Tec Kinase Research Articles

Pulmonary arterial hypertension associated with protein kinase inhibitors: a pharmacovigilance–pharmacodynamic study

The pathophysiology of pulmonary arterial hypertension (PAH) induced by protein kinase inhibitors (PKIs) remains unclear. To gain knowledge into this rare and severe pathology we performed a study combining a pharmacovigilance approach and the pharmacodynamic properties of PKIs.A disproportionality analysis on the World Health Organization pharmacovigilance database VigiBase using the reporting odds ratio (ROR) and 95% confidence interval was first performed. Then, we identified the most relevant cellular targets of interest through a systematic literature review and correlated the pharmacovigilance signals with the affinity for the different PKIs. We further performed a hierarchical cluster analysis to assess patterns of binding affinity.A positive disproportionality signal was found for dasatinib, bosutinib, ponatinib, ruxolitinib and nilotinib. Five non-receptor protein kinases significantly correlate with disproportionality signals: c-Src (r=0.79, p=0.00027), c-Yes (r=0.82, p=0.00015), Lck (r=0.81, p=0.00046) and Lyn (r=0.80, p=0.00036), all belonging to the Src protein kinase family, and TEC (r=0.85, p=0.00006). Kinases of the bone morphogenetic protein signalling pathway also seem to play a role in the pathophysiology of PKI-induced PAH. Interestingly, the dasatinib affinity profile seems to be different from that of other PKIs in the cluster analysis.The study highlights the potential role of the Src protein kinase family and TEC in PAH induced by PKIs. This approach combining pharmacovigilance and pharmacodynamics data allowed us to generate some hypotheses about the pathophysiology of the disease; however, the results have to be confirmed by further studies.

The Tec kinase ITK is essential for ILC2 survival and epithelial integrity in the intestine

Innate lymphoid cells (ILC) are lymphocytes that lack an antigen-specific receptor and are preferentially localized in non-lymphoid tissues, such as mucosal barriers. In these locations ILC respond to tissue perturbations by producing factors that promote tissue repair and improve barrier integrity. We show that mice lacking the Tec kinase ITK have impaired intestinal tissue integrity, and a reduced ability to restore homeostasis after tissue damage. This defect is associated with a substantial loss of Type 2 ILC (ILC2) in the intestinal lamina propria. Adoptive transfer of bone marrow ILC2 precursors confirms a cell-intrinsic role for ITK. Intestinal ILC2 numbers in Itk-/- mice are restored by the administration of IL-2 complexes, also leading to improved intestinal tissue damage repair. Reduced Bcl-2 expression in intestinal Itk-/- ILC2 is also restored to WT levels after IL-2 complex treatment, indicating a tissue-specific role for ITK in ILC2 survival in the intestine.

Effects of Tec Tyrosine Kinase Inhibition on the Inflammatory Response of Severe Acute Pancreatitis-Associated Acute Lung Injury in Mice.

The Tec kinase family is involved in acute and chronic inflammatory diseases, but its relationship with severe acute pancreatitis (SAP) remains unclear. To investigate whether Tec tyrosine kinase can be used as a target for severe acute pancreatitis-associated acute lung injury (PALI). A total of 90 mice were randomly assigned into four groups: SAP (n = 15), control (n = 15), SAP + α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13) (pretreated with Tec kinase inhibitor LFM-A13, n = 15), and SAP + Tec siRNA (pretreated with PBS/negative control siRNA/Tec siRNA, n = 45). SAP was induced by caerulein and lipopolysaccharide. Animals were sacrificed at 0, 3, 24, 48, and 72h, respectively. Pathological changes and scores of the lung and pancreas were determined using hematoxylin-eosin staining. Expression of Tec and phosphorylated Tec (p-Tec) were examined by real-time polymerase chain reaction, Western blot, and immunoprecipitation. Serum levels of amylase, myeloperoxidase, and pro-inflammatory cytokines were measured by ELISA. The expression of Tec in lung tissue was significantly higher in the SAP group than in the control group (p < 0.05), and p-Tec expression gradually increased with time. Furthermore, p-Tec expression was significantly lower in the SAP + LFM-A13 group than in the SAP group (p < 0.05); however, Tec expression did not vary. Tec inhibitors, LFM-A13 and Tec siRNA, alleviated pathological damage and release of inflammatory cytokines (p < 0.05). Tec tyrosine kinase plays a key role in PALI, and is therefore a potential target for clinical treatment.

