Selective Btk inhibition by PRN1008/PRN473 blocks human CLEC-2 & PRN473 reduces venous thrombosis formation in mice.

Platelet CLEC-2 is a hemITAM-containing receptor which has a critical role in venous thrombosis, but minimal involvement in haemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk however do not bleed, suggesting selective Btk inhibition is a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signalling and function mediated by CLEC-2 and GPVI. We used healthy donor and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on GPCR-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin positive vessels following Salmonella infection and the presence of IVC thrombosis following vein stenosis. The potent inhibition of human platelet CLEC-2, and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib treated immune thrombocytopenia patients, suggest Btk inhibition as a promising antithrombotic strategy.

The interleukin-4/interleukin-13 pathway in type 2 inflammation in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is predominantly a type 2 inflammatory disease associated with type 2 (T2) cell responses and epithelial barrier, mucociliary, and olfactory dysfunction. The inflammatory cytokines interleukin (IL)-4, IL-13, and IL-5 are key mediators driving and perpetuating type 2 inflammation. The inflammatory responses driven by these cytokines include the recruitment and activation of eosinophils, basophils, mast cells, goblet cells, M2 macrophages, and B cells. The activation of these immune cells results in a range of pathologic effects including immunoglobulin E production, an increase in the number of smooth muscle cells within the nasal mucosa and a reduction in their contractility, increased deposition of fibrinogen, mucus hyperproduction, and local edema. The cytokine-driven structural changes include nasal polyp formation and nasal epithelial tissue remodeling, which perpetuate barrier dysfunction. Type 2 inflammation may also alter the availability or function of olfactory sensory neurons contributing to loss of sense of smell. Targeting these key cytokine pathways has emerged as an effective approach for the treatment of type 2 inflammatory airway diseases, and a number of biologic agents are now available or in development for CRSwNP. In this review, we provide an overview of the inflammatory pathways involved in CRSwNP and describe how targeting key drivers of type 2 inflammation is an effective therapeutic option for patients.

Defining disease severity in atopic dermatitis and psoriasis for the application to biomarker research: an interdisciplinary perspective.

More severe atopic dermatitis and psoriasis are associated with a higher cumulative impact on quality of life, multimorbidity and healthcare costs. Proactive, early intervention in those most at risk of severe disease may reduce this cumulative burden and modify the disease trajectory to limit progression. The lack of reliable biomarkers for this at-risk group represents a barrier to such a paradigm shift in practice. To expedite discovery and validation, the BIOMarkers in Atopic Dermatitis and Psoriasis (BIOMAP) consortium (a large-scale European, interdisciplinary research initiative) has curated clinical and molecular data across diverse study designs and sources including cross-sectional and cohort studies (small-scale studies through to large multicentre registries), clinical trials, electronic health records and large-scale population-based biobanks. We map all dataset disease severity instruments and measures to three key domains (symptoms, inflammatory activity and disease course), and describe important codependencies and relationships across variables and domains. We prioritize definitions for more severe disease with reference to international consensus, reference standards and/or expert opinion. Key factors to consider when analysing datasets across these diverse study types include explicit early consideration of biomarker purpose and clinical context, candidate biomarkers associated with disease severity at a particular point in time and over time and how they are related, taking the stage of biomarker development into account when selecting disease severity measures for analyses, and validating biomarker associations with disease severity outcomes using both physician- and patient-reported measures and across domains. The outputs from this exercise will ensure coherence and focus across the BIOMAP consortium so that mechanistic insights and biomarkers are clinically relevant, patient-centric and more generalizable to current and future research efforts.

