Role of FcγRIII in the nasal cavity of BALB/c mice in the primary amebic meningoencephalitis protection model.

Different mechanisms of the host immune response against the primary amebic meningoencephalitis (PAM) in the mouse protection model have been described. It has been proposed that antibodies opsonize Naegleria fowleri trophozoites; subsequently, the polymorphonuclear cells (PMNs) surround the trophozoites to avoid the infection. FcγRs activate signaling pathways of adapter proteins such as Syk and Hck on PMNs to promote different effector cell functions which are induced by the Fc portion of the antibody-antigen complexes. In this work, we analyzed the activation of PMNs, epithelial cells, and nasal passage cells via the expression of Syk and Hck genes. Our results showed an increment of the FcγRIII and IgG subclasses in the nasal cavity from immunized mice as well as Syk and Hck expression was increased, whereas in the in vitro assay, we observed that when the trophozoites of N. fowleri were opsonized with IgG anti-N. fowleri and interacted with PMN, the expression of Syk and Hck was also increased. We suggest that PMNs are activated via their FcγRIII, which leads to the elimination of the trophozoites in vitro, while in the nasal cavity, the adhesion and consequently infection are avoided.

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative.

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.

HIV-1 Nef-Induced Secretion of the Proinflammatory Protease TACE into Extracellular Vesicles Is Mediated by Raf-1 and Can Be Suppressed by Clinical Protein Kinase Inhibitors.

Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable. Strikingly, among the numerous Src-family downstream effectors, serine/threonine kinase Raf-1 was found to be necessary and alone sufficient to trigger the secretion of TACE into EVs. These data reveal the involvement of Raf-1 in regulation of unconventional protein secretion and highlight the importance of Raf-1 as a cellular effector of Nef, thereby suggesting a novel rationale for testing pharmacological inhibitors of the Raf-MAPK pathway to treat HIV-associated immune activation.IMPORTANCE Chronic immune activation contributes to the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and is associated with poor recovery of the immune system despite potent antiretroviral therapy, which is observed in 10% to 40% drug-treated patients depending on the definition of immune reconstitution. We have previously shown that the HIV pathogenicity factor Nef can promote loading of the proinflammatory protease TACE into extracellular vesicles (EVs), and the levels of such TACE-containing EVs circulating in the blood correlate with low CD4 lymphocyte counts in HIV patients receiving antiretroviral therapy. Here, we show that Nef promotes uploading of TACE into EVs by triggering unconventional secretion via activation of the Hck/Raf/mitogen-activated protein kinase (MAPK) cascade. We find that several pharmaceutical inhibitors of these kinases that are currently in clinical use for other diseases can potently suppress this pathogenic deregulation and could thus provide a novel strategy for treating HIV-associated immune activation.

HIV-1 Nef Induces Hck/Lyn-Dependent Expansion of Myeloid-Derived Suppressor Cells Associated with Elevated Interleukin-17/G-CSF Levels.

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

Ethyl-4-(aryl)-6-methyl-2-(oxo/thio)-3,4-dihydro-1H-pyrimidine-5-carboxylates: Silica supported bismuth(III)triflate catalyzed synthesis and antioxidant activity.

Novel ethyl-4-(aryl)-6-methyl-2-(oxo/thio)-3,4-dihydro-1H-pyrimidine-5-carboxylates were synthesized from one-pot, three-component Biginelli reaction of aryl aldehydes, ethyl acetoacetate and urea/ thiourea by catalytic action of silica supported Bismuth(III) triflate, a Lewis acid. All the synthesized compounds were structurally characterized by spectral (IR, 1H NMR & 13C NMR spectroscopic and Mass spectrometric) and elemental (C, H & N) analyses. The present protocol has deserved novel as, formed the products in high yields with short reaction times, involved eco-friendly methodology and reusable heterogeneous Lewis acid catalyst. The title compounds were screened for in vitro DPPH free radical scavenging antioxidant activity and identified 4i, 4j, 4h & 4f as potential antioxidants. The obtained in vitro results were correlated with molecular docking, ADMET, QSAR, Bioactivity & toxicity risk studies and molecular finger print properties and found that in silico binding affinities were identified in good correlation with in vitro antioxidant activity and studied the structure activity relationship. The molecular docking study has disclosed strong hydrogen bonding interactions of title compounds with aspartic acid (ASP197) aminoacid residue of 2HCK, a complex enzyme of haematopoietic cell kinase and quercetin. Results of toxicology study evaluated for potential risks of compounds have revealed title compounds as safer drugs. In ultimate the study has established ligand's antioxidant potentiality as they effectively binds with ASP197 amino acid of Chain A hence confirms the inhibition of growth of reactive oxygen species in vivo. In addition, the title compounds have been identified as potential blood-brain barrier penetrable entities and efficient central nervous system (CNS) active neuro-protective antioxidant agents.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.

