| License: Publishing license for publications excluding print on demand (7MB) |
- URN to cite this document:
- urn:nbn:de:bvb:355-epub-297733
- DOI to cite this document:
- 10.5283/epub.29773
| Item type: | Thesis of the University of Regensburg (PhD) |
|---|---|
| Date: | 9 April 2015 |
| Referee: | PD Dr. Sabine Amslinger |
| Date of exam: | 28 March 2014 |
| Institutions: | Chemistry and Pharmacy > Institut für Organische Chemie > Arbeitskreis Dr. Sabine Amslinger |
| Keywords: | heme oxygenase-1 activity, anti-inflammatory activity, enones |
| Dewey Decimal Classification: | 500 Science > 540 Chemistry & allied sciences |
| Status: | Published |
| Refereed: | Yes, this version has been refereed |
| Created at the University of Regensburg: | Yes |
| Item ID: | 29773 |
Abstract (English)
To fight chronic inflammation in autoimmune diseases such as rheumatic arthritis and multiple sclerosis or cancer is an exigency. Therefore the inhibition of proinflammatory proteins such as inducible NO-synthase (iNOS) and the activation of anti-inflammatory proteins such as heme oxygenase-1 (HO-1) are important measures to be addressed. Many natural products and synthetic compounds with an ...
Abstract (English)
To fight chronic inflammation in autoimmune diseases such as rheumatic arthritis and multiple sclerosis or cancer is an exigency. Therefore the inhibition of proinflammatory proteins such as inducible NO-synthase (iNOS) and the activation of anti-inflammatory proteins such as heme oxygenase-1 (HO-1) are important measures to be addressed. Many natural products and synthetic compounds with an α,β-unsaturated carbonyl moiety reveal a variety of biological properties, including antioxidant, anti-inflammatory and cytoprotective properties. α,β-Unsaturated carbonyl compounds possess Michael acceptor activity and can react with nucleophilic sulfhydryl groups on key cysteins to regulate the inhibition of NF-κB and the activation of Nrf2, which are the transcriptional factors needed for the expression of iNOS and HO-1, respectively.
Heme oxygenase-1 is a redox sensitive, inducible stress protein converting heme to CO, Fe2+ and biliverdin, which is further reduced to bilirubin by biliverdin reductase (BVR). Thereby, the overall anti-inflammatory, chemopreventive and chemoprotective effect connected to the HO-1 activity is a result of the heme degradation. An ELISA-based HO-1 assay was developed and proved to be a reliable and easy cell line-based assay in order to screen compound libraries to find new HO-1 inducers, which could be used as lead structures for drug development. The HO-1 activity assay combines the HO enzyme reaction in presence of recombinant BVR with the quantification of the reaction product bilirubin by an indirect sandwich enzyme-linked immuno-sorbent assay (ELISA) using the murine macrophage cell line RAW264.7. The detection of the total bilirubin level in whole cell lysates requires only a fraction of the total protein amount, that is 10-30 µg per sample. By comparing the bilirubin levels in stimulated cells and control cells, the HO-1 activity can be expressed as ‘fold of control’ based on pmol bilirubin h-1mg-1 total protein. The reliable HO-1 activity assay was developed in a 96-well plate format allowing the screening of multiple compounds in parallel. 18 small molecules, mainly natural products with an α,β-unsaturated carbonyl unit as well as the drugs dexamethasone and oltipraz were tested and the activity of most known HO-1 inducers was confirmed. The diterpene carnosol was found the most potent HO-1 inducer in the screen of HO-1 activity (8.2 ± 1.9 fold after 6 h) followed by rosolic acid with an induction of HO-1 activity of 3.9 ± 0.4 fold after 24 h. Amongst the natural chalcones, cardamonin and flavokawain A were identified as new potent HO-1 inducers. The most potent HO-1 activity inducer was found to be the 2’,4’-dihydroxy-3,4-dimethoxychalcone (DHDMCH), with a maximum induction of HO-1 activity of 6.1 ± 2.3 fold. Moreover, the time dependence of HO-1 protein expression for DHDMCH was compared to its enzyme activity, which was further evaluated in presence of lipopolysaccharide (LPS) and the specific HO-1 inhibitor tin protoporphyrin IX (SnPPIX). Furthermore, the HO-1 activity assay was successfully applied on human primary dendritic cells, were the HO-1 activity of the α-CF3-TMCH was determined.
