81 results on '"atp binding site"'
Search Results
2. The chemical diversity and structure-based discovery of allosteric modulators for the PIF-pocket of protein kinase PDK1
- Author
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Xinyuan Xu, Yingyi Chen, Qiang Fu, Duan Ni, Jian Zhang, Xiaolong Li, and Shaoyong Lu
- Subjects
pi3k ,pdk1 ,allosteric modulators ,allostery ,orthosteric ligands ,atp binding site ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Phosphoinositide-dependent protein kinase-1 (PDK1) is an important protein in mediating the PI3K-AKT pathway and is thus identified as a promising target. The catalytic activity of PDK1 is tightly regulated by allosteric modulators, which bind to the PDK1 Interacting Fragment (PIF) pocket of the kinase domain that is topographically distinct from the orthosteric, ATP binding site. Allosteric modulators by attaching to the less conserved PIF-pocket have remarkable advantages such as higher selectivity, less side effect, and lower toxicity. Targeting allosteric PIF-pocket of PDK1 has become the focus of recent attention. In this review, we summarise the current advances in the structure-based discovery of PDK1 allosteric modulators. We will first present the three-dimensional structure of PDK1 and illustrate the allosteric regulatory mechanism of PDK1 through the modulation of the PIF-pocket. Then, the recent advances of PDK1 allosteric modulators targeting the PIF-pocket will be recapitulated detailly according to the structural similarity of allosteric modulators.
- Published
- 2019
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- View/download PDF
3. On the origins of enzyme inhibitor selectivity and promiscuity : a case study of protein kinase binding to staurosporine
- Author
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Tanramluk, Duangrudee and Blundell, Tom
- Subjects
615.19 ,Kinase ,Staurosporine ,ATP binding site ,Structural analysis ,Selectivity ,Promiscuity ,Inhibitor design ,Shape comparison - Abstract
Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.
- Published
- 2010
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4. The chemical diversity and structure-based discovery of allosteric modulators for the PIF-pocket of protein kinase PDK1.
- Author
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Xu, Xinyuan, Chen, Yingyi, Fu, Qiang, Ni, Duan, Zhang, Jian, Li, Xiaolong, and Lu, Shaoyong
- Subjects
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PROTEIN kinases , *PHOSPHOINOSITIDE-dependent kinase-1 , *ALLOSTERIC regulation , *BINDING sites , *MUSCARINIC acetylcholine receptors , *CATALYTIC activity - Abstract
Phosphoinositide-dependent protein kinase-1 (PDK1) is an important protein in mediating the PI3K-AKT pathway and is thus identified as a promising target. The catalytic activity of PDK1 is tightly regulated by allosteric modulators, which bind to the PDK1 Interacting Fragment (PIF) pocket of the kinase domain that is topographically distinct from the orthosteric, ATP binding site. Allosteric modulators by attaching to the less conserved PIF-pocket have remarkable advantages such as higher selectivity, less side effect, and lower toxicity. Targeting allosteric PIF-pocket of PDK1 has become the focus of recent attention. In this review, we summarise the current advances in the structure-based discovery of PDK1 allosteric modulators. We will first present the three-dimensional structure of PDK1 and illustrate the allosteric regulatory mechanism of PDK1 through the modulation of the PIF-pocket. Then, the recent advances of PDK1 allosteric modulators targeting the PIF-pocket will be recapitulated detailly according to the structural similarity of allosteric modulators. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
5. Cholate Disrupts Regulatory Functions of Cytochrome c Oxidase
- Author
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Rabia Ramzan, Jörg Napiwotzki, Petra Weber, Bernhard Kadenbach, and Sebastian Vogt
- Subjects
cholat ,cytochrome c oxidase ,ATP binding site ,regulatory function ,allosteric ATP-Inhibition ,Cytology ,QH573-671 - Abstract
Cytochrome c oxidase (CytOx), the oxygen-accepting and rate-limiting enzyme of mitochondrial respiration, binds with 10 molecules of ADP, 7 of which are exchanged by ATP at high ATP/ADP-ratios. These bound ATP and ADP can be exchanged by cholate, which is generally used for the purification of CytOx. Many crystal structures of isolated CytOx were performed with the enzyme isolated from mitochondria using sodium cholate as a detergent. Cholate, however, dimerizes the enzyme isolated in non-ionic detergents and induces a structural change as evident from a spectral change. Consequently, it turns off the “allosteric ATP-inhibition of CytOx”, which is reversibly switched on under relaxed conditions via cAMP-dependent phosphorylation and keeps the membrane potential and ROS formation in mitochondria at low levels. This cholate effect gives an insight into the structural-functional relationship of the enzyme with respect to ATP inhibition and its role in mitochondrial respiration and energy production.
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- 2021
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6. Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation
- Author
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Dimitrios T. Trafalis, Sofia Sagredou, Panayiotis Dalezis, Maria Voura, Stella Fountoulaki, Nikolaos Nikoleousakos, Konstantinos Almpanakis, Maria V. Deligiorgi, and Vasiliki Sarli
- Subjects
1,2,4-triazolo[3,4-b]-1,2,4-thiadiazole ,inhibitor of Akt phosphorylation ,anticancer ,MTT assay ,HT-29 human colon tumor xenograft ,ATP binding site ,Pharmacy and materia medica ,RS1-441 - Abstract
The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer properties in vitro against a panel of cancer cells and in vivo efficacy in HT-29 human colon tumor xenograft in CB17 severe combined immunodeficient (SCID) mice. Preliminary mechanistic studies revealed that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising cancer therapeutic agents.
