Entwicklung allosterischer Inhibitoren von AGC-Proteinkinasen und Co-Kristallographie mit den Target-Kinasen
Zusammenfassung der Projektergebnisse
During three funding periods the DFG funded a research project on the rational development of allosteric compounds directed to a regulatory site, the PIF-pocket, present in AGC kinases. One of us, RMB, had discovered the PIF-pocket regulatory site on the kinase PDK1, had characterized the role of the PIF-pocket biochemically and structurally, and, together with other researchers, had characterized the role of the equivalent site in other protein kinases from the AGC family, e.g. PKB/Akt, S6K, SGK, MSK, RSK, etc. With the support of the first funding period we published the first small compounds that targeting an allosteric site on the kinase domain activate a kinase. During the second funding period, we synthesized numerous compound analogs, crystallized PDK1 in complex with two small compound activators and published the molecular details of binding of compounds to the PIF-pocket and the allosteric effects that activate the enzyme. Interestingly, the novel-mode-of-action compounds mimic the conformational changes that activate AGC kinases by phosphorylation. In the third funding period we further synthesized compounds into our focused library and identified additional compound activators that enable to observe additional allosteric conformational changes, fully closing the catalytic domain and also identified new allosteric inhibitors of different AGC kinases. In order to obtain crystal structures of allosteric compounds in complex with the PIF-pocket of other AGC kinases, we developed a chimera technology, using the catalytic domain of PDK1 as a scaffold and mutating the PIF-pocket residues to those of different AGC kinases. Using this technology we identified the binding mode of compounds from our library to aPKCs, SGK and PKB/Akt. As part of the final grant period we also crystallized PDK1 in complex with a series of compounds that promote novel allosteric effects. Our acquired knowledge on PDK1 and the mechanism of allosteric activation and inhibition by small compounds has apparently also influenced research outside of the specific topic of PDK1 and AGC kinases. In a 2010 review published in Nat. Chem. Biol. the authors considered our working model, PDK1, as one of the best characterized mechanisms of activation of enzymes by small compounds and the model was employed to develop a novel methodology for the discovery of covalent modulators of allosteric proteins. The method was later used to develop novel allosteric drugs directed to mutant K-Ras, enabling drug development to a highly important cancer target which was previously not considered druggable. In similar manners, we hope that our work will successfully outreach more widely in the field of innovative drug discovery.
Projektbezogene Publikationen (Auswahl)
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"AGC protein kinases: from structural mechanism of regulation to allosteric drug development for the treatment of human diseases", Biochim Biophys Acta 1834(7):1302-21
Arencibia JM, Pastor-Flores D, Bauer AF, Schulze JO, Biondi RM
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(2006) “Allosteric activation of protein kinase PDK1 with low molecular weight compounds”, EMBO J., 25(23):5469-80
Engel, M., Hindie, V., Lopez-Garcia, L.A., Stroba, A., Schaeffer, F., Adrian, I., Imig, J., Idrissova, L., Nastainczyk, W., Zeuzem, S., Alzari, P.M., Hartmann, R.W., Piiper, A. and Biondi, R.M.
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(2009) “3,5-diphenylpent-2-enoic acids as allosteric eactivators of the protein kinase PDK1: Structure-activity relationships and thermodynamic characterization of binding as paradigms for PIF-binding pocket-targeting compounds”, J. Med. Chem., 52(15):4683-93
Stroba A, Schaeffer F, Hindie V, Lopez-Garcia LA, Adrian I, Frohner W, Hartmann RW, Biondi R.M. and Engel M.
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(2009) “High resolution complex structure and allosteric effects of low molecular weight activators on the protein kinase PDK1”, Nat. Chem. Biol., 5(10), 758-64
Hindie V, Stroba A, Zhang H, Lopez-Garcia LA, Idrissova L, Zeuzem S, Hirschberg D, Schaeffer F, Jorgensen TJ, Engel M, Alzari PM and Biondi R.M.
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(2011) " Allosteric regulation of protein kinase PKCz by the N-terminal C1 domain and small compounds to the PIF-pocket", Chem. Biol., 18(11):1463-7
Lopez-Garcia, L.A., Schulze, J.O., Fröhner, W., Zhang, H., Süß, E., Weber, N., Navratil, J., Amon, S., Hindie, V., Zeuzem, S., Jørgensen, T.J.D., Alzari, P.M., Neimanis, S., Engel, M., and Biondi, R.M.
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(2011) “4-Benzimidazolyl-3-Phenylbutanoic Acids As Novel Pif- Pocket-Targeting Allosteric Inhibitors of Protein Kinase PKCζ”, J. Med. Chem., 54(19):6714-23
Fröhner W, Lopez-Garcia LA, Neimanis S, Weber N, Navratil J, Maurer F, Stroba A, Zhang H, Biondi RM, Engel M
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(2012) “2-(3-Oxo-1,3-diphenylpropyl)malonic Acids as Potent Allosteric Ligands of the PIF Pocket of Phosphoinositide-Dependent Kinase-1: Development and Prodrug Concept”, J. Med Chem. 55 (22):9817-30
Wilhelm A, Lopez-Garcia LA, Busschots K, Fröhner W, Maurer F, Boettcher S, Zhang H, Schulze JO, Biondi RM, Engel M
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(2012) “Substrate-Selective Inhibition of Protein Kinase PDK1 by Small Compounds that Bind to the PIF-Pocket Allosteric Docking Site”, Chem Biol. 19(9):1152-63
Busschots K, Lopez-Garcia LA, Lammi C, Stroba A, Zeuzem S, Piiper A, Alzari PM, Neimanis S, Arencibia JM, Engel M, Schulze JO, Biondi RM
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(2012) “The PIF pocket of AGC kinases: a target site for allosteric modulators and protein–protein interaction inhibitors”, book chapter in "Protein-Protein Interactions in Drug Discovery”, Ed. Alexander Doemling, Wiley
Engel M
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(2013) “The PIF-pocket as a target for C. albicans Pkh selective inhibitors.“ ACS Chem Biol. 8(10):2283-92
Pastor-Flores D, Schulze JO, Bahí A, Giacometti R, Ferrer-Dalmau J, Passeron S, Engel M, Suess E, Casamayor A, Biondi RM
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(2014) "Molecular Mechanism of Regulation of the Atypical Protein Kinase C by N- terminal Domains and an Allosteric Small Compound." Chem. Biol. 21(6):754-65
Zhang H, Neimanis S, Lopez-Garcia LA, Arencibia JM, Amon S, Stroba A, Zeuzem S, Proschak E, Stark H, Bauer AF, Busschots K, Jørgensen TJ, Engel M, Schulze JO, Biondi RM
