Interleukin-1 receptor associated kinase (IRAK)1 and IRAK4 are serine/threonine kinases critical for downstream signaling of most toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs), leading to proinflammatory cytokine production and activation of innate immune and inflammatory responses. Herein, we describe the preclinical characterization and early clinical development of R835, a potent, selective dual inhibitor of IRAK1/IRAK4 and the active metabolite of the prodrug R289, which is currently undergoing clinical evaluation in relapsed/refractory lower-risk myelodysplastic syndrome (LR-MDS). In multiple cell types and in orally dosed mice, R835 potently and selectively inhibited TLR- and IL-1R-dependent proinflammatory cytokine production. In a randomized, placebo-controlled, double-blind, phase 1, first-in-human study in 82 healthy participants, R835 was well tolerated with a favorable pharmacokinetic profile and markedly inhibited lipopolysaccharide-induced cytokine release (TNF, IL-6, IL-8, MIP1α, and MIP1β) compared with placebo, mirroring preclinical data. Taken together, these initial findings indicate that R835 may represent a novel therapeutic for inflammatory diseases.

ABSTRACT Receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) inhibitors are being investigated for chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Ocadusertib is a potent and selective allosteric inhibitor of RIPK1 and is currently being studied in clinical trials. Here, we present data on the development of ocadusertib, including preclinical studies and pharmacokinetic, safety, and target engagement results from phase 1 trials. Preclinically, ocadusertib inhibited RIPK1 enzymatic activity with high potency (half‐maximal inhibitory concentration [IC50] = 12–38 nM) and inhibited necroptotic responses in multiple cell‐based assays. Ocadusertib was highly selective for RIPK1, showing no significant inhibition of more than 100 other kinases representative of the full kinome. In mouse studies, ocadusertib treatment prevented RIPK1‐dependent hypothermia in response to tumor necrosis factor (TNF) challenge and significantly reduced disease severity in a model of chronic proliferative dermatitis. In a first‐in‐human study of ocadusertib in healthy participants, single oral doses exhibited linear pharmacokinetics and dose‐proportional exposure, with a time to maximum concentration of 1–4 h and a half‐life of 13–15 h. Steady state was attained at 4–6 days after multiple once‐daily dose administrations. Ocadusertib was well tolerated, with no deaths, serious adverse events, or significant treatment‐emergent adverse events reported. In a phase 1, double‐blind, randomized, multiple‐dose study in healthy participants, greater than 90% RIPK1 target engagement was achieved at Day 14 with ocadusertib treatment. Taken together, these findings support further assessment of ocadusertib for the treatment of chronic inflammatory diseases.

Background: The inhibition of IRAK signaling may potentially to be beneficial in several dermal indications including plaque psoriasis and palmoplantar psoriasis which are driven by IL-23 and IL-17. The Rigel IRAK1/4 inhibitor project resulted in an extensive portfolio of inhibitors that block pro-inflammatory cytokine release, which we leveraged to identify a molecule that was optimized for topical administration.

Certain somatic mutations provide a fitness advantage to hematopoietic stem cells and lead to clonal expansion of mutant blood cells, known as clonal hematopoiesis (CH). Among the most common CH mutations, ASXL1 mutations pose the highest risk for cardiovascular diseases (CVDs), yet the mechanisms by which they contribute to CVDs are unclear. Here we show that hematopoietic cells harboring C-terminally truncated ASXL1 mutant (ASXL1-MT) accelerate the development of atherosclerosis in Ldlr-/- mice. Transcriptome analyses of plaque cells showed that monocytes and macrophages expressing ASXL1-MT exhibit inflammatory signatures. Mechanistically, we demonstrate that wild-type ASXL1 has an unexpected non-epigenetic role by suppressing innate immune signaling through the inhibition of IRAK1-TAK1 interaction in the cytoplasm. This regulatory function is lost in ASXL1-MT, resulting in NF-kappa B activation. Inhibition of IRAK1/4 alleviated atherosclerosis driven by ASXL1-MT and decreased inflammatory monocytes. The present work provides a mechanistic and cellular explanation linking ASXL1 mutations, CH and CVDs.

ObjectiveSyk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG-mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis.MethodsPhosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)-ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization.ResultsWe identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV.ConclusionThese findings indicate that Syk plays a critical role in MPO-ANCA IgG-induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.