- Published
- 2021
- Full Text
- View/download PDF
7. Linker-switch approach towards new ATP binding site inhibitors of DNA gyrase B.
- Author
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Jukič, Marko, Ilaš, Janez, Brvar, Matjaž, Kikelj, Danijel, Cesar, Jožko, and Anderluh, Marko
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BINDING sites , *DNA topoisomerase II , *ADENOSINE triphosphate , *BIOCHEMICAL mechanism of action , *MOLECULAR docking - Abstract
Due to increasing emergence of bacterial resistance, compounds with new mechanisms of action are of paramount importance. One of modestly researched therapeutic targets in the field of antibacterial discovery is DNA gyrase B. In the present work we synthesized a focused library of potential DNA gyrase B inhibitors composed of two key pharmacophoric moieties linked by three types of sp 3 -rich linkers to obtain three structural classes of compounds. Using molecular docking, molecular dynamics and analysis of conserved waters in the binding site, we identified a favourable binding mode for piperidin-4-yl and 4-cyclohexyl pyrrole-2-carboxamides while predicting unfavourable interactions with the active site for piperazine pyrrole-2-carboxamides. Biological evaluation of prepared compounds on isolated enzyme DNA gyrase B confirmed our predictions and afforded multiple moderately potent inhibitors of DNA gyrase B. Namely trans -4-(4,5-dibromo-1 H -pyrrole-2-carboxamide)cyclohexyl)glycine and 4-(4-(3,4-dichloro-5-methyl-1 H -pyrrole-2-carboxamido)piperidin-1-yl)-4-oxobutanoic acid with an IC 50 value of 16 and 0.5 μM respectively. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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8. Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit
- Author
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Kuznetsov Aleksei and Järv Jaak
- Subjects
single-subunit allostery ,allosteric cooperativity ,enzyme kinetics ,enzyme inhibition ,ligand binding ,interaction factor ,peptide phosphorylation ,camp-dependent protein kinase catalytic subunit ,atp binding site ,peptide binding site ,Biology (General) ,QH301-705.5 - Published
- 2009
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9. Biophysical changes of ATP binding pocket may explain loss of kinase activity in mutant DAPK3 in cancer: A molecular dynamic simulation analysis.
- Author
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Agarwal, Tarun, Annamalai, Nithyanan, Maiti, Tapas Kumar, and Arsad, Hasni
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ATP-binding cassette transporters , *BIOPHYSICS , *PROTEIN kinases , *DISEASE progression , *TUMOR suppressor genes - Abstract
DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42–Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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10. On the ATP binding site of the ε subunit from bacterial F-type ATP synthases.
- Author
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Krah, Alexander and Takada, Shoji
- Subjects
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ADENOSINE triphosphate , *BINDING sites , *ADENOSINE triphosphatase , *ELECTROCHEMICAL analysis , *CONFORMATIONAL analysis - Abstract
F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg 2+ ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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11. Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90.
- Author
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Singh, Salam Pradeep, Deb, Chitta Ranjan, Ahmed, Sharif Udin, Saratchandra, Yenisetti, and Konwar, Bolin Kumar
- Subjects
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MOLECULAR docking , *FLAVONOLS , *HEAT shock proteins - Abstract
Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
12. Expression, purification and structural characterization of the type 1-specific ATP binding site of IP3 receptor (IP3R1-ATPA).
- Author
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Kim, Ha-Neul, Seok, Seung-Hyeon, Chung, Ka Young, Won, Hyung-Sik, Son, Woo Sung, and Seo, Min-Duk
- Subjects
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INOSITOL trisphosphate receptors , *ENDOPLASMIC reticulum , *CALCIUM channels regulation , *HOMEOSTASIS , *HEAT stability in proteins , *CIRCULAR dichroism , *BINDING sites - Abstract
The inositol 1,4,5-triphosphate receptor (IP 3 R), an IP 3 -gated Ca 2+ release channel on the endoplasmic reticulum (ER) membrane, plays a critical role in maintaining cytosolic Ca 2+ homeostasis in cells. Particularly, ATP increases IP 3 R activity by binding to ATPA, a putative glycine-rich Walker A-type motif (GXGXXG) specific to type 1 IP 3 R (IP 3 R1). Here, we established an efficient process to produce the ATPA containing domain of IP 3 R1 (IP 3 R1-ATPA) using a chaperone co-expression system in Escherichia coli . The recombinant protein was well expressed as a soluble form and showed a high thermostability. Circular dichroism results indicated a mainly α-helical conformation of the purified protein. Additionally, model structures of IP 3 R1-ATPA were calculated and validated using different modeling algorithms. The structural models of IP 3 R1-ATPA not only supported the observed high thermostability, but also suggested a potential ATP binding site. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
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13. Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation
- Author
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Stella Fountoulaki, Maria V Deligiorgi, Konstantinos Almpanakis, Sofia Sagredou, Dimitrios T Trafalis, Maria Voura, Panayiotis Dalezis, Vasiliki Sarli, and Nikolaos Nikoleousakos
- Subjects
lcsh:RS1-441 ,Pharmaceutical Science ,AKT2 ,Pharmacology ,anticancer ,Article ,lcsh:Pharmacy and materia medica ,03 medical and health sciences ,inhibitor of Akt phosphorylation ,0302 clinical medicine ,Thiadiazoles ,In vivo ,MTT assay ,Protein kinase B ,030304 developmental biology ,1,2,4-triazolo[3,4-b]-1,2,4-thiadiazole ,0303 health sciences ,Chemistry ,ATP binding site ,In vitro ,HT-29 human colon tumor xenograft ,030220 oncology & carcinogenesis ,Cancer cell ,Phosphorylation - Abstract
The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer properties in vitro against a panel of cancer cells and in vivo efficacy in HT-29 human colon tumor xenograft in CB17 severe combined immunodeficient (SCID) mice. Preliminary mechanistic studies revealed that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising cancer therapeutic agents.
- Published
- 2021
14. Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate analogues as a new scaffold for protein kinase casein kinase 2 inhibitor.
- Author
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Jin, Cheng-Hao, Jun, Kyu-Yeon, Lee, Eunjung, Kim, Seongrak, Kwon, Youngjoo, Kim, Kunhong, and Na, Younghwa
- Subjects
- *
CARBOXYLATES , *GROWTH factors , *SERINE/THREONINE kinases , *ENZYME inhibitors , *GENE expression , *CANCER cells - Abstract
Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/threonine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000 chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased) and performed in vitro kinase assay-based screening using hits found from the first screening. We identified compound 24 (C 24 )[( Z )-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-oxo-3,5-dihydro-2 H -thiazolo[3,2- a ] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half maximal inhibitory concentration (IC 50 ) = 0.56 μM] inhibited PKCK2 2.2-fold more efficiently than did TBB (IC 50 = 1.24 μM), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31 human protein kinases. The K i values of compound 24 and TBB for PKCK2 were 0.78 μM and 2.70 μM, respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular docking of compound 24 bound to PKCK2α versus previously reported complexes of PKCK2 with other inhibitors, our findings suggest a new scaffold for specific PKCK2α inhibitors. Thus, compound 24 appears to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
15. ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.
