The ubiquitin-proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C-terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α-ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.
The in vivo diagnosis of Alzheimer's disease (AD) is of high socioeconomic interest and remains a demanding field of research. The biopathological hallmarks of the disease are extracellular plaques consisting of aggregated β-amyloid peptides (Aβ) and tau protein derived intracellular tangles. Here we report the synthesis and evaluation of fluorescent pyrazine, pyrimidine,and pyridazine derivatives in vitro and in vivo aiming at a tau-based diagnosis of AD. The probes were pre-evaluated on human brain tissue by fluorescence microscopy and were found to label all known disease-related alterations at high contrast and specificity. To quantify the binding affinity, a new thiazine red displacement assay was developed and selected candidates were toxicologically profiled. The application in transgenic mouse models demonstrated bioavailability and brain permeability for one compound. In the course of histological testing, we discovered an AD-related deposition of tau aggregates in the Bowman's glands of the olfactory epithelium, which holds potential for an endoscopic diagnosis of AD in the olfactory system.
The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off-target-related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA-approved proteasome inhibitors bortezomib (1) or carfilzomib (2). A systematic comparison of electrophilic headgroups recently identified the class of α-keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure-activity relationship (SAR)-based approach for rational ligand design using an electronic parameter (Hammett's σ) and in silico molecular modeling. This resulted in the tripeptidic α-keto phenylamide BSc4999 [(S)-3-(benzyloxycarbonyl-(S)-leucyl-(S)-leucylamino)-5-methyl-2-oxo-N-(2,4-dimethylphenyl)hexanamide, 6 a], a highly potent (IC50 = 38 nM), cell-permeable, and slowly reversible covalent inhibitor which targets both the primed and non-primed sites of the proteasome's substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α-keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.
Das Ubiquitin-Proteasom-System (UPS) wurde in akademischer wie pharmazeutischer Forschung erfolgreich für onkologische und immunologische Anwendungen adressiert. Typische Proteasominhibitoren basieren auf einem peptidischen Rückgrat, das mit einem elektrophilen C-Terminus zur Bindung an die aktiven proteolytischen Zentren gekuppelt ist. Obwohl der peptidische Anteil viel Interesse bezüglich der Untereinheitenselektivität hervorgerufen hat, wird die Wirkspezifität und biologische Stabilität der Substanzen entscheidend durch die reaktiven Kopfgruppen geprägt. In dieser Studie wurde daher eine systematische Untersuchung mit Invitro-, In-vivo-und strukturbiologischen Methoden durchgeführt, um die Auswirkungen unterschiedlicher Funktionalitä-ten und chemischer Reaktivitäten aufzudecken. Dies ermçg-lichte die Einführung und Charakterisierung der Klasse der aKetoamide als potenteste reversible Inhibitoren mit mçglichen Anwendungen für die Behandlung von soliden Tumoren und Autoimmunerkrankungen. Die Zulassung von Carfilzomib (Kyprolis) für multiplesMyelom und Mantelzelllymphom als Proteasominhibitor der zweiten Generation durch die FDA im Jahr 2012 hat das 20S-Proteasom (Kernpartikel; KP) aus dem nichtlysosomalen Proteinabbauweg erneut als mçgliches Angriffsziel für Krebserkrankungen bestätigt.[1] Zudem wurde durch die Entwicklung des entzündungshemmenden KP-Inhibitors PR-957 der Weg für die Behandlung von Autoimmunerkrankungen durch die Modulierung der proteasomalen Signalgebungsfunktion bereitet.[2] Carfilzomib und PR-957 tragen als gemeinsames Strukturmerkmal das a',b'-Epoxyketon-Elektrophil, welches von dem Naturstoff Epoxomicin ableitet. [3] Dieses Leitmotiv hat sich gegen die hochreaktive Boronsäure des ersten klinisch zugelassenen Inhibitors Bortezomib (Velcade) [4] durchgesetzt, indem es die Bivalenz des katalytisch aktiven Thr1 in den proteasomalen Substratbindekanä-len adressiert (Schema 1).[5] Durch diesen Bindemodus werden Cystein-, Aspartat und Metalloproteasen von Epoxyketonen nicht betroffen, was sich in einem stark verbesserten Nebenwirkungsprofil niederschlägt.[6] Dennoch weist auch diese zweite Generation an Proteasominhibitoren einen irreversiblen Bindemodus ähnlich der Reaktivität von Bortezomib auf, [5] was mutmaßlich die Durchdringung von festem Gewebe mindert und die Einsetzbarkeit auf verschiedene Arten von Blutkrebs beschränkt. [7] Daher war das Ziel dieser Forschungsarbeit, die bivalente Leitstruktur von Epoxyketonen zu einer reversiblen Funktionalität umzuformen, indem wir die Verbindungsklasse der a-Ketoaldehyde einführten. [8] Unerwarteterweise zeigten spätere Experimente, dass a-Ketoaldehyde sowohl ein wesentlich geringeres Inhibitionspotenzial als auch eine verminderte Zytotoxizität gegen Krebszelllinien aufweisen, was diese Substanzklasse bereits im Vorfeld von klinischen Studien ausschließt.Um diesen Reaktivitätsverlust näher zu beleuchten, wurde eine systematische Analyse der biochemischen Auswirkungen verschiedener C-terminaler Elektrophile in vitro Schema 1. Reaktionsmechani...
The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.
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