Current Understanding of Immune Thrombocytopenia: A Review of Pathogenesis and Treatment Options.

The management of immune thrombocytopenia (ITP) and the prediction of patient response to therapy still represent a significant and constant challenge in hematology. ITP is a heterogeneous disease with an unpredictable evolution. Although the pathogenesis of ITP is currently better known and its etiology has been extensively studied, up to 75% of adult patients with ITP may develop chronicity, which represents a significant burden on patients' quality of life. A major risk of ITP is bleeding, but knowledge on the exact relationship between the degree of thrombocytopenia and bleeding symptoms, especially at a lower platelet count, is lacking. The actual management of ITP is based on immune suppression (corticosteroids and intravenous immunoglobulins), or the use of thrombopoietin receptor agonists (TPO-RAs), rituximab, or spleen tyrosine kinase (Syk) inhibitors. A better understanding of the underlying pathology has facilitated the development of a number of new targeted therapies (Bruton's tyrosine kinase inhibitors, neonatal Fc receptors, strategies targeting B and plasma cells, strategies targeting T cells, complement inhibitors, and newer TPO-RAs for improving megakaryopoiesis), which seem to be highly effective and well tolerated and result in a significant improvement in patients' quality of life. The disadvantage is that there is a lack of knowledge of the predictive factors of response to treatments, which would help in the development of an optimized treatment algorithm for selected patients.

Synthesis of BI 894416 and BI 1342561, two potent and selective spleen tyrosine kinase inhibitors, labeled with carbon 14 and with deuterium.

(R)-4-((R)-1-((6-(1-[tert-butyl]-1H-pyrazol-4-yl)-2-methyl-2H-pyrazolo[3,4-d]pyridin-4-yl)oxy)ethyl)pyrrolidin-2-one (BI 894416, 1) and (R)-4-((R)-1-((6-(1-[tert-butyl]-1H-pyrazol-4-yl)-2,3-dimethyl-2H-indazol-4-yl)oxy)ethyl)pyrrolidin-2-one (BI 1342561, 2) are two new potent and selective spleen tyrosine kinase inhibitors developed to treat severe asthma. Both compounds have similar structures and they differ only in the bicyclic moiety 2-methyl-2H-pyrazolo[4,3-c]pyridine in 1 versus 2,3-dimethyl-2H-indazole in 2. In the carbon 14 synthesis, 1-(1-[tert-butyl]-1H-pyrazol-4-yl)ethan-1-one-1-14 C ([14 C]-8) was prepared from the cyanation of 4-bromopyrazole using zinc [14 C]cyanide followed by methyl lithium addition on the nitrile group. The enolate of [14 C]-8 was then used to access these two bicyclic moieties via pyrano-pyrazoles [14 C]-11 and [14 C]-12, which were further transformed in few more steps to [14 C]-(1) and [14 C]-2. Both inhibitors contain a tert-butyl group. Introducing tert-butyl-d9 will not only provide internal standards for bioanalytical studies, but it is also expected to slow down the metabolism of these two compounds. Most of the metabolites of compound 1, for example, are the result of tert-butyl oxidation, like alcohol 3, acid 4, and the further N-demethylation of 4 to 5. The detailed preparation of these deuterium-labeled metabolites is also described.

Discovery of a Natural Syk Inhibitor from Chinese Medicine through a Docking-Based Virtual Screening and Biological Assay Study.

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 ß-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 µM) and exhibited anti-mast cell degranulation activity in vitro (IC50 = 2.76 µM). 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.

The kinase inhibitors R406 and GS-9973 impair T cell functions and macrophage-mediated anti-tumor activity of rituximab in chronic lymphocytic leukemia patients.

Small molecules targeting kinases involved in B cell receptor signaling are showing encouraging clinical activity in chronic lymphocytic leukemia (CLL) patients. Fostamatinib (R406) and entospletinib (GS-9973) are ATP-competitive inhibitors designed to target spleen tyrosine kinase (Syk) that have shown clinical activity with acceptable toxicity in trials with CLL patients. Preclinical studies with these inhibitors in CLL have focused on their effect in patient-derived leukemic B cells. In this work we show that clinically relevant doses of R406 and GS-9973 impaired the activation and proliferation of T cells from CLL patients. This effect could not be ascribed to Syk-inhibition given that we show that T cells from CLL patients do not express Syk protein. Interestingly, ζ-chain-associated protein kinase (ZAP)-70 phosphorylation was diminished by both inhibitors upon TCR stimulation on T cells. In addition, we found that both agents reduced macrophage-mediated phagocytosis of rituximab-coated CLL cells. Overall, these results suggest that in CLL patients treated with R406 or GS-9973 T cell functions, as well as macrophage-mediated anti-tumor activity of rituximab, might be impaired. The potential consequences for CLL-treated patients are discussed.

Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors.

A series of pyrazolylpyrimidine scaffold based Syk inhibitors were synthesized and evaluated for their biological activities and selectivity. Lead optimization efforts provided compounds with potent Syk inhibition in both enzymatic and TNF-α release assay.

Discovery of benzylidene derivatives as potent Syk inhibitors: synthesis, SAR analysis, and biological evaluation.