Atopic dermatitis (AD), and psoriasis are long-term skin conditions that can significantly affect people's lives, especially when symptoms are severe. Approximately 10% of adults and 20% of children are affected by AD, while psoriasis affects around 5% of people in the UK. Both conditions are associated with debilitating physical symptoms (such as itch) and have been linked to depression and anxiety. Biomarkers are naturally occurring chemicals in the human body and have potential to enhance the longer-term management of AD and psoriasis. Currently, there are no routinely used biomarkers that can identify people who experience or will go on to develop severe AD and psoriasis. For this reason, research is under way to understand which biomarkers are linked to severity. In this study, a multidisciplinary team of skin researchers from across Europe, along with patient groups, discussed the complexities of studying severity-related biomarkers. We identified a number of severity measurement approaches and there were recommendations for future biomarker research, including (i) considering multiple measures as no single measure can encompass all aspects of severity, (ii) exploring severity measures recorded by both healthcare professionals and patients, as each may capture different aspects, and (iii) accounting for influencing factors, such as different treatment approaches, that may impact AD and psoriasis severity, which make it challenging to compare findings across studies. Overall, we anticipate that the insights gained from these discussions will increase the likelihood of biomarkers being effectively applied in real-world settings, to ultimately improve outcomes for people with AD and psoriasis.

A cancer personalised activity and lifestyle tool (CAN-PAL): A codesign study with patients and healthcare professionals.

AIMS: To codesign a cancer personalised activity and lifestyle tool (CAN-PAL) based on an existing tool. To help cancer care workers support people affected by cancer to plan and integrate physical activity into lifestyles. DESIGN: Mixed-methods codesign study. METHODS: Phase 1: Focus groups with people affected by cancer (n = 10) or interviews (n = 2) to discuss suitable physical activities and adaptation of the existing tool. Data were recorded, transcribed and analysed thematically. Themes informed the design of the prototype CAN-PAL and user guide. Phase 2: Healthcare professionals considered the potential use of the CAN-PAL prototype and completed an online survey including the system usability scale and free text responses. RESULTS: Phase 1: Identified suitable physical activities and four themes were identified including: Capability, benefits, barriers and resources which informed the prototype CAN-PAL and user guide. Phase 2: The user survey was completed by 12 healthcare professionals. Median (range) system usability scale was 80 (50-95) (best score 100), scores >68 indicate good or better usability. Themes from the free text comments included strengths, amendments, considerations and limitations. Results were used to finalise CAN-PAL and the user guide. CONCLUSION: The codesigned CAN-PAL tool had good usability. Further work is needed to evaluate the impact of CAN-PAL on activity levels and behaviour in people affected by cancer. RELEVANCE TO CLINICAL PRACTICE: People affected by cancer need support to undertake physical activity. The purpose of CAN-PAL is to assist cancer care workers to support people affected by cancer to plan and integrate physical activity into lifestyles. PATIENT OR PUBLIC CONTRIBUTION: Public partners considered the findings from Phase 1 and 2 and informed the design of the prototype, final CAN-PAL and user guide and coauthored the paper. REPORTING METHOD: The study adhered to relevant EQUATOR guidelines; the study was reported according to the COREQ checklist.

Cellular mechanisms and effects of IL-4 receptor blockade in experimental conjunctivitis evoked by skin inflammation.

Ocular surface diseases, including conjunctivitis, are recognized as common comorbidities in atopic dermatitis (AD) and occur at an increased frequency in patients with AD treated with biologics targeting IL-4 receptor α (IL-4Rα) or IL-13. However, the inflammatory mechanisms underlying this pathology are unknown. Here, we developed a potentially novel mouse model of skin inflammation-evoked conjunctivitis and showed that it is dependent on CD4+ T cells and basophils. Blockade of IL-4Rα partially attenuated conjunctivitis development, downregulated basophil activation, and led to a reduction in expression of genes related to type 2 cytokine responses. Together, these data suggest that an IL-4Rα/basophil axis plays a role in the development of murine allergic conjunctivitis. Interestingly, we found a significant augmentation of a number of genes that encode tear proteins and enzymes in anti-IL-4Rα-treated mice, and it may underlie the partial efficacy in this model and may represent candidate mediators of the increased frequency of conjunctivitis following dupilumab in patients with AD.

cAMP-induced decrease in cell-surface laminin receptor and cellular prion protein attenuates amyloid-ß uptake and amyloid-ß-induced neuronal cell death.