Introduction: We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of tients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells.Methods: This study was a double-blind, randomized, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomized to receive placebo, fosta-matinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors. The primary end point was reduction of proteinuria. Secondary points included change from baseline in estimated glomerular filtration rate (eGFR) and kidney histology.Results: Although we could not detect significant reduction in proteinuria with fostamatinib overall, predetermined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27%, and 36% in the placebo, fostamatinib 100 mg, and 150 groups, respectively) in patients with baseline urinary protein-to-creatinine ratios (UPCR) more than mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well-tolerated. Side effects included diarrhea, hypertension, and increased liver enzymes. Thirty-nine patients underwent repeat opsy showing reductions in SYK staining associated with therapy at low dose (-1.5 vs. 1.7 SYK+ glomerulus in the placebo group, P < 0.05).Conclusions: There was a trend toward reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 Further study may be warranted.

Dimethyl fumarate 1 is approved for the treatment of multiple sclerosis but is also associated with off-target activation of the niacin receptor. By using a tetrazolone or triazolone bioisostere approach to the fumarate and vinyl sulfone series of Nrf2 activators, we have optimized the electrophilicity of the double bond to tune the on-target Nrf2 activation with PK properties to achieve efficacy in animal models of multiple sclerosis. The study linked highly potent, highly electrophilic molecules to low plasma stability and, subsequently, limited efficacy. By contrast, a sulfonylvinyltriazolone 17 retains on-target potency but shows much weaker electrophilic potential. As a consequence, in vivo high exposures of 17 are obtained, resulting in efficacy in the EAE model similar to that observed for DMF. 17 (R079) is Ames negative, is not cytotoxic to cells, and shows little inhibition of either the niacin receptor or a panel of off-target receptors.

Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG–mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis. Phosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)–ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. These findings indicate that Syk plays a critical role in MPO-ANCA IgG–induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.

Background NETosis is a key neutrophil response to tissue damage and pathogens. It is a form of programmed cell death resulting in chromatin decondensation, histone citrullination and controlled extracellular release of chromosomal DNA. This DNA complexed to a variety of noxious proteins such as elastase, myeloperoxidase and citrullinated histones is called neutrophil extracellular traps (NETs). NETs are largely responsible for anti-viral and anti-microbial effects of neutrophils during infection. NETs can cause significant tissue damage in various pathological conditions such as severe infections (COVID-19 and others) and multiple autoimmune and autoinflammatory diseases characterized by hyperactivation of innate and adaptive immune systems. Together with concurrent platelet and coagulation cascade activation, NETs formation is known to drive coagulopathy and thrombosis often associated with sterile inflammation in autoimmune and autoinflammatory diseases and in severe COVID-19 (Y. Zhou et al.). Strong correlation between NETosis biomarkers and thrombosis has been established in patients with many thrombogenic diseases (Mołek et al.; Martos et al.). Inhibition of NETs using DNAse treatment, protein arginine deiminase 4 (PAD4) inhibitors, or PAD4 knockout results in decrease in thrombi formation in vitro and in vivo (Franck et al.; Martinod et al.; Yan et al.). NETs release induced by plasma from severe COVID-19 patients in normal human neutrophils can be blocked in the presence of the highly selective SYK inhibitor R406, an active metabolite of the immune thrombocytopenia drug fostamatinib (Strich et al.). The inhibitor is thought to block antibody- and viral particle-mediated neutrophil activation through FcgRIIA and C-type lectin receptors (CLRs) controlled by SYK. Methods/Results To further elucidate the role of SYK kinase in NETosis, we have analyzed healthy human neutrophil responses to various stimuli in the presence of multiple commercially available SYK inhibitors using the Incucyte Live-Cell Analysis System. As expected, R406 potently inhibited immune complex induced NETosis mediated by FcgRIIA. To our surprise, every SYK inhibitor we've tested selectively blocked seemingly unrelated LPS-mediated TLR4-dependent NETs release. The effect was clearly specific as SYK inhibition had no effect on SYK-independent LPS-induced cytokine secretion or on cell proliferation at concentrations far exceeding those fully blocking NETosis. In contrast to SYK inhibitors, neither a selective JAK inhibitor nor dexamethasone had a significant effect on NETs release under the same assay conditions. Moreover, R406 potently inhibited NETs production in response to hydrogen peroxide and monosodium urate (MSU) crystal treatment of healthy neutrophils. The mechanisms behind the ability of SYK inhibitors to block NETs formation in response to such a plethora of unrelated stimuli will be further discussed. Conclusions The ability of SYK inhibitors to block both NETosis and platelet activation (in case of platelets, via CLEC2 and GPVI - two receptors involved in thrombosis yet dispensable for hemostasis) is consistent with possibility that, in addition to anti-inflammatory properties, SYK inhibition has potential anti-thrombotic effects (Harbi et al.). Indeed, the latter might be behind the lower than expected rates of thrombotic events in ITP patients observed in clinical trials of fostamatinib (Cooper et al.). References Yilu Zhou et al. Front Cardiovasc Med. 2021 Dec 2;8:786387. Laura Martos et al. Int J Mol Sci. 2020 Aug 6;21(16):5651. Patrycja Mołek et al. Thromb Res. 2022 May;213:1-7. Grégory Franck et al. Circ Res. 2018 June 22;123(1): 33-42. Kimberly Martinod et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8674-8679. Yanyan Yan et al. Aging (Albany NY). 2019 Sep 15;11(17):6951-6959. Jeffrey R Strich et al. J Infect Dis. 2021 Mar 15;223(6):981-984. Maan H. Harbi et al. Int J Mol Sci. 2022 Jul;23(13):6982. Nichola Cooper et al. Ther Adv Hematol. 2021 Apr 30;12:20406207211010875