Vero Cell Proteomic Changes Induced by Zika Virus Infection.

The re-emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV-associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer-based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV-induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25-fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.

Liquid Chromatography Mass Spectrometry (LC-MS) analysis in determining the saliva protein of orthodontic patients during retention phase

BackgroundThe biological responses involved during retention phase have been studied for many years but little is known about the effect of saliva proteome during retention phase of post-orthodontic treatment. This study aims to identify the protein profiles during retention phase in relation to biological processes involved by Liquid Chromatography Mass Spectrometry (LC-MS) approach.Material and MethodsA total of 5 ml of unstimulated saliva was collected from each subject (10 non-orthodontic patients and 15 post-orthodontic patients with 6-months retention phase). Samples were then subjected to LC-MS analysis. The expressed proteins were identified and compared between groups. Incisor irregularity for both maxilla and mandible were determined with Little’s Irregularity Index at 6-months retention phase.Results146 proteins and 135 proteins were expressed in control and 6-months retention phase group respectively. 15 proteins were identified to be co-expressed between groups. Immune system process was only detected in 6-months retention phase group. Detected protein in immune system process was identified as Tyrosine-protein kinase Tec. Statistical significant of incisor irregularity was only found in mandible at 6-months retention phase.ConclusionsOur study suggests that immune system process protein which is Tyrosine-protein kinase Tec could be used as biomarker for prediction of stability during retention phase of post-orthodontic treatment. Key words:Orthodontics, proteomics, retention, LC-MS, saliva.

The Effect of Btk Inhibitors on Haemostasis and Thrombosis

▪IntroductionThe Btk inhibitor, Ibrutinib (Imbruvica) which has proven to be efficacious in achieving remission of lymphocytosis and lymph node enlargement in B-CLL, it does have adverse side effects of bleeding, including major haemorrhages. The bleeding associated with Ibrutinib use is thought to be due to a combination of on-target Btk inhibition (as Btk is a key component of platelet GPVI signalling) as well as off targeted inhibition of other kinases including EGFR, ITK, JAK3 and Tec kinase. The major next generation Btk inhibitors in clinical development include Zanubrutinib (BGB-3111). Zanubrutinib shows improved selectivity for Btk compared with Ibrutinib, and thus may have reduced bleeding effects. Our study aims to determine in detail differential platelet effects between Ibrutinib and Zanubrutinib in human and mouse models using in vitro, exvivo and in vivo approaches.MethodsIntravital microscopy was used to determine thrombus formation and growth after Btk inhibitors treatment in vitro and ex vivo using micro-slides or inside the mesenteric arterioles after injury by ferric chloride (FeCl3). Z-stack digital Axiocam mRm camera (Carl Zeiss) and Zeiss Axiovision software was used to capture images. Three dimensional (3D) deconvolved reconstructions of thrombi formed were analysed for surface coverage of platelet aggregates (μm2), thrombus height (μm) and thrombus volume (μm3). Flow cytometry analysis was also used to determine the release of agonist-induced platelet P-selectin exposure and dense granule after treatment with Btk inhibitors.ResultsIn vitro experiments demonstrated that Btk inhibitors did not affect alpha or dense granule secretion mediated by GPCRs agonists, thrombin, PAR1 or PAR4. However, they inhibited alpha granule secretion mediated by GPVI selective agonists, CRP-XL or Rhodocytin. Ibrutinib inhibited human thrombus formation on type I collagen, fibrinogen or von Willebrand factor under arterial shear with 3 fold reduction whereas Zanubrutinib had no effect over a dose dependent range of concentrations. Ibrutinib treated PRP significantly delayed the kinetics of clot retraction at all-time points over the 2 hour time frame compared to Zanubrutinib treated and vehicle control. The studies also showed that Ibrutinib but not Zanubrutinib inhibited ex vivo human thrombus formation on type I collagen under arterial shear using B-CLL patient samples. The data demonstrated that treatment of C57BL/6 mouse whole blood with 0.5-2.0 µM of ibrutinib significantly inhibited thrombus growth on type I collagen under in vitro flow conditions whereas Zanubrutinib was comparable to the vehicle control. Consequently, pre-treatment of C57BL/6 mice with ibrutinib (10 mg/kg), but not Zanubrutinib (10 mg/kg) markedly inhibited platelet thrombus growth and formation on type I collagen under ex vivo arterial flow conditions. Intravital microscopy of vascular injury of mesenteric arterioles induced by ferric chloride (FeCl3) demonstrated that Ibrutinib (10 mg/kg), but not Zanubrutinib (10 mg/kg) inhibited in vivo murine thrombus formation and growth over time.ConclusionBtk inhibitors used in the treatment of B-cell malignancies have differential effects on platelet function and thrombosis. Zanubrutinib is superior to ibrutinib as it showed no effect on platelet thrombus formation, thus reduces risk of bleeding. DisclosuresTam:AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Beigene: Honoraria.