Natural and synthetic hydroxy- and methoxychalcones were characterized towards their anti-inflammatory and antioxidant behavior in RAW264.7 murine macrophages. Particularly, their influence on the induction of the anti-inflammatory and cytoprotective protein HO-1 and the inhibition of the proinflammatory protein iNOS, more specifically the reduction of NO production was investigated. Additionally, a cell-free assay performed under physiological conditions was used to determine the radical scavenging properties of the chalcones and establish an antioxidant capacity compared to the vitamin E derivative Trolox. The overall biological activity of chalcones depends on their Michael acceptor reactivity, which can be influenced by different substitution patterns on the aromatic rings, as well as on the presence of free hydroxy groups on the aromatic rings of the chalcone. By these means, introducing methoxy groups in 2’,4’-position on the A-ring or in 3,4-positions on the B-ring enhances the anti-inflammatory activity, while an increasing number of hydroxy groups leads to a higher radical scavenging activity of chalcones. The observations demonstrate that an indirect influence on the electrophilicity of Michael acceptors can indeed determine the potency of chalcones as inducers of HO-1 activity and as inhibitors of inflammatory NO production. On the other hand, a hydroxy-rich chalcone like butein, which is a relatively weak Michael acceptor, can act as a powerful antioxidant determined by its radical scavenging properties.
A direct change at the Michael system can also alter the reactivity and thus the biological activity of enones. The approach of modifying the α-position of the α,β-unsaturated carbonyl system is a promising concept, because it should lead to a direct and straightforward influence on its reactivity. The chemical reactivity of α-X-enones depends on the nature of the α-substituent, thus activating or deactivating the Michael acceptor reactivity toward thiols responsible for a biological response. The influence of different α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCHs) on the induction of HO-1 protein expression and HO-1 enzymatic activity and on the other hand the inhibition of NO production, regulated by iNOS was determined in RAW264.7 murine macrophages. A clear correlation could be established between the reactivity of α-X-TMCHs, demonstrated by their thia-Michael addition reaction with cysteamine (k2 values) and their biological activity. The results demonstrate that a rather moderate electrophilicity of α-X-TMCHs is a crucial factor determining their potency to induce HO-1 activity or inhibit NO production. This could be observed for the most electrophilic TMCHs in our screen, α-CN-TMCH and α-NO2-TMCH, displaying relatively high k2 values in the thia-Michael addition reaction. Both TMCHs were only weak inhibitors of iNOS activity and showed no induction of HO-1 activity or protein expression, which may suggest an alternative pathway of the chalcones in the cell, such as a consumption by GSH, thus preventing them to reach their target SH-groups. The more intermediate electrophiles (with lower k2 values), α-CF3-TMCH, α-Br-TMCH and α-Cl-TMCH however, displayed a high activity in the biological screening of the α-X-TMCHs. Here, α-CF3-TMCH was the most active chalcone, with an IC50 value of NO production of 120 ± 62 nM and a 2.7 ± 0.5 fold induction of HO-1 activity in RAW264.7 macrophages. The importance of a sufficient Michael acceptor activity of α-X-modified enones required for a reasonable biological activity was demonstrated by the model compounds α-X-Limno-CPs, derived from the natural product limnophilaspiroketone. The α-X-Limno-CPs exerted a weak anti-inflammatory activity in inhibiting the NO production in RAW264.7 macrophages, due to their overall electron rich enone system making them weak electrophiles compared to the α-X-TMCHs.
