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Design, Synthesis and Biological Evaluation of Rhomboid Protease Specific Inhibitors

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Design, Synthesis and Biological Evaluation of Rhomboid Protease Specific Inhibitors Design, synthesis and biological evaluation of rhomboid protease specific inhibitors Inaugural dissertation for the attainment of the title of doctor in the Faculty of Mathematics and Natural Sciences at the Heinrich Heine University Duesseldorf presented by Parul Goel from Gohana, India Duesseldorf, August 2016 from the Institute for Neuropathology, University Hospital at the Heinrich Heine University Duesseldorf Published by permission of the Faculty of Mathematics and Natural Sciences at Heinrich Heine University Duesseldorf Supervisor: Prof Dr. Sascha Weggen Co-supervisor: Prof. Dr. Jörg Pietruzska Date of the oral examination: 24.11.16 Summary Rhomboids are intramembrane serine proteases present in prokaryotic, archaeal and eukaryotic organisms. Rhomboids are composed of 6 core transmembrane helices and feature a serine-histidine catalytic dyad. They hydrolyze the peptide-bond of substrate membrane proteins within the lipid bilayer and control diverse biological processes; e.g. EGF-receptor signaling in Drosophila melanogaster , quorum sensing in Providencia stuartii , host cell invasion of the malaria parasite Plasmodium falciparum , and mitochondrial integrity in mammals. Due to this remarkable range of biological functions, rhomboids might have great potential as drug targets. Previously, small molecules such as isocoumarins, fluorophosphate, β-lactams, β-lactones and chloromethylketones have been found to inhibit rhomboid proteases but most of them had low potency or insufficient selectivity over soluble serine proteases such as chymotrypsin. Hence, the objective of this thesis was the design, synthesis and biological evaluation of new and improved rhomboid inhibitors. Based on computer-aided approaches, candidate-based screening and large molecular library virtual screening, suitable drug-like candidates (peptidic and non-peptidic) were selected and screened against rhomboid proteases. Molecular modeling experiments were performed with the Molecular Operating Environment (MOE) software and a co-crystal structure of the E. coli rhomboid GlpG and the phosphate inhibitor Cbz-AlaP(OiPr)F or CAPF (PDB: 3UBB). Subsequently, the best compounds were selected for synthesis or purchasing and subjected to biological analysis. With the candidate- based screening, we discovered two new classes of rhomboid inhibitors: aryl acid substituted N- methylated saccharins and 2-styryl benzoxazinones. In an in vitro activity assay, 12 saccharin derived inhibitors were found to inhibit the E. coli rhomboid GlpG with IC50 values below 1 µM. The IC50 of the best derivative (BSc5195) was 0.2 µM. All saccharin inhibitors had at least a 20-fold window of selectivity for inhibition of the rhomboid GlpG over the soluble serine protease α-chymotrypsin. Furthermore, we were able to demonstrate that the saccharin inhibitors did not indiscriminately inhibit all rhomboids opening up the possibility to develop inhibitors selective for the evolutionary older bacterial and archaeal rhomboids. With respect to the mechanism of inhibition, we confirmed the release of the aryl acid leaving group during the enzymatic reaction. However, the predicted formation of a cross-linked enzyme-inhibitor complex was not observed. Indeed, our findings suggest that only the active site serine is covalently modified by the saccharin inhibitors. The 2-styryl benzoxazinones were also found active against GlpG but their potency was comparatively low (best compound BSc5172 with IC50 ≈ 25 µM). Finally, we performed a virtual screen of a large molecular library using the DOCKTITE workflow and identified a new phosphate inhibitor (VS5). VS5 was slightly more potent than known phosphate inhibitors with good selectivity over α-chymotrypsin proving that a virtual screening strategy can be successfully employed to discover novel protease inhibitors. In summary, N-methylated saccharins are a novel and promising class of mechanism-based inhibitors against rhomboid proteases with inhibitory potency in the submicromolar range and clear potential for further improvement. Zusammenfassung Rhomboide sind Intramembranproteasen und kommen sowohl in Archaeen und Prokaryoten als auch in Eukaryoten vor. Rhomboide weisen ein Grundgerüst aus 6 Transmembranhelices auf und besitzen im aktiven Zentrum eine katalytische Dyade aus einem Serin und einem Histidin. Rhomboide hydrolysieren die Peptidbindung ihrer Substrate innerhalb der Lipiddoppelschicht einer biologischen Membran und kontrollieren auf diese Weise vielfältige biologische Prozesse. Dazu gehören z.B. der EGF-Rezeptor vermittelte Signalweg in Drosophila melanogaster , das Quorum Sensing in Providencia stuartii , die Zellinvasion durch den Malaria Parasiten