Combinatorial inhibition of Tec kinases BTK and ITK is beneficial in ameliorating murine sclerodermatous chronic graft versus host disease.

Graft-versus-host disease (GVHD) is the major factor limiting the widespread use of potentially curative allogeneic hematopoietic stem cell transplant (allo-HSCT). Chronic GVHD is characterized by the activation of alloreactive donor immune cells, especially B- and T-cells, leading to tissue damage and pathogenic fibrosis. In this study, we used highly specific next-generation inhibitors of ITK (PCYC-274), BTK (PCYC-804), and ibrutinib-like BTK/ITK inhibitors (PCYC-914 and PCYC-401) in the B10.D2 → BALB/C model of murine sclerodermatous cGVHD. From the third week onward, allogeneic recipients in each group of respective Tec kinase inhibitors were treated three times weekly with inhibitors at doses of 10 and 30 mg/kg or with saline control via oral gavage. Overall, we found that selective BTK inhibition was less effective than combined ITK/BTK or ITK inhibition in lengthening survival and reducing symptoms of cGVHD. ITK inhibition was most efficacious, with PCYC-274 and PCYC-401 demonstrating a nearly 50 percent reduction in GVHD scoring even at the 10 mg/kg dose, while 30 mg/kg of these compounds almost completely ameliorated GVHD symptomology. BTK/ITK and ITK-treated mice showed significant reductions in overall pathology. Significant reductions in dermal thickness and fibrosis were shown for all treatment groups. There was evidence of mixed Th1 and Th2 cytokine profiles in the skin of mice with dermal cGVHD, as both IFN-gamma and IL-4 were upregulated in the allogeneic control group, while kinase inhibition significantly reduced levels of these cytokines. Using an in vitro model of T-cell polarization, Th1 cell production of TNF-alpha and IFN-gamma were partially blocked by ITK. Th2 cell production of IL-4 was almost completely blocked synergistically by ITK and BTK inhibition. BTK-specific inhibition was unable to block either Th1 or Th2 cytokine production. Taken together, these results confirm previous reports that ITK-focused inhibition inhibits Th1 and Th2 cells. Additionally, the compound's effects on T-cell proliferation were tested by CFSE assay. Pure ITK inhibition was most effective at blocking T-cell proliferation, with no proliferation in PCYC-274-treated cells even at 0.1uM. PCYC-401 and PCYC-914 showed some inhibition at lower doses, with complete inhibition evident at 10uM. PCYC-804 was only partially able to block proliferation even at 10uM. In conclusion, we observed substantial benefit for differential inhibition of Tec kinases in GVHD, with ITK being most efficacious and Th1 cells being more resistant to inhibition, matching the previously reported findings of a Th2 to Th1 selective pressure in cells treated with ibrutinib. Our data warrants the further development of ITK and ITK/BTK inhibitors with specific inhibitory ratios to improve the treatment of GVHD and other T-cell mediated diseases.

Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Open trial of Bruton's tyrosine kinase inhibitor (PRN1008) in the treatment of canine pemphigus foliaceus.

BACKGROUND: Bruton's tyrosine kinase (BTK) is important in B-cell signalling. Efficacy has been reported for BTK inhibitors (BTKi) in human autoimmune diseases. Canine pemphigus foliaceus (cPF) is one of the most common canine autoimmune skin diseases. OBJECTIVES: To determine the safety and efficacy of the BTKi PRN1008 in the treatment of cPF. ANIMALS: Four privately owned dogs. METHODS AND MATERIALS: Four dogs diagnosed with PF were administered BTKi PRN1008. Initial dosages approximated to 15 mg/kg once daily, increased to twice daily if inadequate response was seen. Treatment continued for 20 weeks, attempting to decrease to every other day. Dogs were monitored with complete blood counts, serum biochemistry panels and urinalyses, and evaluated with a modified version of a validated human Pemphigus Disease Activity Index (cPDAI). Serum anti-desmocollin-1 (DSC-1) and desmoglein-1 (DSG-1) immunoglobulin (Ig)G titres were performed before and after the treatment period. Drug bound to target was measured in peripheral blood mononuclear cells (PBMC). RESULTS: All four dogs showed reduction in lesions and cPDAI score during the first two weeks of treatment. Three dogs continued to improve and sustained near complete remission by 20 weeks, at which point three responses were considered "good" and one "fair". Final daily dosages were in the range 17-33 mg/kg. Anti-DSC-1 IgG titre decreased dramatically in one dog, was undetectable in two and was uninterpretable in one dog. No dogs had detectable IgG to DSG1. A possible adverse event occurred in one dog. CONCLUSIONS AND CLINICAL IMPORTANCE: BTKi PRN1008 monotherapy may have some beneficial effects in some cases of cPF.

