Inhibitory Potential of the Ocimum sanctum Phytochemicals on Bruton's Tyrosine Kinase, a Well-Known Drug Target for Treatment of Chronic Lymphocytic Leukemia: An In Silico Investigation.

Chronic lymphocytic leukemia (CLL) is an incurable neoplasm of B-lymphocytes, which accounts for about one-third of all leukemias. Ocimum sanctum, an herbaceous perennial, is considered as one of the important sources of drugs for the treatment of various diseases, including cancers and autoimmune diseases. The present study was designed to screen various phytochemicals of O. sanctum for discovering their potential to inhibit Bruton's tyrosine kinase (BTK), a well-known drug target of CLL. Various phytochemicals of O. sanctum were screened for their potential to inhibit BTK using several in silico protocols. First, the molecular docking approach was used to calculate the docking scores of the selected phytochemicals. Then, the selected top-ranked phytochemicals were screened for their physicochemical characteristics using ADME analysis. Finally, the stability of the selected compounds in their corresponding docking complexes with BTK was analysed using molecular dynamics simulations. Primarily, our observations revealed that, out of the 46 phytochemicals of O. sanctum, six compounds possessed significantly better docking scores (ranging from -9.2 kcal/mol to -10 kcal/mol). Their docking scores were comparable to those of the control inhibitors, acalabrutinib (-10.3 kcal/mol), and ibrutinib (-11.3 kcal/mol). However, after ADME analysis of these top-ranked six compounds, only three compounds (Molludistin, Rosmarinic acid, and Vitexin) possessed drug likeliness characteristics. During the MD analysis, the three compounds Molludistin, Rosmarinic acid, and Vitexin were found to remain stable in the binding pocket in their corresponding docking complexes with BTK. Therefore, among the 46 phytochemicals of O. sanctum tested in this study, the three compounds, Molludistin, Rosmarinic acid, and Vitexin are the best inhibitors of BTK. However, these findings need to be confirmed by biological experiments in the laboratory.

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors.

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (BTK) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro. The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.

Geniposide protects depression through BTK/JAK2/STAT1 signaling pathway in lipopolysaccharide-induced depressive mice.

The purpose of this study was to investigate the antidepressant mechanism of GEN (geniposide) on depression mice induced by LPS. The mice were intragastrically treated with GEN (10 mg/kg/d or 40 mg/kg/d) or ibrutinib for continuous 7 days prior to LPS injection. The anxiety- and depression-like behaviors of mice were assessed via behavioral tests (sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open-field test (OFT)). Microglial BV2 cells were treated with GEN or/and ibrutinib and stimulated with LPS. The productions of pro-inflammatory cytokines IL-6 and TNF-α in hippocampus, serum, and supernatant were detected by ELISA. The correlative proteins BTK, p-BTK, JAK2, p-JAK2, STAT1, p-STAT1, BDNF, TrkB, and p-TrkB were assessed through western blot. As a result, GEN ameliorated the anxiety- and depression-like behaviors of mice in behavioral tests. GEN treatment also regulated microglia polarization towards anti-inflammatory phenotype M2 and inhibited the production of pro-inflammatory cytokines IL-6 and TNF-α. In addition, with the application of ibrutinib, the selective inhibitor of BTK, it was proclaimed that the administration of GEN restrained the activation of JAK2/STAT1 pathway via attenuating the hyperphosphorylation of BTK both in mice and BV2 cells. Furthermore, it was also found that GEN activated BDNF/TrkB neuroprotective signaling pathway through the reduction of BTK phosphorylation. From the overall results, we suggested that GEN exerted a beneficial effect on LPS-induced depression in mice possibly through the modulation of BTK/JAK2/STAT1 signaling.

Clinical outcomes of newly diagnosed primary CNS lymphoma treated with ibrutinib-based combination therapy: A real-world experience of off-label ibrutinib use.

Ibrutinib-based combination therapy with high-dose methotrexate (HD-MTX) has recently shown clinical activity against relapse/refractory (R/R) primary central nervous system lymphoma (PCNSL). Herein, we report our real-world experience of treating 11 newly diagnosed PCNSL patients with the ibrutinib/MTX combination. HD-MTX was given at 3.5 g/m2 every 2-week for eight doses. Ibrutinib was held upon HD-MTX infusion until clearance and was administered daily post-induction until disease progression, intolerable toxicity, or death. Nine out of 11 patients completed the induction phase and received ibrutinib as maintenance therapy. An objective response rate (ORR) of 82% (9/11) was observed including complete response (64%) and partial response (18%). The median progression-free survival (PFS) was 7.4 months while the median overall survival (OS) was not reached. The ibrutinib/MTX combination was well tolerated in these treatment-naïve PCNSL patients with an acceptable safety profile. Moreover, the longitudinal analysis of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) revealed that CSF ctDNA detection was closely associated with tumor response, and sustained tumor responses correlated with the clearance of ctDNA from the CSF. In sum, our data not only demonstrated the clinical benefit of the ibrutinib and HD-MTX combination regimen in treating newly diagnosed PCNSL patients in a real-world setting, but also highlighted the significance of liquid biopsy including CSF ctDNA in tracing tumor burden and assessing treatment response.