- Author
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Blayney, Lynda, Beck, Konrad, MacDonald, Ewan, D'Cruz, Leon, Nomikos, Michail, Griffiths, Julia, Thanassoulas, Angelos, Nounesis, George, and Lai, F. Anthony
- Subjects
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ADENOSINE triphosphate , *PROTEIN-protein interactions , *CATECHOLAMINES , *VENTRICULAR tachycardia , *GENETIC mutation , *RYANODINE receptors , *CARRIER proteins - Abstract
Abstract: Background: This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Methods: Wild-type (WT) RyR2 central domain constructs (G2236to G2491) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. Results: The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200–400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. Conclusions: The RyR2 central domain, expressed as a ‘correctly’ folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. General significance: Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. [Copyright &y& Elsevier]
- Published
- 2013
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16. Discovery of the first inhibitors of bacterial enzyme d-aspartate ligase from Enterococcus faecium (Aslfm).
- Author
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Škedelj, Veronika, Perdih, Andrej, Brvar, Matjaž, Kroflič, Ana, Dubbée, Vincent, Savage, Victoria, O'Neill, Alex J., Solmajer, Tom, Bešter-Rogač, Marija, Blanot, Didier, Hugonnet, Jean-Emmanuel, Magnet, Sophie, Arthur, Michel, Mainardi, Jean-Luc, Stojan, Jure, and Zega, Anamarija
- Subjects
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LIGASES , *ENZYME inhibitors , *ENTEROCOCCUS faecium , *ANTIBACTERIAL agents , *MULTIDRUG resistance , *CONFORMATIONAL analysis , *OXAZOLES , *ISOTHERMAL titration calorimetry - Abstract
Abstract: The d-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a K i value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with K i values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium. [Copyright &y& Elsevier]
- Published
- 2013
- Full Text
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17. Heat shock protein 60 of filarial parasite Brugia malayi: cDNA cloning, expression, purification and in silico modeling and analysis of its ATP binding site
- Author
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Misra, R.C., Verma, A.K., Verma, S.K., Kumar, V., Siddiqui, W.A., Siddiqi, M.I., and Murthy, P.K.
- Subjects
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HEAT shock proteins , *FILARIAL worms , *BRUGIA malayi , *ANTISENSE DNA , *ADENOSINE triphosphate , *BINDING sites , *MOLECULAR cloning , *AFFINITY chromatography - Abstract
Abstract: We report here cloning and expression of full length mitochondrial HSP60 gene of Brugia malayi adult worm (mtHSP60bm), purification of the gene product by affinity chromatography, its in silico 3D structure and the sequence homology of the protein with Escherichia coli GroEL/ES and human HSP60. The ATP binding pocket of human HSP60 and mtHSP60bm were analyzed and compared using in silico models. The distribution of HSP60 in different life-stages of the parasite was determined using antibodies raised against recombinant mtHSP60bm (rmtHSP60bm). mtHSP60bm was present in all life-stages of the parasite except third stage infective larvae, in which it could be induced by heat-shock, and showed high degree of homology with E. coli GroEL/ES. The ATP binding pocket of HSP60 in humans, E. coli and B. malayi were also found structurally conserved. This similarity between human and mtHSP60bm might be useful in understanding the host-parasite interactions. This is the first ever report on distribution, cloning, sequence homology and ATP binding site of mtHSP60bm. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
18. The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor.
- Author
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Kostenko, Sergiy, Khan, Mahmud, Sylte, Ingebrigt, and Moens, Ugo
- Subjects
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DITERPENES , *ALKALOIDS , *MITOGEN-activated protein kinases , *ENZYME inhibitors , *ATP-binding cassette transporters , *BINDING sites , *ENZYME kinetics - Abstract
The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC = 37.5 μM; K = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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19. Analysis of c-Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of ATP-Competitive Ligands.
- Author
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Asses, Yasmine, Leroux, Vincent, Tairi-Kellou, Safia, Dono, Rosanna, Maina, Flavio, and Maigret, Bernard
- Subjects
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PROTEIN-tyrosine kinases , *PROTEIN binding , *ADENOSINE triphosphate , *LIGANDS (Biochemistry) , *ALLOSTERIC proteins , *ALLOSTERIC regulation - Abstract
The receptor tyrosine kinase c-Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c-Met catalytic site could guide structure-based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c-Met complexed with adenosine triphosphate (ATP)-competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c-Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c-Met catalytic site involves seven identified structurally-distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c-Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
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20. Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit.
- Author
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Kuznetsov, Aleksei and Järv, Jaak
- Abstract
Protein kinase A (cAMP dependent protein kinase catalytic subunit, EC 2.7.11.11) binds simultaneously ATP and a phosphorylatable peptide. These structurally dissimilar allosteric ligands influence the binding effectiveness of each other. The same situation is observed with substrate congeners, which reversibly inhibit the enzyme. In this review these allosteric effects are quantified using the interaction factor, which compares binding effectiveness of ligands with the free enzyme and the pre-loaded enzyme complex containing another ligand. This analysis revealed that the allosteric effect depends upon structure of the interacting ligands, and the principle “better binding: stronger allostery” observed can be formalized in terms of linear free-energy relationships, which point to similar mechanism of the allosteric interaction between the enzyme-bound substrates and/or inhibitor molecules. On the other hand, the type of effect is governed by ligand binding effectiveness and can be inverted from positive allostery to negative allostery if we move from effectively binding ligands to badly binding compounds. Thus the outcome of the allostery in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if appropriate ligands bind. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
21. Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors.
