking to SYK protein kinase with ATP in the active site (PDB ID 4FL2) revealed two putative heme binding sites – near Tyr64 and near His243, but when ATP was removed from pdb-file, the heme occupied a nucleotide binding site in the cavity near Lys402 and His531. LYN protein kinase (PDB ID 5XY1) had a heme binding site near Tyr321 in the structure with inhibitor molecule (piperazine derivative). After removing the inhibitor, the heme occupied its area near Glu290 and Ala371. In most model complexes, the band 3 protein was revealed to be a more likely heme binding site than LYN and SYK protein kinases, but kinases with free active sites can apparently bind a heme instead of substrate, which will interfere phosphorylation. Disruption of band 3 protein under heme accumulation can inhibit an anion transport or complicate the formation of SLC4A1 complexes with cytoskeletal proteins that together with the effects on phosphorylation could be a mechanism for reducing erythrocyte stability.

Impact of Interindividual Differences in Plasma Fraction Unbound on the Pharmacokinetics of a Novel Syk Kinase Inhibitor in Beagle Dogs.

Inconsistencies in pharmacokinetic parameters between individual animals in preclinical studies are a common occurrence. Often such differences between animals are simply accepted as experimental variability rather than as indications of specific differences in animal phenotype that could lead to a different interpretation of the data. The fraction unbound in plasma is one factor influencing pharmacokinetic parameters and is typically determined using pooled plasma from multiple animals, making the assumption that there is limited population variance. However, this assumption is not often tested and may not hold true if there are polymorphisms affecting binding or variation in the concentrations of individual plasma proteins that could give rise to different fraction unbound phenotypes in individual animals. During profiling of a novel Syk inhibitor, AZ8399, striking interindividual differences in total plasma clearance and volume of distribution were observed between dogs consistent with differences in fraction unbound between animals. Determination of the fraction unbound showed a ∼5-fold difference in fraction unbound between the animals in the study. Broader analysis of individual dogs across a colony demonstrated a correlation between individual animal fraction unbound with total plasma clearance and volume of distribution. The concentrations of the common drug-binding proteins albumin and α1-acid glycoprotein in plasma were determined, and α1-acid glycoprotein levels were found to correlate with fraction unbound. Finally, single-nucleotide polymorphisms were identified at c.502 and c.522 of exon 5 of the dog α1-acid glycoprotein gene that may be correlated to the α1-acid glycoprotein concentration phenotype observed. SIGNIFICANCE STATEMENT: The current work demonstrates the potential for significant interindividual differences in plasma fraction unbound in beagle dogs and goes on to examine the underlying cause for the compound described. The findings suggest that the application of a population mean value of fraction unbound generated from a pooled sample may not always be appropriate and could introduce significant errors in scaling of in vitro clearance values, PBPK understanding, and interpretation of PKPD or toxicokinetic data in the context of unbound concentrations.

A Novel Strategy for P. falciparum Malaria based on Syk Kinase Inhibitors to Design Triple Artemisininbased Combination Therapies (TACTs) to Counteract Delayed Parasite Clearance (Drug Resistance) Following Standard ACT Treatment

A Novel Strategy for P. falciparum Malaria based on Syk Kinase Inhibitors to Design Triple Artemisininbased Combination Therapies (TACTs) to Counteract Delayed Parasite Clearance (Drug Resistance) Following Standard ACT Treatment

Tryptophanyl-tRNA Synthetase 1 Signals Activate TREM-1 via TLR2 and TLR4.

Tryptophanyl-tRNA synthetase 1 (WARS1) is an endogenous ligand of mammalian Toll-like receptors (TLR) 2 and TLR4. Microarray data, using mRNA from WARS1-treated human peripheral blood mononuclear cells (PBMCs), had indicated WARS1 to mainly activate innate inflammatory responses. However, exact molecular mechanism remains to be understood. The triggering receptor expressed on myeloid cells (TREM)-1 is an amplifier of pro-inflammatory processes. We found WARS1 to significantly activate TREM-1 at both mRNA and protein levels, along with its cell surface expression and secretion in macrophages. WARS1 stimulated TREM-1 production via TLR2 and TLR4, mediated by both MyD88 and TRIF, since targeted deletion of TLR4, TLR2, MyD88, and TRIF mostly abrogated TREM-1 activation. Furthermore, WARS1 promoted TREM-1 downstream phosphorylation of DAP12, Syk, and AKT. Knockdown of TREM-1 and inhibition of Syk kinase significantly suppressed the activation of inflammatory signaling loop from MyD88 and TRIF, leading to p38 MAPK, ERK, and NF-κB inactivation. Finally, MyD88, TRIF, and TREM-1 signaling pathways were shown to be cooperatively involved in WARS1-triggered massive production of IL-6, TNF-α, IFN-β, MIP-1α, MCP-1, and CXCL2, where activation of Syk kinase was crucial. Taken together, our data provided a new insight into WARS1′s strategy to amplify innate inflammatory responses via TREM-1.