Discovery of Tricyclic Pyranochromenone as Novel Bruton's Tyrosine Kinase Inhibitors with in Vivo Antirheumatic Activity.

Bruton's tyrosine kinase (BTK) is an attractive target for treating patients with B cell malignancies and autoimmune diseases. Many BTK inhibitors have been identified; however, like other kinase inhibitors, they lack diversity in their core structures. Therefore, it is important to secure a novel scaffold that occupies the adenine-binding site of BTK. We screened an in-house library of natural products and their analogs via a biochemical assay to identify a novel scaffold for targeting BTK. A pyranochromenone scaffold, derived from a natural active component decursin, was found to be effective at targeting BTK and was selected for further optimization. A series of pyranochromenone analogs was synthesized through the modification of pyranochromenone at the C7 position. Pyranochromenone compounds with an electrophilic warhead exhibited promising BTK inhibitory activity, with IC50 values in the range of 0.5-0.9 µM. A docking study of the representative compound 8 provided a reasonable explanation for compound activity. Compound 8 demonstrated good selectivity over other associated kinases and decreased the production of proinflammatory cytokines in THP cells. Moreover, compound 8 presented significant in vivo efficacy in a murine model of collagen-induced arthritis.

Targeted Therapy for Chronic Spontaneous Urticaria: Rationale and Recent Progress.

Chronic spontaneous urticaria (CSU) is characterized by the presence of wheals, angioedema, or both for at least 6 weeks. It may persist for a long time-up to 50% of the patients have been reported to be symptomatic 5 years after the onset. Some patients can suffer more than one episode of CSU during their lifetime. Considering the recurrences, disabling symptoms, and significant impact on quality of life, proper and effective treatment of CSU is critical. The use of antihistamines (AHs) is still the mainstay of treatment. However, given the low rates of response to AHs (38.6% and 63.2% to standard doses and higher doses, respectively), the complete control of symptoms seems difficult to attain. The use of omalizumab for CSU has been a major breakthrough in the care of patients with CSU. However, the partial response and lack of response to omalizumab in a subgroup of patients, as high as 70% in some studies, make the development of alternative treatments desirable. Ever-increasing knowledge on the pathogenesis is making new target molecules available and enabling drug development for CSU. In addition to drug repurposing as in anti-IL-4/13, IL-5, and IL-17 antibodies, novel targeted therapy options such as ligelizumab and Bruton's tyrosine kinase inhibitors are currently undergoing clinical trials and will be available in the near future. This article reviews the current challenges in the treatment of CSU, the pathogenesis and potential target molecules, and the rationale for novel treatments and their rapidly developing status.

Discovery of BIIB068: A Selective, Potent, Reversible Bruton's Tyrosine Kinase Inhibitor as an Orally Efficacious Agent for Autoimmune Diseases.

Autoreactive B cell-derived antibodies form immune complexes that likely play a pathogenic role in autoimmune diseases. In systemic lupus erythematosus (SLE), these antibodies bind Fc receptors on myeloid cells and induce proinflammatory cytokine production by monocytes and NETosis by neutrophils. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that signals downstream of Fc receptors and plays a transduction role in antibody expression following B cell activation. Given the roles of BTK in both the production and sensing of autoreactive antibodies, inhibitors of BTK kinase activity may provide therapeutic value to patients suffering from autoantibody-driven immune disorders. Starting from an in-house proprietary screening hit followed by structure-based rational design, we have identified a potent, reversible BTK inhibitor, BIIB068 (1), which demonstrated good kinome selectivity with good overall drug-like properties for oral dosing, was well tolerated across preclinical species at pharmacologically relevant doses with good ADME properties, and achieved >90% inhibition of BTK phosphorylation (pBTK) in humans.

Inhibition of Bruton tyrosine kinase in patients with severe COVID-19.