- Author
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Marquez-Klaka, Benjamin, Rettinger, Jürgen, and Nicke, Annette
- Subjects
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CATIONS , *CYSTEINE proteinases , *BINDING sites , *DIMERS , *PROTEINASES - Abstract
P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X1 subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X2 (K69C, F289C), P2X3 (K63C, F280C), and P2X4 (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X2K69C and P2X2F289C mutants. Likewise, co-expression of P2X1K68C and P2X2F289C, but not P2X1F291C and P2X2K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X3 or P2X4 mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X1 are preserved in P2X2 receptors and at one of two possible interfaces in heteromeric P2X1/2 receptors but appears to be redundant for P2X3 and P2X4 receptor function. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
22. Validation of BKV large T-antigen ATP-binding site as a target for drug discovery
- Author
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Zeng, Gang, Bueno, Marta, Camachos, Carlos J., Ramaswami, Bala, Luo, Chunqing, and Randhawa, Parmjeet
- Subjects
- *
DNA helicases , *HYDROLYSIS , *DRUG development , *ADENOSINE triphosphatase - Abstract
Abstract: BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. Pilot screening of 300 compounds from the Tim Tec ActiTarg K library identified a compound, STO18584, with selectivity index of 19.2. [Copyright &y& Elsevier]
- Published
- 2009
- Full Text
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23. Structure-based drug design of novel and highly potent pyruvate dehydrogenase kinase inhibitors.
- Author
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Bessho, Yuki, Akaki, Tatsuo, Hara, Yoshinori, Yamakawa, Maki, Obika, Shingo, Mori, Genki, Ubukata, Minoru, Yasue, Katsutaka, Nakane, Yoshitomi, Terasako, Yasuo, Orita, Takuya, Doi, Satoki, Iwanaga, Tomoko, Fujishima, Ayumi, Adachi, Tsuyoshi, Ueno, Hiroshi, and Motomura, Takahisa
- Subjects
- *
PYRUVATE dehydrogenase kinase , *KINASE inhibitors , *DRUG design , *DRUG target , *PYRUVATE kinase , *INTRAMOLECULAR forces , *PYRUVATES , *KINASES - Abstract
[Display omitted] Pyruvate dehydrogenase kinases (PDHKs) are fascinating drug targets for numerous diseases, including diabetes and cancers. In this report, we describe the result of our structure-based drug design from tricyclic lead compounds that led to the discovery of highly potent PDHK2 and PDHK4 dual inhibitors in enzymatic assay. The C3-position of the tricyclic core was explored, and the PDHK2 X-ray structure with a representative compound revealed a novel ATP lid conformation in which the phenyl ring of Phe326 mediated the interaction of the Arg258 sidechain and the compound. Compounds with amide linkers were designed to release the ATP lid by forming an intramolecular pi-pi interaction, and these compounds showed single-digit nM IC 50 values in an enzymatic assay. We also explored the C4-position of the tricyclic core to reproduce the interaction observed with the C3-position substitution, and the pyrrolidine compound showed the same level of IC 50 values. By optimizing an interaction with the Asn255 sidechain through a docking simulation, compounds with 2-carboxy pyrrole moiety also showed single-digit nM IC 50 values without having a cation-pi interaction with the Arg258 sidechain. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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- View/download PDF
24. Identification of an Intersubunit Cross-Link between Substituted Cysteine Residues Located in the Putative ATP Binding Site of the P2X1 Receptor.
- Author
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Marquez-Klaka, Benjamin, Rettinger, Jürgen, Bhargava, Yogesh, Eisele, Thomas, and Nicke, Annette
- Subjects
- *
CYSTEINE proteinases , *ADENOSINE triphosphate , *ION channels , *AMINO acids , *XENOPUS laevis - Abstract
P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, two-electrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X2-1 chimera showed only small ATP-activated currents that, however, increased ∼60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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- View/download PDF
25. ATP binding site in the plant ADP-glucose pyrophosphorylase large subunit
- Author
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Hwang, Seon-Kap, Hamada, Shigeki, and Okita, Thomas W.
- Subjects
- *
GLUCOSE , *SUCROSE , *ENZYME kinetics , *CHEMICAL kinetics - Abstract
Abstract: The ATP binding region in the catalytically inactive large subunit (LS) of the potato tuber ADP-glucose pyrophosphorylase was identified and investigated. Mutations at the ATP binding significantly affected not only the apparent affinities for ATP and Glc-1-P, and catalytic rate but also in many instances, sensitivity to 3-phosphoglycerate. The catalytic rates of the LS mutant enzymes correlated most strongly with changes in the affinity toward ATP, a relationship substantiated by photoaffinity labeling studies with azido-ATP analog. These results indicate that the LS, although catalytically defective, interacts cooperatively with the catalytic small subunit in binding substrates and effectors and, in turn, influencing net catalysis. [Copyright &y& Elsevier]
