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.

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 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>

scholarly journals New Mutation in ParE in a Pneumococcal In Vitro Mutant Resistant to Fluoroquinolones

2001 ◽  
Vol 45 (3) ◽  
pp. 952-955 ◽  
Author(s):  
Claire Janoir ◽  
Emmanuelle Varon ◽  
Marie-Dominique Kitzis ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Topoisomerase Iv ◽  
New Mutation ◽  
Atp Binding Site

ABSTRACT For an in vitro mutant of Streptococcus pneumoniaeselected on moxifloxacin four- to eightfold-increased MICs of new fluoroquinolones, only a twofold-increased MIC of ciprofloxacin, and a twofold-decreased MIC of novobiocin were observed. This phenotype was conferred by two mutations: Ser81Phe in GyrA and a novel undescribed His103Tyr mutation in ParE, outside the quinolone resistance-determining region, in the putative ATP-binding site of topoisomerase IV.

scholarly journals Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

2005 ◽  
Vol 187 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Philippe Dupont ◽  
Alexandra Aubry ◽  
Emmanuelle Cambau ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Atp Binding Site ◽  
Open Conformation ◽  
A Subunit

ABSTRACT In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.

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 In vitro functional rescue by ivacaftor of an ABCB11 variant involved in PFIC2 and intrahepatic cholestasis of pregnancy

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Elodie Mareux ◽  
Martine Lapalus ◽  
Amel Ben-Saad ◽  
Isabelle Callebaut ◽  
Thomas Falguières ◽  
...  
Keyword(s):  
Binding Site ◽  
Intrahepatic Cholestasis ◽  
Mdck Cells ◽  
In Vitro Study ◽  
Missense Variant ◽  
Atp Binding ◽  
Intrahepatic Cholestasis Of Pregnancy ◽  
Atp Binding Site ◽  
Cholestasis Of Pregnancy

Abstract Background ABCB11 variations are responsible for a spectrum of rare liver diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) and intrahepatic cholestasis of pregnancy (ICP). Current medical treatment of these conditions mostly relies on ursodeoxycholic acid with limited efficacy. We report on the in vitro study of the p.A257V missense variant of ABCB11 identified in a PFIC2 patient and in her mother who experienced ICP. Results The Ala257 residue is located outside the ATP-binding site of ABCB11. We show that the p.A257V variant of ABCB11 is correctly expressed at the canalicular membrane of HepG2 cells but that its function significantly decreased when studied in MDCK cells. This functional defect can be fully rescued by Ivacaftor. Conclusion Ivacaftor could be considered as a new pharmacological tool able to respond to an unmet medical need for patients with ICP and PFIC2 due to ABCB11 variations affecting ABCB11 function, even when the residue involved is not located in an ATP-binding site of ABCB11.

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>

Baicalin-Induced Apoptosis is Mediated by Bcl-2-Dependent, but not p53-Dependent, Pathway in Human Leukemia Cell Lines

2006 ◽  
Vol 34 (02) ◽  
pp. 245-261 ◽  
Author(s):  
Den-En Shieh ◽  
Hua-Yew Cheng ◽  
Ming-Hong Yen ◽  
Lien-Chai Chiang ◽  
Chun-Ching Lin
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Chemotherapeutic Agents ◽  
Human Leukemia ◽  
Potential Candidate ◽  
Scutellaria Baicalensis Georgi

Acute lymphoblastic leukemia (ALL), especially T-acute lymphoblastic leukemia (T-ALL), is a common childhood malignant neoplastic disorder. Chemotherapy agents, particularly those that can induce apoptosis, are the major intervening strategy in the treatment of ALL. In this study, we investigated in T-ALL cell line, CCRF-CEM, the in vitro cytotoxic effect and the mechanism of action of baicalin, a compound extracted from Scutellaria baicalensis Georgi and S. rivularis Benth (Labiateae). Results demonstrated that baicalin displayed a remarkable cytotoxic effect in CCRF-CEM, with an IC50value of 10.6 μg/ml. It triggered apoptotic effect by fragmentizing cellular DNA and arrested the cell cycle at G0/ G1phase. Baicalin (37.5 μg/ml)had not effected the expression of p53 and Fas protein. It was shown to decline the expression of Bcl-2 (22.0 pg/ml), which consequently caused the loss (52.7%)of transmembrane potential (ΔΨm) in the mitochondria after 72 hours of treatment. Baicalin (37.5 μg/ml) also elevated the amount of cytosolic cytochrome c (19.2 μg/ml), which finally triggered the activation of caspase-3 (50.1 pmol/min). In conclusion, baicalin was found to induce apoptosis in T-ALL cell lines through multiple pathways. This finding encourages further investigation of baicalin in its role as a potential candidate for chemotherapeutic agents in T-ALL.

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