Patients with severe COVID-19 have a hyperinflammatory immune response suggestive of macrophage activation. Bruton tyrosine kinase (BTK) regulates macrophage signaling and activation. Acalabrutinib, a selective BTK inhibitor, was administered off-label to 19 patients hospitalized with severe COVID-19 (11 on supplemental oxygen; 8 on mechanical ventilation), 18 of whom had increasing oxygen requirements at baseline. Over a 10-14 day treatment course, acalabrutinib improved oxygenation in a majority of patients, often within 1-3 days, and had no discernable toxicity. Measures of inflammation - C-reactive protein and IL-6 - normalized quickly in most patients, as did lymphopenia, in correlation with improved oxygenation. At the end of acalabrutinib treatment, 8/11 (72.7%) patients in the supplemental oxygen cohort had been discharged on room air, and 4/8 (50%) patients in the mechanical ventilation cohort had been successfully extubated, with 2/8 (25%) discharged on room air. Ex vivo analysis revealed significantly elevated BTK activity, as evidenced by autophosphorylation, and increased IL-6 production in blood monocytes from patients with severe COVID-19 compared with blood monocytes from healthy volunteers. These results suggest that targeting excessive host inflammation with a BTK inhibitor is a therapeutic strategy in severe COVID-19 and has led to a confirmatory international prospective randomized controlled clinical trial.

Discovery of 4-Aminoquinoline-3-carboxamide Derivatives as Potent Reversible Bruton's Tyrosine Kinase Inhibitors for the Treatment of Rheumatoid Arthritis.

A structure-hopping strategy was applied to discover a series of novel 4-aminoquinoline-3-carboxamide derivatives as potent, reversible BTK inhibitors. Compared to the previously described cinnoline scaffold compounds, the 4-aminoquinoline analogues showed significantly improved drug-like properties, especially in their aqueous solubility. The most potent compound, 25, displayed a stronger inhibitory effect on both BTKWT (IC50 = 5.3 nM) and BTKC481S (IC50 = 39 nM). In a rodent collagen-induced arthritis model, compound 25 efficiently reduced paw swelling without a loss in body weight. On the basis of potency, drug-like properties, stability, and noncovalent mode of inhibition, our representative inhibitors could have a promising profile to be treatments for a wide range of autoimmune diseases.

Design, synthesis and evaluation of novel 7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as potent, selective and reversible Bruton's tyrosine kinase (BTK) inhibitors for the treatment of rheumatoid arthritis.

A series of 7H-pyrrolo[2,3-d]pyrimidine derivatives were designed and synthesized as reversible BTK inhibitors, and evaluated their kinase selectivity, anti-proliferation against the B-cell lymphoma cell lines (Ramos, Jeko-1) and cell line BTK enhanced (Daudi) in vitro. Among them, compound 28a exhibited the most excellent potency (IC50 = 3.0 nM against BTK enzyme, 8.52 µM, 11.10 µM and 7.04 µM against Ramos, Jeko-1, Daudi cells, respectively), good kinase selectivity and inhibited BTK Y223 auto-phosphorylation and PLCγ2 Tyr1217 phosphorylation. Importantly, 28a showed efficacy anti-arthritic effect on collagen-induced arthritis (CIA) model in vivo. 28a 60 mg/kg dose level once a day group displayed markedly reduced joint damage and cellular infiltration without any bone and cartilage morphology change.

Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

Evolutionary relationship between the cysteine and histidine rich domains (CHORDs) and Btk-type zinc fingers.

Summary: Cysteine and histidine rich domains (CHORDs), implicated in immunity and disease resistance signaling in plants, and in development and signal transduction in muscles and tumorigenesis in animals, are seen to have a cylindrical three-dimensional structure stabilized by the tetrahedral chelation of two zinc ions. CHORDs are regarded as novel zinc-binding domains and classified independently in Pfam and ECOD. Our sequence and structure analysis reveals that both the zinc-binding sites in CHORD possess a zinc ribbon fold and are likely related to each other by duplication and circular permutation. Interestingly, we also detect an evolutionary relationship between each of the CHORD zinc fingers (ZFs) and the Bruton's tyrosine kinase (Btk)-type ZF of the zinc ribbon fold group. Btk_ZF is found in eukaryotic Tec kinase family proteins that are also implicated in signaling pathways in several lineages of hematopoietic cells involved in mammalian immunity. Our analysis suggests that the unique zinc-stabilized fold seen only in the CHORD and Btk_ZFs likely emerged specifically in eukaryotes to mediate diverse signaling pathways. Supplementary information: Supplementary data are available at Bioinformatics online.

Combined pharmacophore, virtual screening and molecular dynamics studies to identify Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine Kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase expressed in hematopoietic cells. BTK plays a critical role in many cellular signalling pathways making it a potential target to treat autoimmune diseases and cancer. BTK signalling is important for the production of arthritis-associated antibodies, and inhibiting BTK will help the system to block the production of disease-associated antibodies. In this study, we have implemented ligand-based pharmacophore modelling and virtual screening against natural compounds followed by molecular docking, density functional theory and molecular dynamics studies for 50 ns. Four compounds with high affinity towards BTK were identified, and it could be used as a potent lead molecule for designing BTK inhibitor.