- Published
- 2006
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- View/download PDF
26. Selectivity and potency of cyclin-dependent kinase inhibitors.
- Author
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Sridhar, Jayalakshmi, Akula, Nagaraju, and Pattabiraman, Nagarajan
- Abstract
Members of the cyclin-dependent kinase (CDK) family play key roles in various cellular processes. There are 11 members of the CDK family known till now. CDKs are activated by forming noncovalent complexes with cyclins such as A-, B-, C-, D- (D1, D2, and D3), and E-type cyclins. Each isozyme of this family is responsible for particular aspects (cell signaling, transcription, etc) of the cell cycle, and some of the CDK isozymes are specific to certain kinds of tissues. Aberrant expression and overexpression of these kinases are evidenced in many disease conditions. Inhibition of isozymes of CDKs specifically can yield beneficiary treatment modalities with minimum side effects. More than 80 3-dimensional structures of CDK2, CDK5, and CDK6 complexed with inhibitors have been published. This review provides an understanding of the structural aspects of CDK isozymes and binding modes of various known CDK inhibitors so that these kinases can be better targeted for drug discovery and design. The amino acid residues that constitute the cyclin binding region, the substrate binding region, and the area around the adenosine triphosphate (ATP) binding site have been compared for CDK isozymes. Those amino acids at the ATP binding site that could be used to improve the potency and subtype specificity have been described. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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- View/download PDF
27. Design, synthesis and structure–activity relationship studies of novel indazole analogues as DNA gyrase inhibitors with Gram-positive antibacterial activity
- Author
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Tanitame, Akihiko, Oyamada, Yoshihiro, Ofuji, Keiko, Kyoya, Yoko, Suzuki, Kenji, Ito, Hideaki, Kawasaki, Motoji, Nagai, Kazuo, Wachi, Masaaki, and Yamagishi, Jun-ichi
- Subjects
- *
DNA topoisomerase II , *GRAM-positive bacteria , *ANTIBACTERIAL agents , *STAPHYLOCOCCUS aureus , *DRUG resistance in microorganisms - Abstract
In this study, we report the design, synthesis and structure–activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). [Copyright &y& Elsevier]
- Published
- 2004
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28. Kinomics—structural biology and chemogenomics of kinase inhibitors and targets
- Author
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Vieth, Michal, Higgs, Richard E., Robertson, Daniel H., Shapiro, Michael, Gragg, Ellen A., and Hemmerle, Horst
- Subjects
- *
HUMAN genome , *MOLECULES , *GENOMES , *HUMAN chromosomes , *PROTEIN kinases , *HOMOLOGY (Biology) - Abstract
Classifying kinases based entirely on small molecule selectivity data is a new approach to drug discovery that allows scientists to understand relationships between targets. This approach combines the understanding of small molecules and targets, and thereby assists the researcher in finding new targets for existing molecules or understanding selectivity and polypharmacology of molecules in related targets. Currently, structural information is available for relatively few of the protein kinases encoded in the human genome (7% of the estimated 518); however, even the current knowledge base, when paired with structure-based design techniques, can assist in the identification and optimization of novel kinase inhibitors across the entire protein class. Chemogenomics attempts to combine genomic data, structural biological data, classical dendrograms, and selectivity data to explore, define, and classify the medicinally relevant kinase space. Exploitation of this information in the discovery of kinase inhibitors defines practical kinase chemogenomics (kinomics). In this paper, we review the available information on kinase targets and their inhibitors, and present the relationships between the various classification schema for kinase space. In particular, we present the first dendrogram of kinases based entirely on small molecule selectivity data. We find that the selectivity dendrogram differs from sequence-based clustering mostly in the higher-level groupings of the smaller clusters, and remains very comparable for closely homologous targets. Highly homologous kinases are, on average, inhibited comparably by small molecules. This observation, although intuitive, is very important to the process of target selection, as one would expect difficulty in achieving inhibitor selectivity for kinases that share high sequence identity. [Copyright &y& Elsevier]
- Published
- 2004
- Full Text
- View/download PDF
29. Characterization of quercetin binding site on DNA gyrase
- Author
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Plaper, Andreja, Golob, Mojca, Hafner, Iva, Oblak, Marko, Šolmajer, Tomaž, and Jerala, Roman
- Subjects
- *
DNA topoisomerase II , *PROKARYOTES - Abstract
Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a KD value of 15 μM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin–gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
- View/download PDF
30. Analysis of Conformational Changes at the Unique Loop Adjacent U the ATP Binding Site of Smooth Muscle Myosin Using a Fluorescent Probe.
- Author
-
Maruta, Shinsaku, Saitoh, Junya, and Asakura, Tsuyoshl
- Subjects
MYOSIN ,MUSCLE proteins ,SMOOTH muscle ,ADENOSINE triphosphatase ,AMINO acids - Abstract
Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320–327), loop M (687–699), and loop N (125–134), aimilar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124,528–533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3′-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N3-ADP), and then measured the fluorescent polarization. When Mant-8-.N3-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N3-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N3-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin SI (SMO-S1) labeled at only loop N (125–134) increased the polarization due to the viscosity of actin. In contrast, SI labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677–689) in skeletal muscle myosin, as previously demonstrated in our laboratory. [ABSTRACT FROM AUTHOR]
- Published
- 2000
- Full Text
- View/download PDF
31. The chemical diversity and structure-based discovery of allosteric modulators for the PIF-pocket of protein kinase PDK1
- Author
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Duan Ni, Xiaolong Li, Yingyi Chen, Qiang Fu, Jian Zhang, Shaoyong Lu, and Xinyuan Xu
- Subjects
animal structures ,Structural similarity ,Allosteric regulation ,Carboxylic Acids ,Computational biology ,Review Article ,Protein Serine-Threonine Kinases ,01 natural sciences ,PI3K ,Alkaloids ,Allosteric Regulation ,Drug Discovery ,Humans ,Disulfides ,Binding site ,Protein kinase A ,PI3K/AKT/mTOR pathway ,Pharmacology ,Sulfonamides ,allostery ,010405 organic chemistry ,Chemistry ,ATP binding site ,lcsh:RM1-950 ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,General Medicine ,Azepines ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,lcsh:Therapeutics. Pharmacology ,Protein kinase domain ,PDK1 ,Chemical diversity ,Structure based ,Benzimidazoles ,allosteric modulators ,orthosteric ligands - Abstract
Phosphoinositide-dependent protein kinase-1 (PDK1) is an important protein in mediating the PI3K-AKT pathway and is thus identified as a promising target. The catalytic activity of PDK1 is tightly regulated by allosteric modulators, which bind to the PDK1 Interacting Fragment (PIF) pocket of the kinase domain that is topographically distinct from the orthosteric, ATP binding site. Allosteric modulators by attaching to the less conserved PIF-pocket have remarkable advantages such as higher selectivity, less side effect, and lower toxicity. Targeting allosteric PIF-pocket of PDK1 has become the focus of recent attention. In this review, we summarise the current advances in the structure-based discovery of PDK1 allosteric modulators. We will first present the three-dimensional structure of PDK1 and illustrate the allosteric regulatory mechanism of PDK1 through the modulation of the PIF-pocket. Then, the recent advances of PDK1 allosteric modulators targeting the PIF-pocket will be recapitulated detailly according to the structural similarity of allosteric modulators.
- Published
- 2019
32. Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.