Assessing Hematologist's Knowledge of the Use of Ibrutinib and Acalabrutinib in B-Cell Malignancies

Background: The objective of this study was to assess current clinical practices of hematology/oncology (hem/onc) specialists related to BTK inhibitor use in the treatment of patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), in order to identify knowledge, competency, and practice gaps and barriers to optimal care.Methods: A continuing medical education (CME)-certified clinical practice assessment consisting of 25 multiple choice questions that were designed to measure knowledge, skills, attitudes, and competence of hem/onc specialists regarding BTK inhibitor use. The survey instrument was made available online to physicians without monetary compensation or charge. Respondent confidentiality was maintained and responses were de-identified and aggregated prior to analyses. The activity launched on May 31, 2018 (after the approval of acalabrutinib for MCL) and participant responses are still being collected at the time of abstract submission.Results: At the time of this report there are 124 hem/onc activity participants, collection is on-going. Demographics are listed in Table 1 and levels of confidence and barriers to incorporating BTK inhibitors are listed in Table 2.•Differentiating Between Ibrutinib and Acalabrutinib•Very low recognition of the differences between first and second generation BTK inhibitors.•Only 16% identified that both ibrutinib and acalabrutinib target BTK at the same amino acid residue.•Barely over a quarter (27%) understand that acalabrutinib irreversibly inhibits BTK, similar to ibrutinib.Higher level of knowledge related to the specificity of second generation BTK inhibitors and the differences in dosing frequency compared to first generation BTK inhibitors, but still room for improvement.•Half correctly identified acalabrutinib is potentially better tolerated than ibrutinib due to greater relative selectivity for BTK over EGFR, ITK, and TEC kinases.•Over half (56%) demonstrated knowledge of the dosing schedule for ibrutinib and acalabrutinib, while 33% incorrectly think both BTK inhibitors are dosed once a day.Strong recognition of the FDA-approved indication for ibrutinib in MCL (74%) with much less recognition of the FDA-approved indication for acalabrutinib in MCL (35%).BTK Mutations•Lack of knowledge about how the C481S mutation causes resistance to ibrutinib and acalabrutinib.•A little over half (54%) understand that a BTK mutation at position 481 from cysteine to serine results in resistance to both ibrutinib and acalabrutinib.•Only 33% demonstrated knowledge of the C481S resistance mechanism.Clinical Trial Data•More familiarity with pivotal phase 3 clinical trial data for ibrutinib, indicating a need for further education about phase 2 acalabrutinib data.•69% correctly identified the OS outcome with ibrutinib in previously treated CLL•43% correctly identified the ORR with acalabrutinib in previously treated CLL•26% correctly identified the ORR with acalabrutinib in previously treated MCLConclusions: This activity indicates there are significant gaps in knowledge and confidence as well as barriers impeding hem/onc ability to incorporate BTK inhibitors into the treatment plans for their patients with B-cell malignancies. There is sub-optimal awareness of differences between first and second generation BTK inhibitors as well as trial data for second generation BTK inhibitors. Additional education is needed to improve the knowledge and confidence of academic and community hem/onc specialists who care for patients with CLL and MCL so they are better equipped to utilize BTK inhibitors. DisclosuresFurman:Acerta: Consultancy, Research Funding; Sunesis: Consultancy; Verastem: Consultancy; TG Therapeutics: Consultancy; Genentech: Consultancy; Incyte: Consultancy, Other: DSMB; Gilead: Consultancy; Loxo Oncology: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; Janssen: Consultancy; AbbVie: Consultancy.