Plasmodium falciparum und die Aufrechterhaltung der mitochondrialen Integrität in Säugerzellen. Aufgrund dieser beeindruckenden Vielfalt an Aufgaben, stellen Rhomboide ein interessantes Ziel für die Entwicklung neuartiger Wirkstoffe dar. Zu den bisher identifizierten Wirkstoffklassen, die als Rhomboid Inhibitoren fungieren, zählen Isocoumarine, Fluorophosphonate, β-Lactame, β-Lactone und Chloromethylketone. Diese Substanzklassen weisen allerdings entweder eine niedrige Potenz auf oder sind im Hinblick auf lösliche Serin-Proteasen wie z.B. Chymotrypsin wenig selektiv. Daher bestand das Ziel dieser Arbeit im Design, der Synthese und der funktionalen biologischen Charakterisierung neuer und verbesserter Rhomboid-Inhibitoren. Auf der Basis Computer-gestützter Ansätze, Kandidaten-basiertem Screening und dem virtuellen Screening einer großen Molekül-Datenbank wurden vielversprechende Wirkstoff- ähnliche Kandidaten (Peptid-basierte und nicht-Peptid basierte) ausgewählt und ihre Aktivität im Hinblick auf die Inhibition von Rhomboiden untersucht. Die Experimente zum Molekularen Modeling wurden mit der Software Molecular Operating Environment (MOE) durchgeführt. Als Ausgangsbasis wurde eine Ko-Kristallstruktur des E. coli Rhomboids GlpG im Komplex mit den Phosphat- Inhibitoren Cbz-AlaP(OiPr)F oder CAPF (PDB: 3UBB) verwendet. Im Anschluss wurden die für aussichtsreich befundenen Substanzen entweder synthetisiert oder käuflich erworben und in einem biologischen Assay auf ihre Aktivität untersucht. Beim Kandidaten-basierten Screening wurden auf diese Weise zwei neue Klassen von Rhomboid-Inhibitoren identifiziert: Arylsäure-substituierte N- methylierte Saccharine und 2-Styryl Benzoxazinone. In einem in vitro Aktivitätsassay konnten für 12 Saccharin-basierte Inhibitoren IC50-Werte im Bereich von unter 1 µM für die Inhibition von GlpG identifiziert werden. Der IC50-Wert der potentesten Substanz lag bei 200 nM (BSc5195). Alle Saccharin-basierten Inhibitoren wiesen ein mindestens 20-faches Selektivitätsfenster gegenüber Chymotrypsin auf. Weiterhin konnten gezeigt werden, dass Saccharin-basierte Inhibitoren nicht alle Rhomboide gleichermaßen inibieren. Dies eröffnet die Möglichkeit Inhibitoren darzustellen, die selektiv für die evolutiv älteren Rhomboide aus Bakterien und Archaeen sind. Der zugrundeliegende Mechanismus der Inhibition von Rhomboid Proteasen basiert auf der Arylsäure-Abgangsgruppe und der damit verbundenen Modifikation des Enzyms während der Katalysereaktion. Der vorhergesagte kovalent quervernetzte Komplex aus Enzym und Inhibitor konnte jedoch nicht bestätigt werden. Die vorliegenden Ergebnisse weisen allerdings darauf hin, dass die Serin-Seitenkette im aktiven Zentrum von Saccharin-basierten Inhibitoren kovalent modifiziert wird. Für die 2-Styryl Benzoxazinone konnte ebenfalls eine inhibitorische Aktivität in Bezug auf GlpG nachgewiesen werden. Allerdings hatten diese Substanzen eine vergleichbar geringe Potenz – die beste Substanz BSc5172 wies einen IC50- Wert von ca. 25 µM auf. Abschließend wurde ein virtuelles Screening einer großen Molekülbibliothek mit Hilfe des DOCKTITE Algorithmus durchgeführt. Das Screening identifizierte den Phosphat- Inhibitor VS5, der potenter als bisher beschriebene Phosphatinhibitoren war, und eine gute Selektivität gegenüber Chymotrypsin aufwies. Somit konnte gezeigt werden, dass die Strategie, neue Protease- Inhibitoren über ein virtuelles Screening zu entdecken, durchaus vielversprechend sein kann. Zusammenfassend lässt sich festhalten, dass die identifizierten N-methylierten Saccharine eine neue und aussichtsreiche Substanzklasse Mechanismus-basierter Inhibitoren für Rhomboid Proteasen sind. deren Potenz schon zum jetzigen Zeitpunkt im submikromolaren Bereich liegt und die deutliches Potential für weitere Verbesserungen aufweisen. Contents LIST OF FIGURES ................................................................................................................................................ I LIST OF TABLES ................................................................................................................................................ IV ABBREVIATIONS ............................................................................................................................................... V 1 INTRODUCTION ............................................................................................................................................. 1 1.1 PROTEASES ....................................................................................................................................................... 1 1.1.1 Proteases classification ......................................................................................................................... 1 1.1.2 Intramembrane proteases ....................................................................................................................
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