- Author
-
Akaki, Tatsuo, Bessho, Yuki, Ito, Takashi, Fujioka, Shingo, Ubukata, Minoru, Mori, Genki, Yamanaka, Kenji, Orita, Takuya, Doi, Satoki, Iwanaga, Tomoko, Ikegashira, Kazutaka, Hantani, Yoshiji, Nakanishi, Isao, and Adachi, Tsuyoshi
- Subjects
- *
BINDING sites , *PYRUVATES , *KINASES , *HYDROGEN bonding interactions , *LEAD compounds - Abstract
[Display omitted] A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
33. Cholate Disrupts Regulatory Functions of Cytochrome c Oxidase.
- Author
-
Ramzan, Rabia, Napiwotzki, Jörg, Weber, Petra, Kadenbach, Bernhard, and Vogt, Sebastian
- Subjects
- *
CYTOCHROME oxidase , *MITOCHONDRIA formation , *SODIUM cholate , *CYTOCHROME c , *MEMBRANE potential - Abstract
Cytochrome c oxidase (CytOx), the oxygen-accepting and rate-limiting enzyme of mitochondrial respiration, binds with 10 molecules of ADP, 7 of which are exchanged by ATP at high ATP/ADP-ratios. These bound ATP and ADP can be exchanged by cholate, which is generally used for the purification of CytOx. Many crystal structures of isolated CytOx were performed with the enzyme isolated from mitochondria using sodium cholate as a detergent. Cholate, however, dimerizes the enzyme isolated in non-ionic detergents and induces a structural change as evident from a spectral change. Consequently, it turns off the "allosteric ATP-inhibition of CytOx", which is reversibly switched on under relaxed conditions via cAMP-dependent phosphorylation and keeps the membrane potential and ROS formation in mitochondria at low levels. This cholate effect gives an insight into the structural-functional relationship of the enzyme with respect to ATP inhibition and its role in mitochondrial respiration and energy production. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
34. Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation.
- Author
-
Trafalis, Dimitrios T., Sagredou, Sofia, Dalezis, Panayiotis, Voura, Maria, Fountoulaki, Stella, Nikoleousakos, Nikolaos, Almpanakis, Konstantinos, Deligiorgi, Maria V., Sarli, Vasiliki, and Tomašič, Tihomir
- Subjects
- *
THIADIAZOLES , *COLON tumors , *BINDING sites , *PHOSPHORYLATION , *CANCER cells , *HETEROCYCLIC compounds - Abstract
The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer properties in vitro against a panel of cancer cells and in vivo efficacy in HT-29 human colon tumor xenograft in CB17 severe combined immunodeficient (SCID) mice. Preliminary mechanistic studies revealed that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising cancer therapeutic agents. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
35. A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.
- Author
-
Fox, Ted, Coll, Joyce T., Xie, Xiaoling, Ford, Pamella J., Germann, Ursula A., Porter, Margaret D., Pazhanisamy, S., Fleming, Mark A., Galullo, Vincent, Su, Michael S. S., and Wilson, Keith P.
- Abstract
Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways.Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G, T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation. [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
- View/download PDF
36. A Unique Loop Contributing to the Structure of the ATP-Binding Cleft of Skeletal Muscle Myosin Communicates with the Actin-Binding Site.
- Author
-
Maruta, Shinsaku and Homma, Kazuaki
- Subjects
ACTOMYOSIN ,MUSCLE proteins ,ADENOSINE triphosphatase ,IMMUNOGLOBULIN idiotypes ,RADIOACTIVITY - Abstract
Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677–689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently cross-linked to loop M (Mant-Sl), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677–689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational moblity was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-Sl to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677–689) probably results from a conformational change in loop M (677–689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
- View/download PDF
37. A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.
- Author
-
Galperin, Michael Y. and Koonin, Eugene V.
- Abstract
The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to d-alanine-d-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis. [ABSTRACT FROM AUTHOR]
- Published
- 1997
- Full Text
- View/download PDF
38. Recent Advances in c-Jun N-Terminal Kinase (JNK) Inhibitors.
- Author
-
Li G, Qi W, Li X, Zhao J, Luo M, and Chen J
- Subjects
- Humans, MAP Kinase Signaling System drug effects, JNK Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
c-Jun N-Terminal Kinases (JNKs), members of the Mitogen-Activated Protein Kinase (MAPK) signaling pathway, play a key role in the pathogenesis of many diseases including cancer, inflammation, Parkinson's disease, Alzheimer's disease, cardiovascular disease, obesity, and diabetes. Therefore, JNKs represent new and excellent target by therapeutic agents. Many JNK inhibitors based on different molecular scaffolds have been discovered in the past decade. However, only a few of them have advanced to clinical trials. The major obstacle for the development of JNK inhibitors as therapeutic agents is the JNKisoform selectivity. In this review, we describe the recent development of JNK inhibitors, including ATP competitive and ATP non-competitive (allosteric) inhibitors, bidentatebinding inhibitors and dual inhibitors, the challenges, and the future direction of JNK inhibitors as potential therapeutic agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
- Published
- 2021
- Full Text
- View/download PDF
39. The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor
- Author
-
Ingebrigt Sylte, Sergiy Kostenko, Mahmud Tareq Hassan Khan, and Ugo Moens
- Subjects
Models, Molecular ,Aconitine ,Active Transport, Cell Nucleus ,MK3 ,MK5 ,Protein Serine-Threonine Kinases ,VDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Biokjemi: 476 ,Mitogen-activated protein kinase kinase ,Biology ,Binding, Competitive ,PC12 Cells ,p38 Mitogen-Activated Protein Kinases ,MAP2K7 ,VDP::Mathematics and natural science: 400::Basic biosciences: 470::Biochemistry: 476 ,Cellular and Molecular Neuroscience ,Alkaloids ,Animals ,ASK1 ,Enzyme Inhibitors ,Phosphorylation ,Kinase activity ,Protein kinase A ,Molecular Biology ,Pharmacology ,MAP kinase kinase kinase ,ATP binding site ,Cyclin-dependent kinase 2 ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Rats ,Cell biology ,Biochemistry ,Molecular docking ,Mitogen-activated protein kinase-activated protein kinase MK5 ,biology.protein ,Molecular Medicine ,Cyclin-dependent kinase 9 ,Diterpenes ,Diterpenoid alkaloid ,Signal Transduction ,Research Article ,Noroxoaconitine - Abstract
The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC50 = 37.5 μM; Ki = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0452-1) contains supplementary material, which is available to authorized users.