Updated Safety and Activity of the Investigational Bruton Tyrosine Kinase Inhibitor Zanubrutinib (BGB-3111) in Patients with Mantle Cell Lymphoma

Updated Safety and Activity of the Investigational Bruton Tyrosine Kinase Inhibitor Zanubrutinib (BGB-3111) in Patients with Mantle Cell Lymphoma

Incidence, Type, and Management of Venous and Arterial Thrombosis during Ibrutinib Treatment

BACKGROUNDIbrutinib is an irreversible inhibitor of BTK in the B-cell receptor signaling cascade and is widely used to treat chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Ibrutinib also inhibits the tyrosine kinase Tec in platelets, which may be one of the mechanisms of its bleeding toxicity. This makes concomitant use of anticoagulation (AC) or antiplatelet agents challenging, which is a common delimma as many patients taking ibrutinib are elderly and have increased risks of venous and arterial thromboses. The incidence of thrombosis in patients taking ibrutinib is unknown, and we hypothesized that the risk of thrombosis may be reduced during ibrutinib treatment. Therefore, we conducted a single-institution retrospective cohort study to determine the incidence and type of both arterial and venous thromboses during ibrutinib treatment and their management. METHODSWe reviewed medical records of all patients treated with ibrutinib for a hematological malignancy at the Ohio State University between 6/1/2010 and 3/31/2016. Baseline patient and disease characteristics were captured at time of starting ibrutinib. All thrombotic events occurring at any time during treatment with ibrutinib and within three days of its discontinuation were recorded. Time to thrombosis was calculated from the date of starting ibrutinib to the date of thrombosis or censored at the last assessment date, treating discontinuation of ibrutinib or death prior to thrombosis as competing risks. The cumulative incidence of thrombosis was estimated and the Fine and Gray regression models accounting for competing riskes were used to examine the association between patient characteristics and risk of thrombosis. RESULTSThe cohort included 565 patients. Median age was 65 (range 23->89) years and 70.3% (397/565) were men. The majority of patients had CLL (73.6%, 416/565). Other diagnoses included mantle cell lymphoma (9.9%, 56/565), indolent B-cell malignancies (8.1%, 46/565), and aggressive lymphomas (8.3%, 47/565). Median number of prior treatments was 3 (range 0-18) and 6.5% (37/565) of patients were treatment naïve. Prior to ibrutinib, 144 of 565 patients (25.5%) had a history of thrombosis. Sixty-four (11.3%, 64/565) patients had only venous thromboses, 66 (11.7% 66/565) had only arterial thromboses, and 14 patients had both. Concurrently with ibrutinib, 193 (34.2%) patients received antiplatelet agents, 16 (2.8%) patients received AC, and 31 (5.5%) patients received both. Total ibrutinib exposure for the cohort was 1,429 person-years with a median exposure of 2.39 (range 0-7.36) years per patient. A second antineoplastic agent was given with ibrutinib in 30.8% (174/565) of cases, including an immunomodulatory drug in 24 (4.2%, 24/565) patients.During ibrutinib treatment, 22 of 565 (3.9%) patients experienced 24 acute thrombotic events, mostly arterial (Table 1). The incidence of thrombosis was 1.7 (95% CI 1.1-2.5) per 100 person-years of ibrutinib exposure. Of the venous thromboses, 87.5% (7/8) were deep vein thromboses and developed at a median of 7.5 (range 0.5-75.3) months after starting ibrutinib. Of the arterial thromboses, the majority were acute cerebrovascular accidents (37.5%, 6/16) and developed at a median of 27.4 (range 0.4 - 56.6) months after starting ibrutinib.Thrombosis treatment is summarized in Table 1. After thrombosis, ibrutinib was discontinued or held in the majority of cases (75%, 18/24). One patient developed a recurrent thrombosis while on ibrutinib and AC. There were six bleeding events, 3 major (based on ISTH criteria) and 3 minor: all were taking ibrutinib and most were on AC (2 patients on antiplatelet, 1 on AC, 2 on both, 1 on neither). On univariable analysis, the only factors associated with significant (p<0.05) and substantial (HR >2) increased risk of venous thrombosis were prior venous (HR 4.73, CI: 1.06-21.11) and arterial (HR 15.66, CI: 3.07-79.87) thromboses. Antiplatelet use was not significantly associated with either thrombus type. CONCLUSIONSThe cumulative incidence of thrombosis during ibrutinib treatment was low (1.7 per 100 person-years), with the majority being arterial. Prior thrombosis was associated with increased venous thrombosis risk. There are more bleeding than thrombotic complications after patients develop thromboses on ibrutinib, and optimal treatment strategies for this population requires further investigation. [Display omitted] DisclosuresKander:AstraZeneca: Consultancy. Wang:Daiichi Sankyo: Consultancy, Other: Travel.