Janus kinases (JAK) play a critical role in JAK/signal transducer and activator of transcription (STAT) signaling pathways that mediate immune response and cell growth. From high-throughput screening (HTS) hit to lead optimization, a series of pyrimidine compounds has been discovered as potent JAK1 inhibitors with selectivity over JAK2. Cell-based assays were used as primary screening methods for evaluating potency and selectivity, the results were further assessed and confirmed by biochemical and additional cellular assays for lead molecules. Also discussed is the unique correlation between a trifluomethyl group and CYP3A4 inhibition in the presence of NADPH, the activity of which was successfully decreased with the reduction of fluoro-atoms, increasing IC50 from 0.5 μM to >10 μM. The development of novel and scalable synthetic routes for amino-phenyl intermediates was essential for the discovery of late-stage lead molecules, including clinical candidate R507 (33). In preclinical studies, 33 exhibited great efficacy in mouse studies by inhibiting IFNγ expression induced by IL-2 and in a rat collagen-induced arthritis disease model.

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.

IRAK4 kinase plays a key role in TLR/IL-1R signaling pathways that regulate innate immune responses, and if uncontrolled, it is responsible for various inflammatory disorders. By high-throughput screening (HTS) and hit-to-lead optimization, compounds with a 5-aryl-2,4-diaminopyrimidine core structure have been identified as potent IRAK4 inhibitors. A cocrystal structure of IRAK4 protein with an early lead molecule helped with understanding the structure-activity relationship and the design of the new compounds. Initial HTS hits from this series of compounds were also found to inhibit TAK1 kinase, which would cause liver toxicity and potentially bone marrow failure. Optimization of this series resulted in improved selectivity over TAK1 kinase. The TAK1 selectivity was found to be closely associated with different sizes and types of substituents at the 5-position of the pyrimidine. The impact of other pyrimidine substituents on the potency and selectivity was also explored. A few representative compounds were evaluated in IL-1β-induced IL-6 inhibition animal model studies and showed modest efficacy.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in transduction of IL-1R/TLR signaling which is responsible for innate immune response. From HTS campaign, bicyclic-pyrimidine compounds have been identified as potent IRAK4 inhibitors, exhibiting good potency in both IRAK4 biochemical and LPS induced IL-23 inhibition cell-based assays. The SAR efforts were focused on further improving on-target potency, reducing PAD activities of HTS hit molecule and improving in vivo PK profiles of early lead compounds. When different aromatic rings were fused to the pyrimidine core, and with various substituents at 2- or 4-position of the pyrimidine, the impact on potency and PK properties were observed and are discussed. Selected compounds were further evaluated in IL-1β induced IL-6 inhibition acute animal model and rodent arthritis disease model, of which compounds 33 and 39 showed good efficacy in both studies.

