Ki23819 (KRN383•HCl) Inhibits Kinase Activity of Wild Type and Mutant FLT3 Receptor Tyrosine Kinase In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1168-1168
Author(s):  
Yukiko Komeno ◽  
Mineo Kurokawa ◽  
Yoichi Imai ◽  
Masataka Takeshita ◽  
Tomoko Matsumura ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blotting ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Assay ◽  
Wild Type ◽  
Direct Inhibition ◽  
Flt3 Inhibitor ◽  
In Vitro Kinase Assay

Abstract [Background] FLT3 is a class III receptor tyrosine kinase which is widely expressed on hematopoietic stem/progenitor cells. Two types of constitutively active FLT3 mutations have been reported to be expressed on a subset of leukemic cells; internal tandem duplications (ITD) and kinase domain mutations. The former are associated with poor prognosis in acute myeloid leukemia (AML) patients. Although several inhibitors targeting FLT3-ITD are tested in clinical trials, their cytotoxic effects are still unsatisfactory. Innate and acquired resistance is also a problem to be solved. [Purpose] To screen a novel potent FLT3 inhibitor and characterize its in vitro activity. [Materials and Methods] MOLM13 and MV4-11 cells, human leukemia cell lines expressing FLT3-ITD, were exposed to candidate compounds for 48 hours, and cytotoxic effect was assessed by colorimetric assay. Inhibitory effect on autophosphorylation was evaluated by immunoprecipitation and Western blotting. These effects were also tested in 32D cells engineered to express wild type FLT3 (FLT3-WT) or FLT3-ITD. FLT3-WT was activated with 50 ng/ml FLT3 ligand for 15 min. Proapoptotic effect was confirmed by flow cytometry with Annexin V staining. In vitro kinase assay was performed to demonstrate direct inhibition of tyrosine kinase activity of FLT3-ITD. Inhibitory effects on downstream signaling molecules, ERK and STAT5, were assessed by Western blotting. [Results] Among candidates for VEGFR inhibitors from a library, a quinoline-urea derivative Ki23819 (KRN383•HCl) was identified to specifically inhibit proliferation and induce apoptosis to MOLM13 and MV4-11 cells. Ki23819 inhibited proliferation of MV4-11 cells more effectively than SU11248, a precedent FLT3 inhibitor (IC50 <1 nM vs 3~10 nM). Similar results were obtained when MOLM13 cells were used. Ki23819 inhibited autophosphorylation of both ligand-activated FLT3-WT and FLT3-ITD (IC50 30 nM and 3 nM, respectively), and abrogated IL-3-independent proliferation of 32D cells expressing FLT3-ITD (IC50 3~10 nM). In vitro kinase assay demonstrated direct inhibition of kinase activity of FLT3-ITD (IC50 7.8 nM). This compound also inhibited ERK and STAT5 constitutively activated by FLT3-ITD. The IC50 for inhibition of phosphorylation in 32D FLT3-ITD cells was 3 nM for both proteins, which is equivalent to that for inhibition of FLT3-ITD autophosphorylation. [Conclusion] Ki23819 is a novel and potent candidate for antileukemic agents against FLT3-ITD positive AML. In vivo activity of KRN383, the free base of Ki23819, is also to be reported in this ASH meeting (Nishiyama et al.).

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

scholarly journals Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

2005 ◽  
Vol 10 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Helmut Mett ◽  
Kerstin Hölscher ◽  
Heidrun Degen ◽  
Christina Esdar ◽  
Birgit Felden De Neumann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Cell Activity ◽  
Kinase Assay ◽  
Activity Assay ◽  
Drug Candidates ◽  
Cellular Context ◽  
In Vitro Kinase Assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 μM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. ( Journal of Biomolecular Screening 2005:36-45)

Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3360-3360
Author(s):  
Daisuke Okamura ◽  
Fumiharu Yagasaki ◽  
Tomoya Maeda ◽  
Maho Ishikawa ◽  
Itsuro Jinnai ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Myeloma Cell ◽  
Kinase Assay ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Tyrosine Kinase 2

Abstract Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.

SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2941-2949 ◽  
Author(s):  
Kevin W. H. Yee ◽  
Anne Marie O'Farrell ◽  
Beverly D. Smolich ◽  
Julie M. Cherrington ◽  
Gerald McMahon ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Leukemic Cell ◽  
Mapk Signaling ◽  
Molecular Defect ◽  
Wild Type ◽  
Type Iii ◽  
Leukemic Cell Lines

Internal tandem duplication (ITD) in the juxtamembrane portion of Fms-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase (RTK), is the most common molecular defect associated with acute myeloid leukemia (AML). The high prevalence of this activating mutation makes it a potential target for molecularly based therapy. Indolinone tyrosine kinase inhibitors have known activity against KIT, another member of the type III RTK family. Given the conserved homology between members of this family, we postulated that the activity of some KIT inhibitors would extend to FLT3. We used various leukemic cell lines (BaF3, MV 4-11, RS 4;11) to test the activity of indolinone compounds against the FLT3 kinase activity of both wild-type (WT) and ITD isoforms. Both SU5416 and SU5614 were capable of inhibiting autophosphorylation of ITD and WT FLT3 (SU5416 concentration that inhibits 50% [IC50], 100 nM; and SU5614 IC50 10 nM). FLT3-dependent activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) was also inhibited by treatment in the same concentration ranges. FLT3 inhibition by SU5416 and SU5614 resulted in reduced proliferation (IC50, 250 nM and 100 nM, respectively) and induction of apoptosis of FLT3 ITD-positive leukemic cell lines. Treatment of these cells with an alternative growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF]) restored MAPK signaling and cellular proliferation, demonstrating specificity of the observed inhibitory effects. We conclude that SU5416 and SU5614 are potent inhibitors of FLT3. Our finding that inhibition of FLT3 induces apoptosis of leukemic cells supports the feasibility of targeting FLT3 as a novel treatment strategy for AML.

scholarly journals The effect of fasting on the activation in vivo of the insulin receptor kinase

1990 ◽  
Vol 265 (3) ◽  
pp. 887-890 ◽  
Author(s):  
I Contreras ◽  
G L Dohm ◽  
S Abdallah ◽  
J A Wells ◽  
N Mooney ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Serum Glucose ◽  
Tyrosine Kinase Activity ◽  
Insulin Receptor Tyrosine Kinase ◽  
And Control

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.

scholarly journals Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503 ◽  
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

The Bcr-Abl SH2-Kinase Domain Interface Is Critical for Leukemogenesis and An Additional Therapeutic Target in CML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 37-37
Author(s):  
Oliver D. Hantschel ◽  
Florian Grebien ◽  
Ines Kaupe ◽  
Boris Kovacic ◽  
John Wojcik ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Critical Factor ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Wild Type ◽  
Tyrosine Kinase Activity ◽  
Downstream Signaling

Abstract Abstract 37 We previously showed that the Abl SH2 domain is an allosteric activator of c-Abl tyrosine kinase activity and substrate phosphorylation (Filippakopoulos et al. (2008) Cell 134(5), 793-803). This effect is exerted directly by docking of the SH2 domain onto the N-lobe of the kinase domain in the active conformation of c-Abl. We also showed that the same structural mechanism is a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. Disruption of binding of the SH2 domain to the kinase domain in Bcr-Abl by the Ile164Glu mutation in the SH2 domain, led to a strong reduction in in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation. Unexpectedly, we observed a differential attenuation of downstream signaling pathways upon disruption of the SH2-kinase domain interface, indicating different activation thresholds of Bcr-Abl downstream signaling pathways. Here, we show that disrupting the SH2-kinase domain interface abrogates the transforming capacity of Bcr-Abl. Cells expressing the Bcr-Abl Ile164Glu mutant were unable to generate cytokine-independent colonies in vitro. Furthermore, mice transplanted with Bcr-Abl Ile164Glu expressing bone marrow cells did not develop the characteristic MPD-like disease that is caused by wild-type Bcr-Abl. Mice that received Bcr-Abl Ile164Glu cells showed normal survival, blood counts and histology after more than 100 days post-transplant, despite the presence of Bcr-Abl Ile164Glu-expressing cells in all blood lineages. This shows that the formation of the SH2-kinase domain interface is strictly necessary for Bcr-Abl to cause CML. Together with our data that show sensitization to imatinib inhibition of Bcr-Abl Ile164Glu as compared to Bcr-Abl wild-type, this argues for the SH2-kinase domain interface as an additional drug target on Bcr-Abl that may synergize with tyrosine kinase inhibitors and may be useful to inhibit tyrosine kinase inhibitor resistant Bcr-Abl clones. To address possibilities to interfere with the SH2-kinase domain interface, we are using an engineered binding protein that binds to the Abl SH2 domain with high-affinity and specificity and supposedly disrupts the interface with the kinase domain, resulting in a decrease in Bcr-Abl kinase activity. In conclusion, we provide strong evidence that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and leukemogenicity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr-Abl. Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia. Disclosures: No relevant conflicts of interest to declare.

Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

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