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.

BMX-Mediated Regulation of Multiple Tyrosine Kinases Contributes to Castration Resistance in Prostate Cancer.

Prostate cancer responds to therapies that suppress androgen receptor (AR) activity (androgen deprivation therapy, ADT) but invariably progresses to castration-resistant prostate cancer (CRPC). The Tec family nonreceptor tyrosine kinase BMX is activated downstream of PI3K and has been implicated in regulation of multiple pathways and in the development of cancers including prostate cancer. However, its precise mechanisms of action, and particularly its endogenous substrates, remain to be established. Here, we demonstrate that BMX expression in prostate cancer is suppressed directly by AR via binding to the BMX gene and that BMX expression is subsequently rapidly increased in response to ADT. BMX contributed to CRPC development in cell line and xenograft models by positively regulating the activities of multiple receptor tyrosine kinases through phosphorylation of a phosphotyrosine-tyrosine (pYY) motif in their activation loop, generating pYpY that is required for full kinase activity. To assess BMX activity in vivo, we generated a BMX substrate-specific antibody (anti-pYpY) and found that its reactivity correlated with BMX expression in clinical samples, supporting pYY as an in vivo substrate. Inhibition of BMX with ibrutinib (developed as an inhibitor of the related Tec kinase BTK) or another BMX inhibitor BMX-IN-1 markedly enhanced the response to castration in a prostate cancer xenograft model. These data indicate that increased BMX in response to ADT contributes to enhanced tyrosine kinase signaling and the subsequent emergence of CRPC, and that combination therapies targeting AR and BMX may be effective in a subset of patients.Significance: The tyrosine kinase BMX is negatively regulated by androgen and contributes to castration-resistant prostate cancer by enhancing the phosphorylation and activation of multiple receptor tyrosine kinases following ADT. Cancer Res; 78(18); 5203-15. ©2018 AACR.

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.

Multidomain Control Over TEC Kinase Activation State Tunes the T Cell Response.

Signaling through the T cell antigen receptor (TCR) activates a series of tyrosine kinases. Directly associated with the TCR, the SRC family kinase LCK and the SYK family kinase ZAP-70 are essential for all downstream responses to TCR stimulation. In contrast, the TEC family kinase ITK is not an obligate component of the TCR cascade. Instead, ITK functions as a tuning dial, to translate variations in TCR signal strength into differential programs of gene expression. Recent insights into TEC kinase structure have provided a view into the molecular mechanisms that generate different states of kinase activation. In resting lymphocytes, TEC kinases are autoinhibited, and multiple interactions between the regulatory and kinase domains maintain low activity. Following TCR stimulation, newly generated signaling modules compete with the autoinhibited core and shift the conformational ensemble to the fully active kinase. This multidomain control over kinase activation state provides a structural mechanism to account for ITK's ability to tune the TCR signal.

Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ.

ABSTRACTTyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo. Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.

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

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 data are available at Bioinformatics online.

STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex.

