The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3544-3544 ◽  
Author(s):  
Daniel B. Lipka ◽  
Linda S. Hoffmann ◽  
Florian Heidel ◽  
Boyka Markova ◽  
Stefan Kasper ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Erythropoietin Receptor ◽  
Kinase Assay ◽  
Atp Binding ◽  
Jak Inhibitor ◽  
Atp Binding Site ◽  
V617f Mutation

Abstract The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was <5μM, and this effect was not reversible using increased ATP-doses. Combination treatment of Ba/F3-EpoR-VF cells using LS104 plus JAK-inhibitor I lead to significantly increased apoptosis as compared to each substance alone. Furthermore, we observed 89% inhibition of in vitro formation of EECs at 10μM LS104 whereas growth of CFU-GM obtained from normal controls was virtually unaffected. Taken together, our data show that LS104 specifically inhibits JAK2 kinase activity and JAK2 downstream signals and thereby induces apoptosis in V617F-positive cells. Our data suggest, that LS104 either irreversibly binds to the ATP-binding site or acts as a substrate kinase inhibitor and may be combined with ATP-competitive JAK2 inhibitors to enhance treatment efficacy. Growth of EECs from patients with MPD is shown to be significantly suppressed by LS104. Based on these data, a phase I/II clinical trial of LS104 for patients with JAK2V617F-positive MPD has been initiated recently.

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

scholarly journals A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779 ◽  
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

2019 ◽  
Vol 19 (11) ◽  
pp. 914-926 ◽  
Author(s):  
Maiara Bernardes Marques ◽  
Michael González-Durruthy ◽  
Bruna Félix da Silva Nornberg ◽  
Bruno Rodrigues Oliveira ◽  
Daniela Volcan Almeida ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Binding Site ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Atp Binding ◽  
Human Leukemia ◽  
Atp Binding Site ◽  
Mechanistic Insight ◽  
Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

scholarly journals Structural and Biochemical Studies Confirming the Mechanism of Action of Asciminib, an Agent Specifically Targeting the ABL Myristoyl Pocket (STAMP)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Peter Schuld ◽  
Stephan Grzesiek ◽  
Johannes Schlotte ◽  
Judith M Habazettl ◽  
Wolfgang Jahnke ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Tumor Regression ◽  
Isothermal Calorimetry ◽  
Kinase Domain ◽  
Ternary Complexes ◽  
Kinase Assay ◽  
Atp Binding ◽  
Publicly Traded ◽  
Atp Binding Site

