Development of a conformation sensitive assay for the detection of inter-domain interactions mediated by allosteric akt inhibitors
Loading...
Date
2013-08-28
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Die Aktivität vieler Proteinkinasen wird durch das Zusammenspiel der Kinasedomäne mit zusätzlichen regulatorischen Domänen gesteuert. Daher besitzen niedermolekulare Verbindungen, die solche Interdomänen-Interaktionen allosterisch adressieren und modulieren können, einen großen Wert in der chemischen Biologie und der Pharmaforschung, da diese Interaktionen oft spezifisch für bestimmte Kinasen bzw. Kinasefamilien sind, und somit im Gegensatz zu klassischen ATP-kompetitiven Inhibitoren eine höhere Selektivität versprechen. Die Proteinkinase B (PKB, auch genannt Akt) ist eine solche Multidomänen-Kinase und stellt ein wichtiges Zielprotein in der Krebsforschung dar. Im Hochdurchsatzscreening wurden in den letzten Jahren eine neue Familie von allosterischen Akt Inhibitoren entdeckt, deren Wirkmechanismus die Anwesenheit einer regulatorischen PH Domäne voraussetzt, da sie in einer Bindungstasche an der Grenzfläche zwischen der Kinase- und PH Domäne binden und dadurch Akt in einer inaktiven Konformation arretieren. Diese einzigartige Bindetasche führt zu einer außergewöhnlich hohen Selektivität und somit in vivo potenziell niedrigen Toxizität durch Reduktion unerwünschter Arzneimittelwirkungen. Allerdings basieren alle bisher bekannten Interdomänen-Inhibitoren auf demselben chemischen Grundgerüst, und die Identifizierung von neuartigen Leitstrukturen wird durch den Mangel an geeigneten Hochdurchsatzmethoden ausgebremst. In der vorliegenden Arbeit wird die Entwicklung von iFLiK (interface-FLiK) beschrieben, einem Fluoreszenz-basierten Assay, der solche Interdomänen-Interaktionen detektieren kann. Die für iFLiK benötigten Proteine wurden anhand kristallographischer Informationen konzipiert, in Insektenzellen exprimiert und schließlich mit verschiedenen Fluorophoren gekuppelt, um ihre Eignung als Sensoren für Proteinkonformationen sys-tematisch zu untersuchen. Zur Etablierung des Assays wurden zudem eine Reihe von Sondenmolekülen synthetisiert und auf ihre biologische Aktivität in vitro und in BT474 Zellen untersucht. Mit iFLiK konnten allosterische Inhibitoren von ATP-kompetitiven unterschieden und somit ein Machbarkeitsbeweis erbracht werden, dass solvatochrome Fluorophore zur Detektion von Protein-Protein-Interaktionen geeignet sind, wenn der Fluorophor an der korrekten Stelle angebracht ist. Nach sorgsamer und eingehender Assayoptimierung sowie Validierung mit orthogonalen Assaysystemen wurde eine interne Substanzbibliothek mit iFLiK durchmustert. Dadurch konnten Verbindungen mit einem neuartigen Grundgerüst gefunden werden, die vorher nicht als allosterische Akt Inhibitoren bekannt waren.
In addition to the catalytically active kinase domain, many kinases feature regulatory domains that govern their activity. Modulating and interfering with these inter-domain interactions presents a major opportunity for understanding biological systems and developing innovative therapeutics. Therefore, small molecule inhibitors that target these interactions with an allosteric mode of action have great value and higher intrinsic selectivity, as these interactions are often unique for a single kinase or kinase family. Protein Kinase B (PKB, also known as Akt) is such a multi-domain kinase and represents an important target in cancer research. A number of allosteric inhibitors have been reported for Akt over the last years, with one of them currently in clinical phase II trials. Their mode of action requires the presence of a regulatory PH domain as they bind at the PH/kinase domain interface, locking Akt into an inactive conformation. This unique binding site enables an extraordinary high selectivity and therefore low off-target toxicity. However, all known inter-domain inhibitors so far are based on the same scaffold, and the discovery of new chemical entities is hampered by the lack of methods to identify such inhibitors in high-throughput screens. This thesis describes the development of iFLiK (interface-FLiK), a fluorescence-based assay that can monitor such inter-domain interactions. Guided by X-ray crystal structures, the assay was designed to sense the conformational changes of the protein associated with these interactions by attaching an environmentally sensitive fluorophore to a suitable site. In order to establish the assay, a series of probe molecules were synthesised and tested for their inhibitory activity in in vitro and cellular assays. Furthermore, the proteins required for iFLiK were expressed in insect cells and labelled with various fluorophores, systematically assessing their suitability to report on protein conformations. iFLiK could easily distinguish allosteric from classic ATP-competitive inhibitors, thereby providing a proof of principle that solvatochromic fluorophores are capable to monitor protein-protein interactions when attaching the sensor to the correct position. After extensive and careful assay optimisation and validation with orthogonal assay systems, an in-house library was screened with iFLiK. Novel compounds with a scaffold that was previously not described as allosteric Akt inhibitors were identified.
In addition to the catalytically active kinase domain, many kinases feature regulatory domains that govern their activity. Modulating and interfering with these inter-domain interactions presents a major opportunity for understanding biological systems and developing innovative therapeutics. Therefore, small molecule inhibitors that target these interactions with an allosteric mode of action have great value and higher intrinsic selectivity, as these interactions are often unique for a single kinase or kinase family. Protein Kinase B (PKB, also known as Akt) is such a multi-domain kinase and represents an important target in cancer research. A number of allosteric inhibitors have been reported for Akt over the last years, with one of them currently in clinical phase II trials. Their mode of action requires the presence of a regulatory PH domain as they bind at the PH/kinase domain interface, locking Akt into an inactive conformation. This unique binding site enables an extraordinary high selectivity and therefore low off-target toxicity. However, all known inter-domain inhibitors so far are based on the same scaffold, and the discovery of new chemical entities is hampered by the lack of methods to identify such inhibitors in high-throughput screens. This thesis describes the development of iFLiK (interface-FLiK), a fluorescence-based assay that can monitor such inter-domain interactions. Guided by X-ray crystal structures, the assay was designed to sense the conformational changes of the protein associated with these interactions by attaching an environmentally sensitive fluorophore to a suitable site. In order to establish the assay, a series of probe molecules were synthesised and tested for their inhibitory activity in in vitro and cellular assays. Furthermore, the proteins required for iFLiK were expressed in insect cells and labelled with various fluorophores, systematically assessing their suitability to report on protein conformations. iFLiK could easily distinguish allosteric from classic ATP-competitive inhibitors, thereby providing a proof of principle that solvatochromic fluorophores are capable to monitor protein-protein interactions when attaching the sensor to the correct position. After extensive and careful assay optimisation and validation with orthogonal assay systems, an in-house library was screened with iFLiK. Novel compounds with a scaffold that was previously not described as allosteric Akt inhibitors were identified.
Description
Table of contents
Keywords
Akt, Allosteric inhibition, Environmentally sensitive fluorophores, Inter-domain interactions, PKB, Protein kinases