TY  - THES
A1  - Vicik, Radim
T1  - Synthese und Eigenschaften N-Acylierter Aziridin-2,3-dicarboxylate als selektive, peptidomimetische Inhibitoren von Cystein-Proteasen der Cathepsin-L-Subfamilie
T1  - Synthesis and Properties N-Acylated Aziridin-2,3-dicarboxylates as selective, peptidomimetic Inhibitors of Cystein Proteases of Cathepsin-L-Subfamily
N2  - Die Cystein-Proteasen der Säuger und Parasiten wurden erst in den letzten zwei Jahrzehnten als pharmazeutisch/medizinisches Target erkannt. Die genauen Aufgaben der einzelnen Enzyme dieser sehr umfangreichen und ständig wachsenden Protease-Familie bleiben zwar teilweise noch unbekannt, es ist jedoch klar, dass ihre Aufgabe nicht nur der unspezifische Protein-Abbau ist. Das Ziel der vorliegenden Arbeit waren die Synthese einer Reihe peptidomimetischer Inhibitoren mit elektrophilem Aziridin-2,3-dicarbonsäure-Baustein und deren Testung an den Proteasen Cathepsin B (human), Cathepsin L (Paramecium tetraurelia), Falcipain-2 (Plasmodium falciparum) und Rhodesain (Trypanosoma brucei rhodesiense). Die Verbindungen sind als irreversible Inhibitoren der Proteasen konzipiert. Der Aziridin-Baustein als Elektrophil wird durch den Cystein-Rest des aktiven Zentrums der Proteasen angegriffen, es erfolgt eine nucleophile Ringöffnung und damit die irreversible Alkylierung der Proteasen. Die Aziridin-Bausteine wurden entweder stereoselektiv aus Tartraten oder als Racemate aus Fumaraten dargestellt. Durch NMR-spektroskopische Versuche wurde der Mechanismus der Epimerisierung der als Intermediate der stereoselektiven Synthese auftretenden Azidoalkohole aufgeklärt. Die N-Acylierung des Aziridin-Bausteins mit den Aminosäuren bzw. Dipeptiden erfolgte über Segmentkopplungen oder über eine schrittweise Anknüpfung der Aminosäuren. Es wurden dabei verschiedenste Methoden der Peptidchemie eingesetzt. Die Hemmkonstanten der synthetisierten Substanzen wurden in einem kontinuierlichen fluorimetrischen Mikrotiterplatten-Assay bei Inhibitor-Konzentrationen von 0.35 - 140 µM ermittelt. Als Substrat diente für alle Enzyme Z-Phe-Arg-AMC. Der Nachweis der Irreversibilität der Hemmung wurde durch Dialyse-Versuche und die Affinitätsmarkierung von Cathepsin L und Falcipain 2 mit Hilfe eines Biotin-markierten Inhibitors erbracht. Bei Inhibitoren, die eine zeitabhängige Hemmung aufweisen, wurden die Alkylierungskonstanten (ki –Werte) ermittelt. Diese sind im Vergleich zu den Konstanten der Epoxysuccinyl-Peptide ca. 1000x kleiner, was frühere Untersuchungen bestätigt. Aus den ermittelten Dissoziationskonstanten (Ki) ist die Selektivität für Cathepsin-L-ähnliche Proteasen eindeutig. Dabei wird die Reihenfolge RD > CL > FP >>> CB gefunden. Der beste Inhibitor für alle Enzyme ist die Substanz 116C (BOC-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)2), für die Hemmkonstanten im unteren micromolaren bzw. sogar nanomolaren Bereich gefunden werden. Unter den Substanzen finden sich auch einige, die für einzelne Enzyme selektiv sind. Für CL sind es die Verbindungen 517C, 105G, Z-023B, 023A; für CB 034A und 013B und für RD 112C, 222C, 105B, 013A. Dabei gibt es zwei Inhibitoren (105A, 517G), die selektiv nur die parasitären Enzyme FP und RD hemmen. Die Analyse der Struktur-Wirkungs-Beziehungen ergab, dass in Abhängigkeit von den Substituenten am Aziridinring (Benzylester, Ethylester, Disäure), von den Substituenten am Aziridin-Stickstoff (Phe-Ala, Leu-Xxx, Gly-Xxx, Xxx = cyclische Aminosäure) und der Stereochemie unterschiedliche Bindungsmodi vorliegen müssen. Erste Docking-Versuche, die in Kooperation mit der Arbeitsgruppe Baumann (Institut für Pharmazie und LMC, Universität Würzburg) durchgeführt wurden, bestätigen dies. Postuliert wird für Inhibitoren, die die Sequenz Leu-Pro enthalten, eine Bindung an die S`- Seite von Cathepsin L. Dies erklärt die Selektivität dieser Inhibitoren, denn innerhalb der S`-Substratbindungstaschen finden sich die größten strukturellen Unterschiede zwischen Cathepsin B und den Cathepsin-L-ähnlichen Proteasen. Im Gegensatz dazu wird für eines der