Four scaffolds of varied benzylidene derivatives were synthesized and evaluated as Syk inhibitors for the treatment of rheumatoid arthritis (RA). Among these 31 compounds, 3-benzylidene pyrrolidine-2,5-dione derivatives (including 12k) universally showed good Syk inhibitory activities in the low micromolar to submicromolar range. In the cellular profiling, compound 12k, the most efficient compound, showed excellent antiproliferative activity against fibroblast-like synoviocytes (FLS)-RA, and demonstrated potencies for suppression of IL-6 and MMP-3 secretion almost equal to R406 (positive control). The oral efficacy of 12k in the murine collagen-induced arthritis model was significant, despite being weaker than R406. Taken together, all preliminary pharmacological results supported 12k as a potential small-molecule inhibitor targeting Syk for the treatment of RA.

Investigational spleen tyrosine kinase (SYK) inhibitors for the treatment of autoimmune diseases.

INTRODUCTION: Autoimmune diseases (ADs) are disorders induced by multiple inflammatory mediators, in which immune system attacks healthy tissues and triggers tissue injury. Targeted regulation of the activity of kinases that influence inflammation is one of the major therapies for ADs. Recently, investigational spleen tyrosine kinase (SYK) inhibitors have shown encouraging results in the AD therapy. AREAS COVERED: This article provides a background on autoimmune diseases and provides an update on investigational SYK inhibitors. This literature review was conducted by searching publications about investigational SYK inhibitors in the treatment of ADs from experimental to clinical studies. The search terms used were SYK inhibitors, R406, fostamatinib (R788), P505-15 (PRT062607), entospletinib (GS-9973), R112, lanraplenib (GS-9876), cerdulatinib, R343, BAY-61-3606, GSK compound 143 (GSK143), R211, SKI-G-618, SKI-O-85, ER-27319, YM193306, RO9021 in conjunction with autoimmune disease using electronic databases including PubMed, EMBASE, MEDLINE and Google Scholar. EXPERT OPINION: SYK inhibitors are promising drugs with unique advantages and acceptable tolerability and safety for the treatment of ADs. However, the difficulties in developing highly selective SYK inhibitors and the unknown effects are challenges. Long-term and real-world data are essential to determine the risk-benefit ratio and true role of SYK inhibitors in the therapy of ADs.

Splenic tyrosine kinase (SYK) inhibitors and their possible use in acute myeloid leukemia.

INTRODUCTION: Splenic tyrosine kinase (SYK) is a non-receptor tyrosine kinase. It is important for downstream signaling from several cell surface receptors, including Fc receptors, complement receptors and integrins. SYK can have either oncogenic or tumor suppressor activity in human malignancies. Recent studies suggest that SYK inhibition may have an antileukemic effect in human acute myeloid leukemia (AML). AREAS COVERED: Relevant publications were identified through literature searches in the PubMed database. We searched for (i) original articles describing the results from clinical studies of SYK inhibition; (ii) published articles describing the importance of SYK in human malignancies, especially AML. EXPERT OPINION: SYK is important for the downstream signaling from several cell surface receptors. There is also a crosstalk between SYK and signaling initiated through ligation of Toll like receptors, and SYK is thereby linked with the NFκB mediated transcriptional regulation. SYK activation will also influence PI3K-Akt-mTOR signaling. Several of these signaling events are important for survival and proliferation of primary human AML cells. In the present review we describe and discuss the role of SYK in human AML, and these data suggest that SYK inhibition is a possible therapeutic strategy in human AML.

T cell tyrosine phosphorylation response to transient redox stress.

Reactive Oxygen Species (ROS) are crucial to multiple biological processes involved in the pathophysiology of inflammation, and are also involved in redox signaling responses. Although previous reports have described an association between oxidative events and the modulation of innate immunity, a role for redox signaling in T cell mediated adaptive immunity has not been described yet. This work aims at assessing if T cells can sense redox stress through protein sulfhydryl oxidation and respond with tyrosine phosphorylation changes. Our data show that Jurkat T cells respond to -SH group oxidation with specific tyrosine phosphorylation events. The release of T cell cytokines TNF, IFNγ and IL2 as well as the expression of a number of receptors are affected by those changes. Additionally, experiments with spleen tyrosine kinase (Syk) inhibitors showed a major involvement of Syk in these responses. The experiments described herein show a link between cysteine oxidation and tyrosine phosphorylation changes in T cells, as well as a novel mechanism by which Syk inhibitors exert their anti-inflammatory activity through the inhibition of a response initiated by ROS.

Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM. METHODS: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235). RESULTS: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs. CONCLUSIONS: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.

Role of spleen tyrosine kinase in the pathogenesis of chronic lymphocytic leukemia.

The antigen-dependent B-cell receptor (BCR) is triggered by binding to external antigens and transmits signals in normal B lymphocytes. Tonic signaling through the BCR plays a crucial role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). Spleen tyrosine kinase (Syk) is a key component of both BCR signals, and regulates multiple physiological functions of B lymphocytes. Studies have defined enhanced gene expression and protein expression of Syk in CLL cells which are closely related to the status of the immunoglobulin heavy chain variable region genes (IgVH). Recently, abrogating the BCR-induced signaling pathway by Syk inhibitors has represented a novel and active therapeutic approach for CLL. Studies of the correlation between Syk and ZAP-70 expression in CLL cells have brought a new perspective to determining the value of Syk in evaluating the effect of therapy and the prognosis of CLL. Therefore, we here review the role of Syk in the pathogenesis of CLL and provide an update of progress in the clinical development of Syk inhibitors.