- Published
- 2010
40. Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit
- Author
-
Aleksei Kuznetsov and Jaak Järv
- Subjects
Enzyme complex ,peptide phosphorylation ,QH301-705.5 ,Stereochemistry ,ligand binding ,Protein subunit ,Allosteric regulation ,peptide binding site ,General Biochemistry, Genetics and Molecular Biology ,camp-dependent protein kinase catalytic subunit ,enzyme kinetics ,Enzyme kinetics ,Biology (General) ,Protein kinase A ,enzyme inhibition ,chemistry.chemical_classification ,single-subunit allostery ,atp binding site ,General Immunology and Microbiology ,biology ,allosteric cooperativity ,General Neuroscience ,Ligand (biochemistry) ,Enzyme ,chemistry ,Allosteric enzyme ,biology.protein ,interaction factor ,General Agricultural and Biological Sciences - Abstract
Protein kinase A (cAMP dependent protein kinase catalytic subunit, EC 2.7.11.11) binds simultaneously ATP and a phosphorylatable peptide. These structurally dissimilar allosteric ligands influence the binding effectiveness of each other. The same situation is observed with substrate congeners, which reversibly inhibit the enzyme. In this review these allosteric effects are quantified using the interaction factor, which compares binding effectiveness of ligands with the free enzyme and the pre-loaded enzyme complex containing another ligand. This analysis revealed that the allosteric effect depends upon structure of the interacting ligands, and the principle “better binding: stronger allostery” observed can be formalized in terms of linear free-energy relationships, which point to similar mechanism of the allosteric interaction between the enzyme-bound substrates and/or inhibitor molecules. On the other hand, the type of effect is governed by ligand binding effectiveness and can be inverted from positive allostery to negative allostery if we move from effectively binding ligands to badly binding compounds. Thus the outcome of the allostery in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if appropriate ligands bind.
- Published
- 2009
41. Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling
- Author
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Cécile Martin, Nurit Hadad, Richard H. Ashley, and Varda Shoshan-Barmatz
- Subjects
Gene isoform ,GTP' ,Ca2+-release channel ,Molecular Sequence Data ,Biophysics ,In Vitro Techniques ,Endoplasmic Reticulum ,Biochemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Structural Biology ,Microsomes ,Genetics ,Animals ,Protein Isoforms ,Nucleotide ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Photoaffinity ,Cytoskeleton ,chemistry.chemical_classification ,Tricine ,Binding Sites ,Sheep ,Photoaffinity labeling ,Nucleotides ,Ryanodine receptor ,Chemistry ,ATP binding site ,Dynein ,Brain ,Dyneins ,Affinity Labels ,Ryanodine Receptor Calcium Release Channel ,Intracellular Membranes ,Cell Biology ,Molecular biology - Abstract
Two high Mr protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (Bz2ATP). The 420 kDa band is labeled by [α-32P]-Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross-reactivity with anti-dynein antibodies. The 440 kDa protein is immunologically identified as the type-2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP>ATP>ADP>AMP>AMP-PNP>GTP>cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.
- Published
- 1999
42. Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90
- Author
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Yenisetti Saratchandra, Salam Pradeep Singh, Sharif Udin Ahmed, Chitta Ranjan Deb, and Bolin Kumar Konwar
- Subjects
0301 basic medicine ,Hsp90 ,Molecular Docking Simulation ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,03 medical and health sciences ,Flavonols ,Non-competitive inhibition ,0302 clinical medicine ,Heat shock protein ,cancer ,Binding site ,chemistry.chemical_classification ,biology ,Chemistry ,ATP binding site ,Active site ,molecular docking ,General Medicine ,flavonols ,030104 developmental biology ,Biochemistry ,biology.protein ,Original Article ,Adenosine triphosphate ,030217 neurology & neurosurgery - Abstract
Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters.
- Published
- 2016
43. The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP
- Author
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Nicholas C. Price, Graeme J. Thomson, and John R. Coggins
- Subjects
Protein subunit ,ATPase ,Molecular Sequence Data ,Biophysics ,Substrate analog ,Biochemistry ,Substrate Specificity ,Chaperonin ,chemistry.chemical_compound ,Adenosine Triphosphate ,Bacterial Proteins ,GroEL protein ,Structural Biology ,2',3' dialdehyde ATP ,Escherichia coli ,Genetics ,Electrospray mass spectrometry ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Heat-Shock Proteins ,GroEL Protein ,Adenosine Triphosphatases ,biology ,ATP binding site ,Serine Endopeptidases ,Affinity labelling ,Affinity Labels ,Chaperonin 60 ,Cell Biology ,GroEL ,chemistry ,Reagent ,biology.protein ,bacteria ,Endopeptidase K - Abstract
The reaction of the E. coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first-order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys-457 and Cys-518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.
- Published
- 1993
44. Analysis of c-Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of ATP-Competitive Ligands
- Author
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Rosanna Dono, Yasmine Asses, Safia Taïri-Kellou, Bernard Maigret, Flavio Maina, Vincent Leroux, Knowledge representation, reasonning (ORPAILLEUR), INRIA Lorraine, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Institut National de Recherche en Informatique et en Automatique (Inria)-Université Henri Poincaré - Nancy 1 (UHP)-Université Nancy 2-Institut National Polytechnique de Lorraine (INPL)-Centre National de la Recherche Scientifique (CNRS)-Université Henri Poincaré - Nancy 1 (UHP)-Université Nancy 2-Institut National Polytechnique de Lorraine (INPL)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Lorraine (INPL)-Université Nancy 2-Université Henri Poincaré - Nancy 1 (UHP)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Lorraine (INPL)-Université Nancy 2-Université Henri Poincaré - Nancy 1 (UHP)
- Subjects
Secondary ,Molecular model ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Protein Structure, Secondary ,Adenosine Triphosphate/*chemistry/metabolism ,Receptor tyrosine kinase ,chemistry.chemical_compound ,Adenosine Triphosphate ,0302 clinical medicine ,Catalytic Domain ,Drug Discovery ,kinase inhibitors ,ComputingMilieux_MISCELLANEOUS ,0303 health sciences ,Crystallography ,biology ,protein receptor model ,ATP binding site ,Proto-Oncogene Proteins c-met ,3. Good health ,030220 oncology & carcinogenesis ,Molecular Medicine ,Antineoplastic Agents/chemistry/pharmacology ,allosteric interactions ,Protein Binding ,Protein Kinase Inhibitors/chemistry/pharmacology ,Protein Structure ,Stereochemistry ,Molecular Sequence Data ,Allosteric regulation ,Antineoplastic Agents ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,03 medical and health sciences ,Allosteric Regulation ,Computer Simulation ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,Binding site ,Protein Kinase Inhibitors ,Proto-Oncogene Proteins c-met/*chemistry/metabolism ,030304 developmental biology ,c-Met ,Pharmacology ,protein-ligand complexes ,molecular modeling ,Organic Chemistry ,ligand binding modes ,Active site ,Allosteric enzyme ,Protein kinase domain ,chemistry ,Drug Design ,X-Ray ,biology.protein ,structure-based drug design ,Adenosine triphosphate - Abstract
International audience; The receptor tyrosine kinase c-Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c-Met catalytic site could guide structure-based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c-Met complexed with adenosine triphosphate (ATP)-competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c-Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c-Met catalytic site involves seven identified structurally-distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c-Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design.