The naturally occurring (+)-arteludovicinolide A and its synthetic enantiomer (-)-arteludo-vicinolide A were shown to display anti-inflammatory activity, demonstrated by their ability to inhibit the NO production and induce HO-1 activity in RAW264.7 macrophages. (+)-Arteludo-vicinolide A was the more active enantiomer, displaying an inhibitory effect of the NO production with an IC50 value of 4.9 ± 1.1 µM and an induction of HO-1 activity of 2.1 ± 0.7 fold after 6 h of incubation. The functional groups on these sesquiterpenes, a α-methylene group and a cyclopentenone ring may be involved in their biological activity, since it was shown that the α methylene group in the lactone ring is crucial for the anti-inflammatory activity of the natural product.
In conclusion, the Michael acceptor functionality of electrophilic enones proved to be crucial for their biological activity, regarding their potency as inducers of the cytoprotective and anti-inflammatory HO-1 activity and protein expression and as inhibitors of proinflammatory iNOS activity. α,β-Unsaturated carbonyl compounds are a very useful class of substances. A systematic manipulation of the Michael acceptor reactivity by modifying the α-position of the enone system leads to a fine-tuned biological activity in chalcone scaffolds. This approach might be highly valuable for drug design in order to predict the biological activity.
Translation of the abstract (German)
Neue Konzepte zur Therapie von chronisch-entzündlichen Krankheiten oder Autoimmun-Erkrankungen wie Multiple Sklerose und rheumatoide Arthritis sowie bei Krebserkrankungen stellen einen bedeutenden Beitrag zur medizinischen Wirkstoffforschung dar. Als wichtige Zielstrukturen zur Entwicklung neuer Entzündungshemmer spielt auf der einen Seite die Inhibierung der proinflammatorischen induzierbaren ...
Translation of the abstract (German)
Neue Konzepte zur Therapie von chronisch-entzündlichen Krankheiten oder Autoimmun-Erkrankungen wie Multiple Sklerose und rheumatoide Arthritis sowie bei Krebserkrankungen stellen einen bedeutenden Beitrag zur medizinischen Wirkstoffforschung dar. Als wichtige Zielstrukturen zur Entwicklung neuer Entzündungshemmer spielt auf der einen Seite die Inhibierung der proinflammatorischen induzierbaren Stickstoffmonoxid-Synthase (iNOS) und auf der anderen Seite die Induktion der antientzündlichen Hämoxygenase-1 (HO-1) eine bedeutende Rolle. α,β-Ungesättigte Carbonylverbindungen (Enone) können aufgrund ihrer Michael-Akzeptor-Reaktivität mit nucleophilen Thiolgruppen regulatorischer Cysteine zu einer Inhibierung von NF-κB oder zu einer Aktivierung von Nrf2 führen, zweier Transkriptionsfaktoren, die für die Expression von iNOS bzw. HO-1 verantwortlich sind.
Ziel dieser Arbeit war es, biologisch aktive bzw. aufgrund ihrer Struktur potentiell aktive Naturstoffe und Naturstoffanaloga, welche eine α,β-ungesättigte Carbonyleinheit besitzen, auf ihr Potential als mögliche Leitstrukturen für neue Entzündungshemmer zu untersuchen. Dabei wurden eine Vielzahl von Verbindungen auf ihre antientzündliche und antioxidative Aktivität in unterschiedlichen biologischen Testsystemen untersucht. In der murinen Makrophagen-Zelllinie RAW264.7 wurde die antientzündliche Wirkung der Verbindungen anhand ihrer Fähigkeit, die HO-1-Aktivität zu induzieren, mit einem neu entwickelten HO-1-Aktivitätsassay ermittelt. Der Einfluss der Substanzen auf die NO-Produktion, aufgrund einer Inhibierung von iNOS wurde mit den sogenannten Grieß-Nitrat-Assay in RAW264.7 Makrophagen, die mit dem bakteriellen Endotoxin Lipopolysaccharid (LPS) stimuliert wurden, bestimmt. Zusätzlich konnte die Radikalfängereigenschaft der Verbindungen, vor allem von Polyphenolen, in einem zellfreien Assay, dem ORAC-Fluorescein-Assay, bestimmt werden und somit ihre antioxidative Wirkung im Vergleich zum bekannten Antioxidans und Vitamin E Derivat Trolox ermittelt werden.