Previous studies have shown that amyloid-ß oligomers (AßO) bind with high affinity to cellular prion protein (PrPC ). The AßO-PrPC complex binds to cell-surface co-receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen-1 cells, 67LR is the major co-receptor involved in the cellular uptake of AßO and AßΟ-induced cell death. Both pharmacological (dibutyryl-cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase-activating polypeptide) cAMP-elevating agents decreased cell-surface PrPC and 67LR, thereby attenuating the uptake of AßO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AßO-induced cytotoxicity by decreasing cell-surface-associated PrPC and 67LR.

The mitochondrial genomes of two Gryllus crickets (Grylloidea: Gryllidae) via RNA-seq.

Here, we used RNA-seq reads to assemble the complete mitochondrial genomes of the spring field cricket, Gryllus veletis, and the variable field cricket, Gryllus lineaticeps. The mitochondrial genomes of G. veletis (15,686 bp, MW322713) and G. lineaticeps (15,607 bp, MW315773) each contain the expected 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a large control (D-loop) region. The arrangements of these features were similar for both species and consistent with other closely related Orthoptera. A phylogenetic analysis of the mitochondrial genome sequences revealed that G. veletis and G. lineaticeps cluster with the other Gryllus species and all reside in a clade with the Gryllidae.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and ßS-globin. Kinetic studies with MetAP2 show that ßS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood-derived erythroid progenitor 2 cells shows more extensive modification of α-globin than ß-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on ßS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor-treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2-affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Arming oHSV with ULBP3 drives abscopal immunity in lymphocyte-depleted glioblastoma.

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSVULBP3 with anti-programmed cell death 1 (anti-PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti-PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti-PD-1-sensitive abscopal effect of oHSVULBP3 warrants further investigation in patients with IDH wild-type glioblastoma.

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system.

While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the 'immune fingerprint' of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis.

8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.

Discovery of novel BTK inhibitors with carboxylic acids.

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Discovery of 8-Amino-imidazo[1,5-a]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.

TLR4, TLR7/8 agonist-induced miR-146a promotes macrophage tolerance to MyD88-dependent TLR agonists.

TLRs facilitate the recognition of pathogens by immune cells and the initiation of the immune response, leading to the production of proinflammatory cytokines and chemokines. Production of proinflammatory mediators by innate immune cells, such as macrophages, is tightly regulated to facilitate pathogen clearance while limiting an adverse impact on host tissue. Exposure of innate immune cells to TLR ligands induces a state of temporary refractoriness to a subsequent exposure of a TLR ligand, a phenomenon referred to as "tolerance." This study sought to evaluate the mechanistic regulation of TLR4 and TLR7/8 ligand-induced tolerance to other TLRs by microRNA-146a. With the use of THP-1 macrophages, as well as human classic and alternative macrophages, we demonstrate that priming with a TLR4 agonist (LPS) or a TLR7/8 agonist (R848) induces homologous and heterologous tolerance to various TLR ligands in macrophages, leading to the impaired production of cytokines and chemokines. We also demonstrate that overexpression of microRNA-146a is sufficient to mimic LPS or R848-induced hyporesponsiveness. Conversely, inhibition of microRNA-146a activity leads to LPS- or R848-induced TLR hyper-responsiveness in TLR signaling tolerance. Furthermore, we demonstrate that microRNA-146a dampens cytokine production following a primary stimulus with MyD88-dependent but not MyD88-independent TLR pathways. Collectively, these data provide comprehensive evidence of the central role of microRNA-146a in TLR signaling tolerance to plasma membrane, as well as endosomal TLR ligands in human macrophages.

Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression.

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked ß-N-acetylglucosamine transferase (OGT), which adds an O-linked ß-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

Development and pharmacological validation of novel methods of B cell activation in rat whole blood.

INTRODUCTION: Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood. METHODS: B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay. RESULTS: Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable. DISCUSSION: Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.