Hematopoietic cell kinase enhances osteosarcoma development via the MEK/ERK pathway.

Osteosarcoma (OS) is a sarcoma with high rates of pulmonary metastases and mortality. The mechanisms underlying tumour generation and development in OS are not well-understood. Haematopoietic cell kinase (HCK), a vital member of the Src family of kinase proteins, plays crucial roles in cancer progression and may act as an anticancer target; however, the mechanism by which HCK enhances OS development remains unexplored. Therefore, we investigated the role of HCK in OS development in vitro and in vivo. Downregulation of HCK attenuated OS cell proliferation, migration and invasion and increased OS cell apoptosis, whereas overexpression of HCK enhanced these processes. Mechanistically, HCK expression enhanced OS tumorigenesis via the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; HCK upregulation increased the phosphorylation of MEK and ERK and promoted epithelial-mesenchymal transition, with a reduction in E-cadherin in vitro. Furthermore, HCK downregulation decreased the tumour volume and weight in mice transplanted with OS cells. In conclusion, HCK plays a crucial role in OS tumorigenesis, progression and metastasis via the MEK/ERK pathway, suggesting that HCK is a potential target for developing treatments for OS.

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes.

Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.

miR-181b and miR-204 suppress the VSMC proliferation and migration by downregulation of HCK.

BACKGROUND: VSMC proliferation and migration pathways play important roles in plaque formation in the vessel stenosis and re-stenosis processes. The microRNAs affect the expression of many genes that regulate these cellular processes. The aim of this study was to investigate the effects of miR-181b, miR-204, and miR-599 on the gene and protein expression levels of hematopoietic cell kinase (HCK) in VSMCs. METHODS: miR-181b, miR-204 were predicted for the suppression of HCK in the chemokine signaling pathway using bioinformatics tools. Then, the VSMCs were transfected by PEI-containing microRNAs. The HCK gene and protein expression levels were evaluated using RT-qPCR and Western blotting techniques, respectively. Moreover, the cellular proliferation and migration were evaluated by MTT and scratch assay methods. RESULTS: The miR-181b and miR-204 decreased significantly the HCK gene and (total and phosphorylated) protein expression levels. Also, the miR-599 did not show any significant effects on the HCK gene and protein levels. The data also showed that miR-181b, miR-204, and miR-599 prevent significantly the proliferation and migration of VSMCs. CONCLUSION: The downregulation of HCK by miR-181b and miR-204 suppressed the VSMC proliferation and migration.

Hunig's base catalyzed synthesis of new 1-(2,3-dihydro-1H-inden-1-yl)-3-aryl urea/thiourea derivatives as potent antioxidants and 2HCK enzyme growth inhibitors.

A series of 1-(2,3-dihydro-1H-indan-1-yl)-3-aryl urea/thiourea derivatives (4a-j) have been synthesized from the reaction of 2,3-dihydro-1H-inden-1-amine (2) with various aryl isocyanates/isothiocyanates (3a-j) by using N,N-DIPEA base (Hunig's base) catalyst in THF at reflux conditions. All of them are structurally confirmed by spectral (IR, 1H &13C NMR and MASS) and elemental analysis and screened for their in-vitro antioxidant activity against DPPH and NO free radicals and found that compounds 4b, 4i, 4h &4g are potential antioxidants. The obtained in vitro results were compared with the molecular docking, ADMET, QSAR and bioactivity study results performed for them and identified that the recorded in silico binding affinities were observed in good correlation with the in vitro antioxidant results. The Molecular docking analysis had unveiled the strong hydrogen bonding interactions of synthesized ligands with ARG 160 residue of protein tyrosine kinase (2HCK) enzyme and plays an effective role in its inhibition. Toxicology studies have assessed the potential risks of 4a-j and inferred that all of them were in the limits of potential drugs. The conformational analysis of 4a-j inferred that the urea/thiourea spacer linking 2,3-dihydro-1H-inden-1-amino and substituted aryl units has facilitated all these molecules to effectively bind with ARG 160 amino acid residue present on the α-helix of the protein tyrosine kinase (2HCK) enzyme specifically on chain A of hemopoetic cell kinase. Collectively this study has established a relationship between the antioxidant potentiality and ligands binding with ARG 160 amino acid residue of chain A of 2HCK enzyme to inhibit its growth as well as proliferation of reactive oxygen species in vivo.