Auf der Suche nach neuen HO-1-Stimulatoren wurde ein zuverlässiger und leistungsfähiger ELISA-basierter Assay zur Aktivitätsmessung von HO-1 in vitro etabliert. Hierbei wurde als Modell die Zelllinie RAW264.7 verwendet, die mit den potentiellen HO-1-Stimulatoren inkubiert wurde. Der HO-1-Aktivitätsassay kombiniert die Enzymaktivität der Hämoxygenase und der Biliverdinreduktase (BVR), die im Zelllysat bestimmt wird, mit der Quantifizierung des Reaktionsproduktes Bilirubin (BR) mittels eines monoklonalen anti-Bilirubin-Antikörpers in einem indirekten Sandwich-ELISA (Enzyme Linked Immunosorbent Assay). Die HO-1-Aktivität kann somit als [pmol Bilirubin/h/mg Protein] bestimmt werden und als x-fache HO-1-Aktivität im Vergleich zu Kontrollzellen angegeben werden. Zur Etablierung des HO-1-Aktivitätsassays wurden in einem Screening 18 verschiedene Verbindungen mit einer Enonstruktur auf ihrer HO-1-Induktion in RAW264.7 Makrophagen getestet und die bekannte Aktivität der meisten Verbindungen wurde bestätigt. Als ein potenter HO-1-Stimulator wurde in der Testreihe das Chalkon DHDMCH (2’,4’-Dihydroxy-3,4-dimethoxychalkon) mit einer maximalen Induktion der HO-1-Aktivität von 6.1 ± 2.3 fach nach 6 h Stimulation gefunden. Zusätzlich konnte die zeitabhängige Induktion von HO-1 in RAW264.7 Zellen in Gegenwart von 10 µM DHDMCH auf der Ebene der Proteinexpression mittels Western Blot-Analyse und der HO-1-Enzymaktivität mittels HO-1-Assay gezeigt werden. In einem Kontrollexperiment konnte weiterhin die HO-1-Aktivität mit dem bekannten HO-1-Enzyminhibitor Zinn-Protoporphyrin IX (SnPPIX) erfolgreich gehemmt werden. Der HO-1-Aktivitätsassay wurde auch in humanen dendritischen Zellen erfolgreich durchgeführt und bestätigt somit dessen Anwendung in unterschiedlichen Zelltypen.
Es konnte gezeigt werden, dass eine Modifikation des α,β-ungesättigten Carbonylsystems in 2’,3,4,4’-Tetramethoxychalkonen (α-X-TMCH) zu einer Modifikation der Michael-Akzeptor-Eigenschaften solcher Verbindungen und damit ihren biologischen Eigenschaften führt. Mit der Einführung von elektronenziehenden bzw. elektronenschiebenden Substituenten in der α-Position des α,β-ungesättigten Carbonylsystems konnte die Reaktivität der α-X-TMCH variiert werden, was sich auch in ihrer biologischen Aktivität bezüglich ihrer HO-1-Induktion der Proteinexpression und Enzymaktivität und ihrer Inhibierung der iNOS-Aktivität in RAW264.7 Makrophagen wiedergespiegelt hat. In der Testreihe wiesen dabei die intermediär reaktiven α-CF3-TMCH, α-Br-TMCH und α-Cl-TMCH eine hohe biologische Aktivität auf. Dabei zeigte α-CF3-TMCH einen IC50-Wert der NO-Produktion von 120 ± 62 nM und eine 2.7 ± 0.5 fache Induktion der HO-1-Aktivität in RAW264.7 Makrophagen. Stark reaktive Verbindungen, wie zum Beispiel α-CN-TMCH zeigten dagegen eine schwache biologische Aktivität, was die Annahme zulässt, dass starke Elektrophile auf anderem Wege in der Zelle neutralisiert werden, wie zum Beispiel durch das Peptid Glutathion (GSH).
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