- Published
- 2009
45. On the binding of ATP to the autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii
- Author
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Patricia Doublet, Christophe Grangeasse, Bertrand Duclos, C. Vincent, Alain J. Cozzone, and Deleage, Gilbert
- Subjects
Recombinant Fusion Proteins ,Molecular Sequence Data ,Biophysics ,Biochemistry ,Phosphates ,Adenosine Triphosphate ,Structural Biology ,Genetics ,medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Nucleotide ,Amino Acid Sequence ,Binding site ,Tyrosine ,Phosphorylation ,Molecular Biology ,Glutathione Transferase ,chemistry.chemical_classification ,Binding Sites ,biology ,Acinetobacter ,Base Sequence ,Kinase ,ATP binding site ,Autophosphorylation ,Cell Biology ,Protein-Tyrosine Kinases ,biology.organism_classification ,Adenosine ,Molecular biology ,Amino acid ,chemistry ,Autophosphorylation mechanism ,Bacterial protein phosphorylation ,Bacteria ,medicine.drug - Abstract
The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5'-p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5'-p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.
- Published
- 1999
46. ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp-335 with C1RATP
- Author
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Svetlana Lutsenko, Theo Wallimann, Ernesto Carafoli, Markus Wyss, and Peter James
- Subjects
Molecular Sequence Data ,Biophysics ,Adenylate kinase ,Biology ,Biochemistry ,Mitochondria, Heart ,Adenosine Triphosphate ,Structural Biology ,Sequence Homology, Nucleic Acid ,Genetics ,Animals ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Creatine Kinase ,Heart metabolism ,chemistry.chemical_classification ,Aspartic Acid ,Binding Sites ,CIRATP analogue ,ATP binding site ,Active site ,Affinity Labels ,Cell Biology ,Molecular biology ,Pepsin A ,Peptide Fragments ,Amino acid ,Isoenzymes ,Enzyme ,chemistry ,biology.protein ,Creatine kinase ,Chickens - Abstract
The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, CIRATP, in which the reactive label was covalently bound to the γ-phosphote group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335–343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by CIRATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.
- Published
- 1990
47. Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90.
- Author
-
Singh SP, Deb CR, Ahmed SU, Saratchandra Y, and Konwar BK
- Abstract
Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters., (© 2016 the Journal of Biomedical Research. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
48. Mechanism of F1-ATPase studied by the genetic approach
- Author
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Futai, Masamitsu, Noumi, Takato, and Maeda, Masatomo
- Published
- 1988
- Full Text
- View/download PDF
49. Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm).
- Author
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Škedelj V, Perdih A, Brvar M, Kroflič A, Dubbée V, Savage V, O'Neill AJ, Solmajer T, Bešter-Rogač M, Blanot D, Hugonnet JE, Magnet S, Arthur M, Mainardi JL, Stojan J, and Zega A
- Subjects
- Dose-Response Relationship, Drug, Enterococcus faecium metabolism, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Ligases metabolism, Models, Molecular, Molecular Structure, Structure-Activity Relationship, D-Aspartic Acid metabolism, Drug Discovery, Enterococcus faecium enzymology, Enzyme Inhibitors pharmacology, Ligases antagonists & inhibitors
- Abstract
The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
50. ATP binding site in the plant ADP-glucose pyrophosphorylase large subunit
- Author
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Shigeki Hamada, Seon-Kap Hwang, and Thomas W. Okita
- Subjects
AGPase ,Protein subunit ,Molecular Sequence Data ,Biophysics ,Glucose 1-phosphate ,Glucose-1-Phosphate Adenylyltransferase ,Photoaffinity Labels ,Glyceric Acids ,Biochemistry ,Catalysis ,Mass Spectrometry ,Substrate Specificity ,chemistry.chemical_compound ,Adenosine Triphosphate ,ADP-glucose pyrophosphorylase ,Structural Biology ,ATP synthase gamma subunit ,Photoaffinity labeling ,Genetics ,Amino Acid Sequence ,Enzyme kinetics ,Binding site ,Site-directed mutagenesis ,Molecular Biology ,Solanum tuberosum ,chemistry.chemical_classification ,Binding Sites ,biology ,ATP binding site ,Cell Biology ,Protein Structure, Tertiary ,Kinetics ,Protein Subunits ,Enzyme ,chemistry ,biology.protein ,Mutant Proteins ,Starch synthesis ,Large subunit - Abstract
The ATP binding region in the catalytically inactive large subunit (LS) of the potato tuber ADP-glucose pyrophosphorylase was identified and investigated. Mutations at the ATP binding significantly affected not only the apparent affinities for ATP and Glc-1-P, and catalytic rate but also in many instances, sensitivity to 3-phosphoglycerate. The catalytic rates of the LS mutant enzymes correlated most strongly with changes in the affinity toward ATP, a relationship substantiated by photoaffinity labeling studies with azido-ATP analog. These results indicate that the LS, although catalytically defective, interacts cooperatively with the catalytic small subunit in binding substrates and effectors and, in turn, influencing net catalysis.
- Full Text
- View/download PDF
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