Optimization of the efflux ratio and permeability of covalent irreversible BTK inhibitors

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.     

Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells

Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML.     

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC₅₀?=?139?nM; PI3Kδ: IC₅₀?=?275?nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Exploration of Novel Inhibitors for Bruton’s Tyrosine Kinase by 3D QSAR Modeling and Molecular Dynamics Simulation

Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase which is expressed in most of the hematopoietic cells and plays an important role in many cellular signaling pathways. B cell malignancies are dependent on BCR signaling, thus making BTK an efficient therapeutic target. Over the last few years, significant efforts have been made in order to develop BTK inhibitors to treat B-cell malignancies, and autoimmunity or allergy/hypersensitivity but limited success has been achieved. Here in this study, 3D QSAR pharmacophore models were generated for Btk based on known IC₅₀ values and experimental energy scores with extensive validations. The five features pharmacophore model, Hypo1, includes one hydrogen bond acceptor lipid, one hydrogen bond donor, and three hydrophobic features, which has the highest correlation coefficient (0.98), cost difference (112.87), and low RMS (1.68). It was further validated by the Fisher’s randomization method and test set. The well validated Hypo1 was used as a 3D query to search novel Btk inhibitors with different chemical scaffold using high throughput virtual screening technique. The screened compounds were further sorted by applying ADMET properties, Lipinski’s rule of five and molecular docking studies to refine the retrieved hits. Furthermore, molecular dynamic simulation was employed to study the stability of docked conformation and to investigate the binding interactions in detail. Several important hydrogen bonds with Btk were revealed, which includes the gatekeeper residues Glu475 and Met 477 at the hinge region. Overall, this study suggests that the proposed hits may be more effective inhibitors for cancer and autoimmune therapy.     

Optimization of novel reversible Bruton’s tyrosine kinase inhibitors identified using Tethering-fragment-based screens

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton’s tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.     

More than just B-cell inhibition

Despite tremendous advances in the therapy of rheumatoid arthritis (RA), there remains interest in oral agents that may offer benefits that are similar to, or better than, those of biologic therapies. In their paper, Chang and colleagues demonstrate the effectiveness of a Bruton tyrosine kinase (Btk) inhibitor in two models of RA. Btk inhibition impacts several pathways affecting both B-cell and macrophage activation, making it a promising target in RA. However, other kinase inhibitors have failed to transition from animal models to human therapy, so it remains to be seen whether a Btk inhibitor will have a role in the RA treatment armamentarium.     

Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk

Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) through the Btk pleckstrin homology (PH) domain, an interaction thought to be required for Btk membrane translocation during B cell receptor signaling. Here, we report that interaction of PtdIns-3,4,5-P(3) with the PH domain of Btk directly induces Btk enzymatic activation in an in vitro kinase assay. A point mutation that reduces interaction of PtdIns-3,4,5-P(3) with the Btk PH domain blocks in vitro PtdIns-3,4,5-P(3)-dependent Btk activation, whereas the PH domain deletion enhances Btk basal activity but eliminates the PtdIns-3,4,5-P(3)-dependent stimulation. Btk kinase activity and the Btk activation loop phosphorylation site are both required for the PtdIns-3,4,5-P(3)-mediated stimulation of Btk kinase activity. Together, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; when interacting with PtdIns-3,4,5-P(3), this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors and Btk catalytically inactive mutants, we demonstrate that in vivo, the activation of Btk is due to both Lyn phosphorylation and PtdIns-3,4,5-P(3)-mediated direct activation. Thus, the Btk-PtdIns-3,4,5-P(3) interaction serves to translocate Btk to the membrane and directly regulate its signaling function.     

Discovery of potent,highly selective covalent irreversible BTK inhibitors from a fragment hit

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.     

Bruton's tyrosine kinase associates with the actin-based cytoskeleton in activated platelets

Bruton's tyrosine kinase (Btk) plays a crucial role in the maturation and differentiation of B-lymphocytes and immunoglobulin synthesis. Recently Btk has been described to be present in significant amount in human platelets. To investigate the regulation of this kinase in the platelets we studied its subcellular redistribution in the resting and activated cells. In the resting platelets Btk was almost absent from the actin-based cytoskeleton. Upon challenge of the platelet thrombin receptor upto 30% of total Btk appeared in the cytoskeleton and the protein underwent phosphorylation on tyrosine. Translocation of Btk to the cytoskeleton but not aggregation was prevented by cytochalasin B, which inhibits actin polymerization. Wortmannin and genistein (inhibitors of phosphoinositide 3-kinase and protein tyrosine kinase, respectively) decreased while phenylarsine oxide (a tyrosine phosphatase inhibitor) increased the cytoskeletal content of Btk. The association of Btk with the cytoskeleton was regulated by integrin alpha(IIb)beta(3) and partly reversible. Taken together, these data suggest that Btk might be a component of a signaling complex containing specific cytoskeletal proteins in the activated platelets.     