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G 0 marker (G 0 M), we narrow down CML LSCs as G 0 M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

BACKGROUND:Patients with immune thrombocytopenia (ITP) are at risk of bleeding and, paradoxically, thromboembolic events (TEEs), irrespective of thrombocytopenia. The risk of thrombosis is increased by advanced age, obesity, and prothrombotic comorbidities: cancer, hyperlipidemia, diabetes, hypertension, coronary artery disease, and chronic kidney disease, among others. Certain ITP treatments further increase the risk of TEE, especially splenectomy and thrombopoietin receptor agonists. Spleen tyrosine kinase (SYK) is a key signaling molecule common to thromboembolic and hemostatic (in addition to inflammatory) pathways. Fostamatinib is an orally administered SYK inhibitor approved in the USA and Europe for treatment of chronic ITP in adults. METHODS:The phase III and extension studies included heavily pretreated patients with long-standing ITP, many of whom had risk factors for thrombosis prior to initiating fostamatinib. This report describes long-term safety and efficacy of fostamatinib in 146 patients with up to 5 years of treatment, a total of 229 patient-years, and assesses the incidence of thromboembolic events (by standardized MedDRA query). RESULTS:Platelet counts ⩾50,000/µL were achieved in 54% of patients and the safety profile was as described in the phase III clinical studies with no new toxicities observed over the 5 years of follow-up. The only TEE occurred in one patient (0.7%, or 0.44/100 patient-years), who experienced a mild transient ischemic attack. This is a much lower rate than might be expected in ITP patients. CONCLUSION:This report demonstrates durable efficacy and a very low incidence of TEE in patients receiving long-term treatment of ITP with the SYK inhibitor fostamatinib. CLINICALTRIALSGOV IDENTIFIERS:NCT02076399, NCT02076412, and NCT02077192.

Fostamatinib is a potent SYK inhibitor that targets activation of both the innate and acquired immune systems caused by tissue damage or pathogen signals via C-type lectin receptors (CLR) as well as by antibody-antigen immune complexes via Fc receptors (Figure 1). SYK is also a key modulator of pathways involved in thrombosis formation. Fostamatinib is FDA-approved for chronic immune thrombocytopenia (ITP) and has been evaluated in over 4000 patients for treatment of allergic, autoimmune, and neoplastic disorders. Fostamatinib is well tolerated, with a consistent safety profile across a number of diseases. In rheumatoid arthritis patients, fostamatinib reduced the plasma levels of IL-6.1 The active metabolite (R406) was protective in mouse models of LPS-induced acute lung injury (ALI)2 and LPS- or antibody-induced acute kidney injury (AKI).3 SYK inhibition decreased the incidence of thrombosis in mouse models of thromboembolism,4 and the incidence of thromboembolic events was decreased in patients treated with fostamatinib compared with other treatments for ITP. These immunomodulatory effects suggest that SYK inhibition represents a new therapeutic strategy for the treatment of COVID-19, which led to design of a clinical study of fostamatinib in hospitalized COVID-19 patients. COVID-19 patients are at risk for a potentially fatal acute respiratory distress syndrome (ARDS), cytokine storm, severe systemic capillary leak syndrome, thromboembolic events, and multi-organ dysfunction/failure. SYK is a master regulator of signal transduction pathways implicated in these COVID-19 associated complications, which involves hyperactivation of both innate and acquired immune systems. Targeting SYK could prevent the cytokine storm, downstream activation of endothelial cells and platelets, and influx of neutrophils and monocytes into lungs or kidney leading to ALI and AKI, respectively. Methods: A randomized, double-blind, placebo-controlled, multi-center, Phase 3 study will evaluate the efficacy and safety of fostamatinib in adult, hospitalized, high-risk COVID-19 patients. Patients will be treated with fostamatinib 150mg BID for up to 28 days plus standard of care. Improvement in clinical status will be assessed using the 8-point ordinal scale. References 1. Weinblatt ME et al.Arth Rheum2008;58:3309-18 2. Nadeem A et al.Int Immunopharm2019;68:39-47 3. Al-Harbi NO, et al.Biochimie2019;158: 102-10 4. Van Eeuwijk JMM et al.Arterioscler Thromb Vasc Biol2016;36:1247-53 Results and Conclusions: Trial in Progress Disclosures Dummer: Rigel:Current Employment, Current equity holder in publicly-traded company.Markovtsov:Rigel:Current Employment, Current equity holder in publicly-traded company.Tong:Rigel:Current Employment, Current equity holder in publicly-traded company.Masuda:Rigel:Current Employment, Current equity holder in publicly-traded company.