Phe-Ala-Derivate eine Bindung an die S-Taschen postuliert, die zwischen den einzelnen Proteasen geringere strukturelle Unterschiede aufweisen. Dieser Inhibitor hemmt, wie fast alle Phe-Ala-Derivate, dementsprechend auch Cathepsin B besser als die Leu-Xxx-Derivate. In Rahmen einer Kooperation mit der Arbeitsgruppe Engels Institut für Organische Chemie, Universität Würzburg) wurden quantenchemische Rechnungen durchgeführt, die u.a. den Einfluss von Substituenten auf die Kinetik und Thermodynamik der nucleophilen Ringöffnung untersuchten. Vorhergesagt wurde, dass Substituenten am Aziridin-Stickstoff, die den Übergangzustand stabilisieren (N-Formyl), zu einer besseren Hemmung führen sollten. Das darauf hin synthetisierte N-Formylaziridin-2,3-dicarboxylat 008B weist eine etwa 5000x bessere Hemmung von CL auf als das nicht-formylierte Diethylaziridin-2,3-dicarboxylat. Die gezielt als "affinity label" entwickelte Biotin-markierte Verbindung 999C wurde zur Identifizierung von Cystein-Proteasen, die von Plasmodium falciparum exprimiert werden, eingesetzt (Kooperation mit der Arbeitsgruppe Gelhaus/Leippe, Institut für Zoologie, Universität Kiel).
N2  - Mammalian and parasitic cysteine proteases have been discovered as potential drug targets within the last two decades. The physiological and pathophysiological functions of this huge and growing family of proteases are not yet known in detail. However, their role is no longer considered to be only unspecific protein degradation. The goal of the present work was the syntheses of a series of peptidomimetic cysteine protease inhibitors containing aziridine-2,3-dicarboxylate as electrophilic fragment, and the testing of the synthesized compounds on the cysteine proteases cathepsin B (human), cathepsin L (Paramecium tetraurelia), falcipain 2 (Plasmodium falciparum), and rhodesain (Trypanosoma brucei rhodesiense. The compounds are designed as irreversible protease inhibitors. The aziridine ring represents an electophilic building block which is attacked by the cysteine residue of the proteases` active sites. As a consequence, the nucleophilic ring opening reaction leads to irreversible enzyme alkylation. The aziridine building blocks were synthesized stereoselectively in a chiral pool synthesis starting from tartrates, and as racemates starting from fumarates, respectively. NMR spectroscopic studies were used to clarify the mechanism of epimerization occurring during the synthesis of the azido alcohols which are intermediates of the stereoselective synthetic route. The N-acylation of the aziridines with amino acids or dipeptides was carried out via segment or subsequent peptide coupling. Various methods of peptide chemistry were used. The inhibition constants were determined in fluorimetric microplate enzyme assays with inhibitor concentrations between 0.35-140 µM. In all cases, the substrate Z-Phe-Arg-AMC was used. The irreversibility of inhibition was proven by dialysis assays, and by affinity labelling of CL and falcipain using a biotinylated inhibitor. The alkylation rate constant ki was determined in cases where time-dependent inhibition could be observed. In comparison to epoxysuccinyl peptides the ki -values are lower by three orders of magnitude confirming previous investigations. The Ki values unambiguously show that the compounds exhibit a selectivity for the CL-like enzymes. The order of inhibition potency is RD > CL > FP >>> CB. The most potent inhibitor is 116C (BOC-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)2) with inhibition constants in the submicromolar and even nanomolar range. Some compounds exhibit selectivity for single enzymes: CL: 517C, 105G, Z-023B, 023A; CB: 034A, 013B; RD: 112C, 222C, 105B, 013A. Compounds 105A and 517G selectively inhibit the parasitic proteases FP and RD. The analysis of the structure-activity-relationship led to the assumption that different binding modes have to exist in dependence on the aziridine ring substituents (benzyl ester, ethyl ester, diacid), of the aziridine nitrogen substituents (Phe-Ala, Leu-Xxx, Gly-Xxx, Xxx = cyclic amino acid), and of the stereochemistry, respectively. First docking experiments, performed in cooperation with Dr. Baumann`s group (Institue of Pharmay and Food Chemistry, University of Wuerzburg), confirm this assumption. Inhibitors containing a Leu-Pro sequence are predicted to bind into the S`-subsites of CL. Since the most striking structural difference between CB and CL-like proteases is found within these S`-subsites the selectivity between the enzymes may be due to binding into these subsites. In contrast, for a Phe-Ala derivative the docking postulates binding into the S-subsites which do not differ much between the enzymes. As a consequence, CB is inhibited much better by Phe-Ala-derivatives than by Leu-Xxx-derivatives. In cooperation with Prof. Engels` group (Institute of Organic Chemistry, University of Wuerzburg) quantumchemical computations were performed analyzing the influence of substituents on the thermodynamics and kinetics of the nucleophilic ring opening. These calculations predicted that substituents stabilizing the transition state (N-formyl) should improve inhibition potency. In order to proof this predicition the compound 008B (N-formyl aziridine-2,3-dicarboxylate) was synthesized and tested. Indeed, the compound is about 5000x more potent on CL than the non-formylated diethyl aziridine-2,3-dicarboxylate. The principal mechanism of inhibition - the nucleophilic ring opening - was proven in a model reaction by means of NMR spectroscopy and mass spectrometry. The biotinylated compound 999C was designed as an affinity labelling inhibitor usable to label and to identify cysteine proteases expressed by Plasmodium falciparum (cooperation with the group of Dr. Gelhaus, Prof. Leippe, Institute of Zoology, University of Kiel).
KW  - Aziridine
KW  - Cysteinproteasen
KW  - Inhibitor
KW  - Cystein
KW  - Protease
KW  - irreversibel
KW  - Aziridin
KW  - Cathepsin
KW  - cystein
KW  - protease
KW  - irreversible
KW  - aziridin
KW  - cathepsin
Y1  - 2004
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-11127
ER  - 
TY  - JOUR
A1  - Oli, Swarna
A1  - Abdelmohsen, Usama Ramadan
A1  - Hentschel, Ute
A1  - Schirmeister, Tanja
T1  - Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation
JF  - MARINE DRUGS
N2  - In this paper, we report new protease inhibitory activity of plakortide E towards cathepsins and cathepsin-like parasitic proteases. We further report on its anti-parasitic activity against Trypanosoma brucei with an IC50 value of 5 mu M and without cytotoxic effects against J774.1 macrophages at 100 mu M concentration. Plakortide E was isolated from the sponge Plakortis halichondroides using enzyme assay-guided fractionation and identified by NMR spectroscopy and mass spectrometry. Furthermore, enzyme kinetic studies confirmed plakortide E as a non-competitive, slowly-binding, reversible inhibitor of rhodesain.
KW  - plakortis halichondroides
KW  - plakortide E.
KW  - protease inhibitor
KW  - slowly-binding reversible inhibitor
KW  - cathepsin
KW  - trypanosoma brucei
KW  - cysteine protease
KW  - malaria parasites
KW  - cathepsin-L
KW  - in-vitro
KW  - rhodesain
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116536
SN  - 1660-3397
VL  - 12
IS  - 5
ER  - 
TY  - JOUR
A1  - Oelschlaegel, Diana
A1  - Weiss Sadan, Tommy
A1  - Salpeter, Seth
A1  - Krug, Sebastian
A1  - Blum, Galia
A1  - Schmitz, Werner
A1  - Schulze, Almut
A1  - Michl, Patrick
T1  - Cathepsin inhibition modulates metabolism and polarization of tumor-associated macrophages
JF  - Cancers
N2  - Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH\(_2\), led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE\(_2\), which correlated with the upregulation of numerous NF\(_k\)B-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.
KW  - cathepsin
KW  - activity-based probes
KW  - tumor-associated macrophage
KW  - autophagy
KW  - lysosome
KW  - lipid metabolism
KW  - inflammation
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213040
SN  - 2072-6694
VL  - 12
IS  - 9
ER  -