BCR engagement induces Fas resistance in primary B cells in the absence of functional Bruton's tyrosine kinase

B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton's tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis. For these reasons, the role of Btk as a mediator of sIg-induced Fas resistance was examined. Dysfunction of Btk through mutation, and absence of Btk through deletion did not interfere with induction of Fas resistance by anti-Ig. This may be due, at least in part, to induction of Btk-dependent Bcl-2 family members by anti-Ig after CD40 ligand treatment. However, the susceptibility to Fas-mediated apoptosis of B cell targets stimulated by CD40 ligand alone was increased in the absence of Btk. These results indicate that Fas resistance produced by sIg triggering does not require Btk, but suggests that in certain situations Btk modulates B cell susceptibility to Fas killing.     

Lethal and sublethal effects of single and double applications of Bacillus thuringiensis variety kurstaki on spruce budworm (Lepidoptera: Tortricidae) larvae

We conducted laboratory experiments to examine the effects of single versus double exposures of spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae) female larvae to various concentrations of a Bacillus thuringiensis variety kurstaki (Btk) commercial formulation (Foray 48B). Our main objective was to document the vulnerability to Btk and the sublethal responses of fifth-instar larvae that survived from a first ingestion of Btk during their fourth stadium and to compare them with insects treated either during their fifth or fourth stadium only. As reported in the literature, fifth-instar larvae were more vulnerable than fourth-instar larvae, but only at low and medium concentrations. Fifth-instar larvae that had survived Btk ingestion during their fourth stadium were more vulnerable to a high concentration of Btk and had a shorter feeding inhibition period than those that had not been exposed during their fourth stadium. Compared with a single treatment at the fourth stadium, a double exposure to Btk further reduced the population by 20-30%, depending on the concentration applied. The second treatment also induced another feeding inhibition period and increased larval development time by 14%. The impact of the different treatments on pupal weight depended on whether treated insects exhibited supernumerary instars. In the absence of developmental polymorphism, a higher concentration, a late, or a double exposure to Btk significantly reduced pupal weight.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.     

A new molecular genetic diagnosis of the Btk(xid) mice.

A new method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of a point mutation of the Bruton's tyrosine kinase (Btk) gene in CBA/N mice bearing an X-linked recessive immunodeficiency (xid). Since restriction site useful for RFLP analysis does not exist in the spontaneous mutant Btk(xid) locus, an artificial restriction site was introduced by PCR amplification with a modified primer. The five genotypes of the Btk locus (Btk(xid)/ Btk(xid), Btk(xid) /Btk+ and Btk+/Btk+ females and Btk(xid)/Btk(null) and Btk+/Btk(null) males) could be distinguished by three patterns clearly and easily. This PCR-RFLP analytic method will be useful as a tool in the production of congenic mice and mice with multiple immunodeficient genes.     

A novel role for Bruton's tyrosine kinase in hepatocyte growth factor-mediated immunoregulation of dendritic cells

The limited success of dendritic cell (DC)-based immunotherapy in multiple myeloma is partly due to hepatocyte growth factor (HGF)-induced DC dysfunction. From a therapeutic standpoint, it is important to understand the molecular events involved in inhibition of DC activation/maturation by HGF. Because Bruton's tyrosine kinase (Btk) negatively regulates maturation and immunostimulatory function of DCs, a role for Btk in HGF-induced inhibition of both murine and human DCs was investigated. We demonstrate that Btk is a novel proximal component of HGF-induced c-MET (HGF receptor) signaling. Following HGF treatment, Btk binds to c-MET and becomes activated. Btk activation in turn blocks the NF-κB pathway and subsequent DC activation via the c-Src-PI3K-AKT-mammalian target of rapamycin (mTOR) pathway. Notably, Btk activation is necessary for HGF-induced association of c-Src and PI3K with c-MET. Furthermore, we provide the first evidence that HGF inhibits DC activation by inducing autocrine interleukin (IL)-10 secretion, which requires activation of Btk. Blocking activation of Btk and its downstream the c-Src-PI3K-AKT-mTOR pathway prevents HGF-induced IL-10 secretion by DCs. In addition, neutralization of IL-10 secretion from DCs impaired the inhibitory effect of HGF on DCs. Thus, our study identifies a novel role for Btk in HGF-induced DC inhibition.     

Bruton's Tyrosine Kinase is involved in innate and adaptive immunity

Btk is a cytoplasmic tyrosine kinase, which is mainly involved in B cell receptor signalling. Gene targeting experiments revealed that Btk is important for B cell development and function. However, Btk is not only expressed in B cells, but also in most other haematopoietic lineages except for T cells and plasma cells. Recently we found that Btk is involved in Toll-like receptor signalling. Toll-like receptors play an important role in innate immunity. They are highly expressed on mast cells, macrophages and dendritic cells, which are essential for the recognition and consequently for the elimination of microbial pathogens. Therefore Btk might play an important role for the function of immunocompetent cells of innate as well as adaptive immunity.