scholarly journals An innovative cell model revealed the inhibitory effect of flavanone structure on peroxynitrite production through interaction with the IKKβ kinase domain at ATP binding site

2020 ◽  
Vol 8 (6) ◽  
pp. 2904-2912
Author(s):  
Supochana Charoensin ◽  
Tzou‐Chi Huang ◽  
Jue‐Liang Hsu
Keyword(s):  
Binding Site ◽  
Cell Model ◽  
Inhibitory Effect ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals An Activating Mutation in the ATP Binding Site of the ABL Kinase Domain

1996 ◽  
Vol 271 (32) ◽  
pp. 19585-19591 ◽  
Author(s):  
Patrick B. Allen ◽  
Leanne M. Wiedemann
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Abl Kinase ◽  
Activating Mutation

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 <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 A conserved mechanism of autoinhibition for the AMPK kinase domain: ATP-binding site and catalytic loop refolding as a means of regulation

2010 ◽  
Vol 66 (2) ◽  
pp. 143-151 ◽  
Author(s):  
Dene R. Littler ◽  
John R. Walker ◽  
Tara Davis ◽  
Leanne E. Wybenga-Groot ◽  
Patrick J. Finerty ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Catalytic Loop

scholarly journals Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

2016 ◽  
Vol 72 (5) ◽  
pp. 682-693 ◽  
Author(s):  
Hongming Chen ◽  
Julie Tucker ◽  
Xiaotao Wang ◽  
Paul R. Gavine ◽  
Chris Phillips ◽  
...  
Keyword(s):  
Binding Site ◽  
Tyrosine Kinases ◽  
Map Kinases ◽  
Complex Structure ◽  
Kinase Domain ◽  
Atp Binding ◽  
Regulation Of Transcription ◽  
Allosteric Site ◽  
Atp Binding Site ◽  
Allosteric Inhibitor

MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

Mitoxantrone Targets the ATP-binding Site of FAK, Binds the FAK Kinase Domain and Decreases FAK, Pyk-2, c-Src, and IGF-1R In Vitro Kinase Activities

2013 ◽  
Vol 13 (4) ◽  
pp. 546-554 ◽  
Author(s):  
Vita M. Golubovskaya ◽  
Baotran Ho ◽  
Min Zheng ◽  
Andrew Magis ◽  
David Ostrov ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals Acquired Mutations within the JAK2 Kinase Domain Confer Resistance to JAK Inhibitors in an in Vitro model of a High-Risk Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Charlotte EJ Downes ◽  
Barbara J McClure ◽  
Jacqueline Rehn ◽  
James Breen ◽  
John B Bruning ◽  
...  
Keyword(s):  
High Risk ◽  
Binding Site ◽  
Lymphoblastic Leukemia ◽  
Computational Modelling ◽  
Kinase Domain ◽  
Atp Binding ◽  
Type I ◽  
Type Ii ◽  
Jak Inhibitors ◽  
Atp Binding Site

Introduction Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL associated with high relapse rates and poor survival. Rearrangements of Janus kinase 2 (JAK2r) are present in approximately 5% and 14% of pediatric and young adult Ph-like ALL cases respectively. The resultant JAK2 gene fusions drive leukemogenesis through constitutive activation of the JAK/STAT signaling pathway and are associated with very poor outcomes in patients with Ph-like ALL. All JAK inhibitors in clinical development are type I inhibitors, which bind in the ATP-binding site of JAK2. A phase II clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-cell ALL cases harboring JAK2 alterations. The development of treatment resistance to targeted inhibitors in other diseases is well documented and often results in disease relapse. Elucidating mechanisms of ruxolitinib resistance in JAK2r ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of therapeutic strategies to overcome or avert resistance. Methods JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 μM ruxolitinib. Sanger sequencing of the RT-PCR amplified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V, and analysis by flow cytometry. Alterations in signaling pathways were determined using phosphoflow cytometry and western blot analysis. Computational modelling of acquired JAK2 mutations and subsequent influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Results In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localized to the ATP/ruxolitinib binding site and conferred resistance to multiple type-I JAK inhibitors, including ruxolitinib, BMS-911543, and AZD-1480 (Table 1). JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868, which binds in an allosteric site of JAK2 in addition to the ATP-binding site. Ruxolitinib resistance correlated with sustained downstream STAT5 activation in the presence of 1 μM ruxolitinib compared with non-mutant ATF7IP-JAK2 Ba/F3 cells. Intracellular phosphoflow cytometry of ruxolitinib-resistant ATF7IP-JAK2 Ba/F3 cells confirmed constitutive activation of JAK/STAT signaling in the presence of 50 nM ruxolitinib, in contrast to non-mutant ATF7IP-JAK2 Ba/F3 cells. Computational modelling suggested that JAK2 p.L983F (Fig. 1D) sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond (Fig. 1B). The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilizing the JAK2 activation loop (Fig1C). Conclusions This study demonstrates that the JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure in the setting of JAK2r ALL, highlighting the importance of monitoring the emergence of mutations within this region. In addition to previously described mutations we identified a novel JAK2 p.G993A mutation that conferred resistance to both type-I and type-II JAK inhibitors. The JAK2 p.G993A mutation was postulated to modulate the stability of a conserved domain. Understanding mechanisms of ruxolitinib resistance, as modelled here, has the potential to inform future drug design and the development therapeutic strategies for this high-risk cohort. Disclosures White: Amgen: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding.

scholarly journals The ATP-binding Site in the 2-Kinase Domain of Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

1996 ◽  
Vol 271 (30) ◽  
pp. 17875-17880 ◽  
Author(s):  
Didier Vertommen ◽  
Luc Bertrand ◽  
Bruno Sontag ◽  
Attilio Di Pietro ◽  
Marc P. Louckx ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site

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 Identification of the ATP binding site in tyrocidine synthetase 1 by selective modification with fluorescein 5'-isothiocyanate.

1994 ◽  
Vol 269 (21) ◽  
pp. 14962-14966
Author(s):  
M. Pavela-Vrancic ◽  
E. Pfeifer ◽  
W. Schröder ◽  
H. von Döhren ◽  
H. Kleinkauf
Keyword(s):  
Binding Site ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

2021 ◽  
Author(s):  
Afsar Ali Mian ◽  
Isabella Haberbosch ◽  
Hazem Khamaisie ◽  
Abed Agbarya ◽  
Larissa Pietsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Binding Pocket ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

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