Efficacy of a Bruton's Tyrosine Kinase Inhibitor (PRN-473) in the treatment of canine pemphigus foliaceus.

BACKGROUND: Bruton's tyrosine kinase (BTK) is important in B-cell signalling. Efficacy has been reported for BTK inhibitors (BTKi) in human autoimmune diseases. Canine pemphigus foliaceus (cPF) is the most common canine autoimmune skin disease. OBJECTIVES: To determine the safety and efficacy of a BTKi in cPF treatment. ANIMALS: Nine privately owned dogs. METHODS AND MATERIALS: Nine dogs diagnosed with PF were administered BTKi PRN473. Initial dosages were ≈15 mg/kg once daily, increased to twice daily if inadequate response was seen. Treatment continued for a maximum of 20 weeks, attempting decrease to every other day. Dogs were monitored with complete blood counts, serum biochemistry panels, urinalyses and evaluated with a modified version of a validated human Pemphigus Disease Activity Index (cPDAI). Anti-desmocollin-1 (DSC-1) and desmoglein-1 (DSG-1) immunoglobulin G (IgG) titres were performed before and after the treatment period. Drug bound to target was measured in peripheral blood mononuclear cells. RESULTS: All nine dogs showed reduction in lesions and cPDAI score during the first two weeks of treatment. At the end of the study, four responses were considered "good", two "fair", two "poor" and one dog withdrawn due to recurrence of a previously excised mast cell tumour. Four dogs continued to improve by Week 4; three sustained near complete remission by study's end. The anti-DSC-1 IgG titre decreased in three dogs, increased in two, was undetected in three and was not performed in the withdrawn dog. No dogs had detectable IgG to DSG1. Possible adverse effects occurred in three dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Bruton's tyrosine kinase inhibitor monotherapy may have beneficial effects in some cases of cPF.

Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib.

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.

PRN473, an inhibitor of Bruton's tyrosine kinase, inhibits neutrophil recruitment via inhibition of macrophage antigen-1 signalling.

BACKGROUND AND PURPOSE: Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways. EXPERIMENTAL APPROACH: In vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions. KEY RESULTS: Pharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: PRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the ß2 -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.

The Irreversible Covalent Fibroblast Growth Factor Receptor Inhibitor PRN1371 Exhibits Sustained Inhibition of FGFR after Drug Clearance.

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668-76. ©2017 AACR.

A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.

Genetic Evidence for Function of the bHLH-PAS Protein Gce/Met As a Juvenile Hormone Receptor.

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. Here, we show that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology.

Prolonged and tunable residence time using reversible covalent kinase inhibitors.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694.

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Structure-based drug design of RN486, a potent and selective Bruton's tyrosine kinase (BTK) inhibitor, for the treatment of rheumatoid arthritis.

Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution, each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1 (RN486) ( J. Pharmacol. Exp. Ther. 2012 , 341 , 90 ), which was selected for advanced preclinical characterization based on its favorable properties.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Šresolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

B-cell antigen receptor signaling in chronic lymphocytic leukemia: therapeutic targets and translational opportunities.