HCK can serve as novel prognostic biomarker and therapeutic target for Breast Cancer patients.

The role of HCK expression in the prognosis of breast cancer patients is unclear. Thus, this study aimed to explore the clinical implications of HCK expression in breast cancer. We assessed HCK expression and genetic variations in breast cancer using Oncomine, GEPIA, UALCAN, and cBioPortal databases. Then, immunochemistry was used to analyze HCK expression in breast cancer specimens, non-cancer tissues and metastatic cancer tissues. Consequently, we evaluated the effect of HCK expression on survival outcomes set as disease-free survival (DFS) and overall survival (OS). Finally, STRING, Coexpedia, and TISIDB database were explored to identify the molecular functions and regulation pathways of HCK. We found that breast cancer tissues have more HCK mRNA transcripts than non-cancer tissues. Patients with HCK expression had significantly shorter DFS and OS. The ratio of HCK expression was higher in cancer tissues than in non-cancer tissues. These results from STRING database, FunRich software, and TISIDB database showed that HCK was involved in mediating multiple biological processes including immune response-regulating signaling pathway, cell growth and maintenance through multiple signaling pathways including epithelial to mesenchymal transition, PI3K/AKT signaling pathway, and focal adhesion. Overall, HCK may be an oncogene in the development of breast cancer and thus may as a novel biomarker and therapeutic target for breast cancer.

Expression of a Constitutively Active Form of Hck in Chondrocytes Activates Wnt and Hedgehog Signaling Pathways, and Induces Chondrocyte Proliferation in Mice.

Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (Hck), which is a member of the Src tyrosine kinase family, in chondrocytes, that Hck expression was high in cartilaginous limb skeletons of wild-type mice but low in those of Runx2-/- mice, and that Runx2 bound the promoter region of Hck. To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of Hck (HckCA) were generated using the Col2a1 promoter/enhancer. The hind limb skeletons were fused, the tibia became a large, round mass, and the growth plate was markedly disorganized. Chondrocyte maturation was delayed until E16.5 but accelerated thereafter. BrdU-labeled, but not terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, chondrocytes were increased. Furthermore, Hck knock-down reduced the proliferation of primary chondrocytes. In microarray and real-time RT-PCR analyses using hind limb RNA from HckCA transgenic mice, the expression of Wnt (Wnt10b, Tcf7, Lef1, Dkk1) and hedgehog (Ihh, Ptch1, and Gli1) signaling pathway genes was upregulated. These findings indicated that Hck, whose expression is regulated by Runx2, is highly expressed in chondrocytes, and that HckCA activates Wnt and hedgehog signaling pathways, and promotes chondrocyte proliferation without increasing apoptosis.

Suppression of hematopoietic cell kinase ameliorates the bone destruction associated with inflammation.

Objectives: To investigate the role of non-receptor tyrosine kinases (NRTKs) in inflammation-induced osteoclastogenesis.Methods: Microarray analyses of global mRNA expression during receptor activator of NF-κB ligand (RANKL) and RANKL plus tumor necrosis factor (TNF)-α-induced osteoclast differentiation were performed. The inhibitory effect on TNF-α-induced osteoclast differentiation of A-419259, a potent inhibitor of hematopoietic cell kinase (Hck), was examined. The in vivo therapeutic effect of A-419259 treatment on lipopolysaccharide (LPS)-induced inflammatory bone destruction was evaluated.Results: We confirmed that Hck expression was selectively increased among the NRTKs during the osteoclast differentiation induced by RANKL and TNF-α, but not by RANKL alone. RANKL and TNF-α-induced osteoclast differentiation and they were dose-dependently inhibited by A-419259 treatment through inhibition of the expression of key regulators of osteoclastogenesis, including Prdm1 and Nfatc1. Notably, LPS-induced inflammatory bone loss in murine calvarial bones was ameliorated by the administration of A-419259.Conclusions: Our results demonstrate that the administration of A-419259 is effective for the inhibition of osteoclast differentiation induced by TNF-α in the presence of RANKL. Therefore, an inhibitor of Hck may be useful as a potent anti-osteoclastogenic agent for the treatment of inflammatory bone destruction.

HB-EGF Improves the Hair Regenerative Potential of Adipose-Derived Stem Cells via ROS Generation and Hck Phosphorylation.