Tyrosine kinase inhibitors (TKIs) that inhibit the transphosphorylation activity of the BCR-ABL1 oncoprotein by binding the ATP-binding site of the catalytic domain of protein kinases are well established as being effective drugs for the treatments of chronic myeloid leukemia (CML). However, the off-target kinase activities of these non-specific TKIs are associated with adverse events that can limit their suitability for the treatment of patients and can negatively impact quality of life. Therefore, a new drug combining high efficacy with minimal side-effects could provide substantial therapeutic advantages. Asciminib is a new investigational agent that at concentrations &lt;10 micromolar has very little effect (residual activities ≥66%) on the transphosphorylation activity of a large panel comprising of 335wild-typeprotein kinases (including ABL1 and ABL2). In this study (33PanQinase® Activity Assay, ProQinase GmbH, Freiburg, Germany) the radiometric kinase assay employed recombinant kinase constructs of the catalytic domains including the ATP-binding site. However, in isothermal calorimetry studies asciminib showed strong binding affinity (KD 0.5 nM) to a much larger construct ofwild-typeABL1 that contains the catalytic, SH2 and SH3 domains (residues 46-534, ABL46-534), with thermodynamic parameters (ΔH = -72.8 kJ/mol, ΔS = -65.3 J/mol/K, resulting in ΔG = -53.2 kJ/mol) indicating strong enthalpy-driven, entropically unfavorable binding. This binding translates to inhibition of tyrosine phosphorylation catalysed by the ABL64-515 construct with a mean IC50 value of 2.6 ± 0.8 nM (radiometric filter binding assay) and 0.5 ± 0.1 nM (fluorescence resonance energy transfer assay). This data shows that asciminib inhibits the kinase activity of ABL1 by an allosteric mechanism which does not involve direct interaction with the ATP-binding pocket. X-ray crystallographic studies of a ternary complex between asciminib, nilotinib and the ABL146-534 protein possessing Thr315Ile and Asp382Asn substitutions show that asciminib binds to ABL1 in a deep-pocket on the C-lobe of the kinase domain, referred to as the myristate (or myristoyl) pocket. Nuclear magnetic resonance studies confirm that asciminib can also form asciminib-ABL1-TKI ternary complexes with thewild-typeABL83-534 protein in solution. Native ABL1 kinase is post-translationally myristoylated at the N-terminal glycine residue and this myristate group plays an important role in autoregulating the kinase by binding to a pocket (myristate pocket) on the catalytic domain of the protein to induce the formation of an inactive conformation of the enzyme. This regulatory mechanism is lost in BCR-ABL1 since the N-terminal region is replaced in the fusion protein with a fragment of the BCR protein, thus rendering the ABL1 kinase constitutively active. The binding of asciminib in this pocket therefore mimics that of myristate, thus stabilizing the inactive state of the ABL1 kinase. Consistent with this binding mode to the ABL1 kinase, asciminib potently inhibits BCR-ABL1 driven proliferation of leukemia-derived cell lines, while having little effect on cells that do not express BCR-ABL1 (Figure 1). Thein vitrodata translates into anti-tumor activity in mouse models of CML where asciminib dose-dependently inhibited the growth of subcutaneous KCL22 cell xenografts, with 30 mg/kg administered twice-daily by oral gavage affording 92% tumor regression (Figure 2). Asciminib, the first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, has several important features as a potential treatment of CML. At physiologically achievable concentrations asciminib can overcome mutations on the ATP-binding site of BCR-ABL1 that impede the binding of TKIs which lead to drug resistance in patients with CML. Secondly, by not inhibiting kinases such as EGFR, KIT, CSF1R, PDGFR or the sSRC-family kinases that are associated with off-target activities of TKIs such as bosutinib, dasatinib, imatinib, nilotinib and ponatinib, asciminib is not expected to be associated with cross-intolerance. Thirdly, asciminib can bind to the ABL1 kinase domain together with ATP-competitive TKIs to form ternary complexes, such that appropriate drug combinations should greatly impede the emergence of drug resistant kinase mutations. Disclosures Schuld: Novartis Pharma AG:Current Employment, Current equity holder in publicly-traded company.Grzesiek:Novartis Pharma:Research Funding.Jahnke:Novartis:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.Barys:Novartis Pharma AG:Current Employment.Cowan-Jacob:Novartis Pharma AG:Current Employment.Loo:Novartis Pharma AG:Current Employment.Wiget:Novartis Pharma AG:Current Employment.Manley:Novartis Pharma AG:Current Employment.

scholarly journals Phosphorylation at Ser26 in the ATP-binding site of Ca2+/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Mehtap Yilmaz ◽  
Samudra S. Gangopadhyay ◽  
Paul Leavis ◽  
Zenon Grabarek ◽  
Kathleen G. Morgan
Keyword(s):  
Catalytic Activity ◽  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Physiological Importance ◽  
Sustained Activity ◽  
Exogenous Substrate ◽  
Autophosphorylation Site

CaMKII (Ca2+/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser26, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser26, we generated a phosphospecific Ser26 antibody and demonstrated an increase in Ser26 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser26 affects the kinase activity, we mutated Ser26 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr287 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser26 of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr287 most probably by blocking ATP binding. We propose that Ser26 phosphorylation constitutes an important mechanism for switching off CaMKII activity.

scholarly journals 1_manuscript_2020-04-14.pdf

2020 ◽  
Author(s):  
Linglan Fang ◽  
Jessica Vilas-Boas ◽  
sujata chakraborty ◽  
zachary potter ◽  
Ames Register ◽  
...  
Keyword(s):  
Binding Site ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Major Influence ◽  
Atp Binding ◽  
Structural Element ◽  
Atp Binding Site ◽  
Regulatory Architecture

<p>Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the glob-al conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix aC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix aC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix aC can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.</p>

scholarly journals How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture

2020 ◽  
Author(s):  
Linglan Fang ◽  
Jessica Vilas-Boas ◽  
sujata chakraborty ◽  
zachary potter ◽  
Ames Register ◽  
...  
Keyword(s):  
Binding Site ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Major Influence ◽  
Atp Binding ◽  
Structural Element ◽  
Atp Binding Site ◽  
Regulatory Architecture

<p>Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the glob-al conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix aC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix aC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix aC can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.</p>

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.

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