B-cell chronic lymphocytic leukemia (CLL) is characterized by clonally expanded and molecularly heterogeneous populations of B lymphocytes with impaired apoptotic mechanisms. This occurs as a result of multiple genetic and epigenetic abnormalities, including chromosomal aberrations and enhancer region hypomethylation, often impinging on intracellular signaling pathways that are essential to normal B-cell activation, proliferation, and survival. The B-cell antigen receptor (BCR) signaling is one such pathway usurped by malignant B cells, as exemplified by the early phase clinical success achieved by small-molecule agents targeting key players involved in the pathway. Such new targeted agents, including those that inhibit the function of Spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), phosphatidylinositol 3-kinases (PI3K), and B-cell lymphoma 2 (BCL-2), along with the current standard therapy comprising chemo-immunotherapies with or without B-cell depleting biologic agent rituximab (anti-CD20 monoclonal antibody), should expand the armamentarium for CLL therapy. We review the therapeutic agents currently in clinical development which target different effectors of the malignant BCR signaling, and discuss their overlapping and discriminating translational opportunities in the context of CLL treatment.

Ecdysone receptors: from the Ashburner model to structural biology.

In 1974, Ashburner and colleagues postulated a model to explain the control of the puffing sequence on Drosophila polytene chromosomes initiated by the molting hormone 20-hydroxyecdysone. This model inspired a generation of molecular biologists to clone and characterize elements of the model, thereby providing insights into the control of gene networks by steroids, diatomic gases, and other small molecules. It led to the first cloning of the EcR subunit of the heterodimeric EcR-USP ecdysone receptor. X-ray diffraction studies of the ligand-binding domain of the receptor are elucidating the specificity of receptor-ecdysteroid interactions, the selectivity of some environmentally friendly insecticides, the evolution of the EcR-USP heterodimer, and indeed Ashburner's classical biochemical evidence for the central role of the ecdysone receptor in his model.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC(50) = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC(50) = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Isoforms of the heteropteran Nezara viridula ecdysone receptor: protein characterisation, RH5992 insecticide binding and homology modelling.

BACKGROUND: Certain bisacylhydrazine compounds such as tebufenozide (RH5992) have been shown to act as order-specific insecticides. Their compatibility with predatory Heteroptera, which are used as biological control agents, has also been demonstrated. However, the molecular mode of action of these ecdysone agonists has not been explored in a heteropteran, much less one that is a significant agricultural pest, such as Nezara viridula. RESULTS: Alternatively spliced ligand-binding regions of the N. viridula ecdysone receptor were expressed, purified and characterised by 2D gel analysis, mass spectrometry, homology modelling and competitive binding of a bisacylhydrazine insecticidal compound (RH5992) and various ecdysteroids. Ligand binding by the two splice isoforms was indistinguishable, and relative affinities were found to occur in the order muristerone A > ponasterone A > 20-hydroxyecdysone > inokosterone > RH5992 > α-ecdysone. CONCLUSION: The predicted difference in amino acid sequence between the ligand-binding domains of the N. viridula ecdysone receptor splice variants was verified by mass spectrometry. Both splice variant isoforms exhibit a greater affinity for the bisacylhydrazine insecticide RH5992 than do the other hemipteran ecdysone receptors characterised to date. Their affinities for a range of ecdysteroids also distinguish them from the ecdysone receptors of other Hemiptera characterised thus far. Homology models of both N. viridula receptor isoforms provide further insight into the bisacylhydrazine- and ecdysteroid-binding properties of these receptors, including their similar affinity for 20-hydroxyecdysone and the postulated pentatomomorphan moulting hormone makisterone A.

3-Amino-pyrazolo[3,4-d]pyrimidines as p38α kinase inhibitors: design and development to a highly selective lead.

Learnings from previous Roche p38-selective inhibitors were applied to a new fragment hit, which was optimized to a potent, exquisitely selective preclinical lead with a good pharmacokinetic profile.