Although adipose-derived stem cells (ASCs) have hair regenerative potential, their hair inductive capabilities are limited. The mitogenic and hair inductive effects of heparin binding-epidermal growth factor-like growth factor (HB-EGF) on ASCs were investigated in this study and the underlying mechanism of stimulation was examined. Cell growth, migration, and self-renewal assays, as well as quantitative polymerase chain reactions and immunostaining, were carried out. Telogen-to-anagen transition and organ culture using vibrissa follicles were also conducted. HB-EGF significantly increased ASC motility, including cell proliferation, migration, and self-renewal activity. The preconditioning of ASCs with HB-EGF induced telogen-to-anagen transition more rapidly in vivo, and injected PKH26-ASCs survived for longer periods of time. Conditioned medium obtained from HB-EGF-treated ASCs promoted hair growth in vivo, upregulating growth factors. In particular, thrombopoietin (THPO) also induced hair growth in vivo, stimulating dermal papilla cells (DPCs). Reactive oxygen species (ROS) appeared to play a key role in ASC stimulation as the inhibition of ROS generation and NOX4 knockout attenuated ASC stimulation and THPO upregulation by HB-EGF. In addition, the Hck phosphorylation pathway mediated the stimulation of ASCs by HB-EGF. In summary, HB-EGF increased the motility and paracrine effects of ASCs releasing THPO growth factor and THPO promoted hair growth-stimulating DPCs. ROS generation and Hck phosphorylation are key factors in HB-EGF-induced ASC stimulation. Therefore, combination therapy involving HB-EGF and ASCs may provide a novel solution for hair-loss treatment.

Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication.

In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the "membranous web" structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

Phosphorylated and non-phosphorylated HCK kinase domains produced by cell-free protein expression.

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE. We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7 Šresolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping.

Genomic Analysis of Kidney Allograft Injury Identifies Hematopoietic Cell Kinase as a Key Driver of Renal Fibrosis.

Renal fibrosis is the common pathway of progression for patients with CKD and chronic renal allograft injury (CAI), but the underlying mechanisms remain obscure. We performed a meta-analysis in human kidney biopsy specimens with CAI, incorporating data available publicly and from our Genomics of Chronic Renal Allograft Rejection study. We identified an Src family tyrosine kinase, hematopoietic cell kinase (Hck), as upregulated in allografts in CAI. Querying the Kinase Inhibitor Resource database revealed that dasatinib, a Food and Drug Administration-approved drug, potently binds Hck with high selectivity. In vitro, Hck overexpression activated the TGF-ß/Smad3 pathway, whereas HCK knockdown inhibited it. Treatment of tubular cells with dasatinib reduced the expression of Col1a1 Dasatinib also reduced proliferation and α-SMA expression in fibroblasts. In a murine model with unilateral ureteric obstruction, pretreatment with dasatinib significantly reduced the upregulation of profibrotic markers, phosphorylation of Smad3, and renal fibrosis observed in kidneys pretreated with vehicle alone. Dasatinib treatment also improved renal function, reduced albuminuria, and inhibited expression of profibrotic markers in animal models with lupus nephritis and folic acid nephropathy. These data suggest that Hck is a key mediator of renal fibrosis and dasatinib could be developed as an antifibrotic drug.

Novel virtual lead identification in the discovery of hematopoietic cell kinase (HCK) inhibitors: application of 3D QSAR and molecular dynamics simulation.

High level of hematopoietic cell kinase (Hck) is associated with drug resistance in chronic myeloid leukemia. Additionally, Hck activity has also been connected with the pathogenesis of HIV-1 and chronic obstructive pulmonary disease. In this study, three-dimensional (3D) QSAR pharmacophore models were generated for Hck based on experimentally known inhibitors. A best pharmacophore model, Hypo1, was developed with high correlation coefficient (0.975), Low RMS deviation (0.60) and large cost difference (49.31), containing three ring aromatic and one hydrophobic aliphatic feature. It was further validated by the test set (r = 0.96) and Fisher's randomization method (95%). Hypo 1 was used as a 3D query for screening the chemical databases, and the hits were further screened by applying Lipinski's rule of five and ADMET properties. Selected hit compounds were subjected to molecular docking to identify binding conformations in the active site. Finally, the appropriate binding modes of final hit compounds were revealed by molecular dynamics (MD) simulations and free energy calculation studies. Hence, we propose the final three hit compounds as virtual candidates for Hck inhibitors.

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage.

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.

Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual inhibitors.

A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds with potent activities against HCK and FLT3-ITD were evaluated in viability studies with acute myeloid leukemia cell line MV4-11. Our structure activity relationship analyses lead to the identification of compound 31, which exhibited potent HCK and FLT3-ITD inhibition and activity against the MV4-11 cell line.