The receptor activator of nuclear factor-κB (RANK) protein activates various protein kinase signaling cascades, including those involving NF-κB, mitogen-activated protein kinase (MAPK), and Bruton tyrosine kinase (Btk)/tyrosine-protein kinase Tec. However, the mechanism underlying the negative regulation of RANK by downstream signaling molecules remains unclear. Here, we report that Src homology 3 domain and cysteine-rich domain-containing protein 2 (STAC2) is a novel RANK ligand-inducible protein that negatively regulates RANK-mediated osteoclast formation. STAC2 physically interacts with RANK and inhibits the formation of the RANK signaling complex, which contains Grb-2-associated binder 2 (Gab2) and phospholipase Cγ2 (PLCγ2), thus leading to the suppression of RANK-mediated NF-κB and MAPK activation. Furthermore, STAC2 overexpression limits Btk/Tec-mediated PLCγ2 phosphorylation via the interaction between STAC2 and Btk/Tec. Taken together, our results reveal a novel mechanism whereby RANK signaling is restricted by its physical interaction with STAC2.

Comparative Proteomic Study of the Antiproliferative Activity of Frog Host-Defence Peptide Caerin 1.9 and Its Additive Effect with Caerin 1.1 on TC-1 Cells Transformed with HPV16 E6 and E7.

Caerin is a family of peptides isolated from the glandular secretion of Australian tree frogs, the genus Litoria, and has been previously shown to have anticancer activity against several cancer cells. In this work, we used two host-defence peptides, caerin 1.1 and caerin 1.9, to investigate their ability to inhibit a murine derived TC-1 cell transformed with human papillomavirus 16 E6 and E7 growth in vitro. Caerin 1.9 inhibits TC-1 cell proliferation, although inhibition is more pronounced when applied in conjunction with caerin 1.1. To gain further insights into the antiproliferative mechanisms of caerin 1.9 and its additive effect with caerin 1.1, we used a proteomics strategy to quantitatively examine (i) the changes in the protein profiles of TC-1 cells and (ii) the excretory-secretory products of TC-1 cells following caerin peptides treatment. Caerin 1.9 treatment significantly altered the abundance of several immune-related proteins and related pathways, such as the Tec kinase and ILK signalling pathways, as well as the levels of proinflammatory cytokines and chemokines. In conclusion, caerin peptides inhibit TC-1 cell proliferation, associated with modification in signalling pathways that would change the tumour microenvironment which is normally immune suppressive.

Negative Regulation of Tec Kinase Alleviates LPS-Induced Acute Kidney Injury in Mice via the TLR4/NF-κB Signaling Pathway

Negative Regulation of Tec Kinase Alleviates LPS-Induced Acute Kidney Injury in Mice via the TLR4/NF-κB Signaling Pathway

A switch in nucleotide affinity governs activation of the Src and Tec family kinases

The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases.

ITK and RLK Inhibitor PRN694 Improves Skin Disease in Two Mouse Models of Psoriasis

The chronic and highly prevalent skin disorder psoriasis vulgaris is characterized by a hyperproliferative epidermis and aberrant immune activity. Many studies have highlighted the role of differentiated T lymphocytes in psoriasis progression. Several biologics are currently available that target proinflammatory cytokines produced by T lymphocytes, but the need for improved therapies persists. The small molecule PRN694 covalently binds ITK and RLK, two Tec kinases activated downstream of T-lymphocyte activation, both of which are up-regulated in psoriatic skin. These Tec kinases are involved in signaling cascades mediating T-lymphocyte proliferation, differentiation, and migration and proinflammatory cytokine production. Invitro analysis showed that PRN694 effectively inhibited IL-17A production from murine T helper type 17-differentiated T lymphocytes. Additionally, PRN694 effectively reduced the psoriasis-like phenotype severity and reduced epidermal proliferation and thickness in both the Rac1V12 and imiquimod mouse models of psoriasis. PRN694 also inhibited CD3+ T-cell and γδ T-cell infiltration into skin regions. Inhibition of ITK and RLK attenuated psoriasis-associated signaling pathways, indicating that PRN694 is an effective psoriasis therapeutic.