A role for the Sts phosphatases in negatively regulating IFNγ‐mediated production of nitric oxide in monocytes

IntroductionThe atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap‐70. Mice lacking Sts expression (Sts −/−) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts −/− mice have not been definitively established, Sts −/− bone marrow‐derived monocytes (BMMs) demonstrate greater clearance of intracellular LVS following ex vivo infection, relative to wild type cells. To determine how the Sts proteins regulate monocyte bactericidal properties, we analyzed responses of infected cells.MethodsMonocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony‐forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse‐transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme‐linked immunosorbent assay.ResultsWe demonstrate that Sts −/− monocyte cultures produce elevated levels of interferon‐γ (IFNγ) after infection, relative to wild type cultures. Sts −/− monocytes also demonstrate heightened responsiveness to IFNγ. Specifically, Sts −/− monocytes produce elevated levels of antimicrobial NO following IFNγ stimulation, and this NO plays an important role in LVS restriction. Additional IFNγ‐stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts −/− cells. Both Sts‐1 and Sts‐2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFNγ‐induced NO in Sts −/− monocytes is abrogated following chemical inhibition of Syk kinase.ConclusionOur results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFNγ.

Syk Kinase Inhibitors Synergize with Artemisinins by Enhancing Oxidative Stress in Plasmodium falciparum-Parasitized Erythrocytes.

Although artemisinin-based combination therapies (ACTs) treat Plasmodium falciparum malaria effectively throughout most of the world, the recent expansion of ACT-resistant strains in some countries of the Greater Mekong Subregion (GMS) further increased the interest in improving the effectiveness of treatment and counteracting resistance. Recognizing that (1) partially denatured hemoglobin containing reactive iron (hemichromes) is generated in parasitized red blood cells (pRBC) by oxidative stress, (2) redox-active hemichromes have the potential to enhance oxidative stress triggered by the parasite and the activation of artemisinin to its pharmaceutically active form, and (3) Syk kinase inhibitors block the release of membrane microparticles containing hemichromes, we hypothesized that increasing hemichrome content in parasitized erythrocytes through the inhibition of Syk kinase might trigger a virtuous cycle involving the activation of artemisinin, the enhancement of oxidative stress elicited by activated artemisinin, and a further increase in hemichrome production. We demonstrate here that artemisinin indeed augments oxidative stress within parasitized RBCs and that Syk kinase inhibitors further increase iron-dependent oxidative stress, synergizing with artemisinin in killing the parasite. We then demonstrate that Syk kinase inhibitors achieve this oxidative enhancement by preventing parasite-induced release of erythrocyte-derived microparticles containing redox-active hemichromes. We also observe that Syk kinase inhibitors do not promote oxidative toxicity to healthy RBCs as they do not produce appreciable amounts of hemichromes. Since some Syk kinase inhibitors can be taken daily with minimal side effects, we propose that Syk kinase inhibitors could evidently contribute to the potentiation of ACTs.

Inhibition of SYK kinase does not confer a pro-proliferative or pro-invasive phenotype in breast epithelium or breast cancer cells.

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Macrophage Syk-PI3Kγ Inhibits Antitumor Immunity: SRX3207, a Novel Dual Syk-PI3K Inhibitory Chemotype Relieves Tumor Immunosuppression.

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8+ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.

Role of Band 3 Tyrosine Phosphorylation in Causing the Vaso-Occlusive Events in Sickle Cell Disease

Background: Sickle cell disease (SCD) derives from a mutation in sickle hemoglobin (HbS) that leads to vaso-occlusive events that eventually cause organ failure. Based on observations from multiple labs, we hypothesize that the events linking HbS denaturation to vaso-occlusion are: HbS denaturation→oxidative stress→inhibition of erythrocyte tyrosine phosphatases→enhanced tyrosine phosphorylation of band 3→dissociation of the spectrin/actin cytoskeleton from the membrane→release of membrane-derived microparticles (MPs) and discharge of cell-free HbS→induction of microthromboses and activation of the vascular endothelium→vascular occlusion. In the studies below, we document each step of this pathway and demonstrate that inhibitors of Band 3 tyrosine phosphorylation block all downstream events. Methods: Band 3 tyrosine phosphorylation, plasma MP counts, plasma cell-free Hb concentration, sickle cell deformability, sickle cell flow through micro-capillaries, and adhesion of sickle erythrocytes to activated endothelial cells are quantitated using fresh sickle blood both before and after incubation with inhibitors of Band 3 tyrosine phosphorylation. Findings: Imatinib and other Syk kinase inhibitors block Band 3 tyrosine phosphorylation, suppress release of membrane MPs, reduce discharge of cell-free HbS, improve sickle cell deformability, enhance flow of sickle cells through microcapillaries, and reduce adherence of sickle cells to heme-activated endothelium. Interpretation: Inhibition of Band 3 Tyrosine phosphorylation restores membrane integrity and thereby suppresses the downstream sequelae that lead to vaso-occlusive events in SCD. Inhibitors of Band 3 tyrosine phosphorylation may therefore constitute a novel effective therapy for SCD. Funding Statement: NIH grants R01GM24417-40 (P.S.L.), 1RF1 NS110049-01 (J.W.), R01HL133574 (U.A.G. and J.A.L.), T32HL134622 (R.A.), and NSF #1552782 (U.A.G.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All sickle cell blood samples were obtained following informed consent using procedures approved by the local IRBs.