Activity-guided isolation of natural products with immunosuppressive activity

Reinhardt, Jakob K.. Activity-guided isolation of natural products with immunosuppressive activity. 2020, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: https://edoc.unibas.ch/79819/

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The worldwide incidence of autoimmune diseases is rising, especially in industrialized countries. Apart from alleviating symptoms, the aim of any treatment is to regain a balance between tolerance and immunity. As T cells play a central role in both processes, the pathways leading to their activation are attractive targets for immunosuppressant drugs. The search for new inhibitors in recent years, however, was mostly focused on biologicals like monoclonal antibodies or fusion proteins. This improved the treatment of autoimmune conditions significantly, but administration and immunogenicity limit their application. Thus, there is a need for new small molecule immunosuppressant drugs with new modes of action.
To pursue new lead compounds, we mined the chemical space contained in a library of extracts from plants used in Traditional Chinese Medicine (TCM). This pre-selection was done to focus on plants used over a long period of time with some evidence for bioactivities. To identify extracts and compounds that inhibit the proliferation of activated primary T lymphocytes isolated from human blood, we used an in vitro assay. Through FACS analysis, the assay also allowed for the direct identification of cytotoxic or necrosis inducing effects.
We screened 435 extracts from plants used in TCM at a concentration of 30 µg/mL. From these, around 40 extracts showed T cell proliferation inhibition and 6 plants in total were selected for further investigations based on the absence of cytotoxicity, availability, batch to batch reproducibility, and a literature survey. Seven extracts from these plants were subjected to HPLC activity profiling, which allowed the identification of regions of activity in four of the extracts. The compounds in these active regions were the focus of subsequent isolation of compounds from these plant extracts. The work on two plants, Artemisia argyi and Toddalia asiatica, resulted in the publication of three peer reviewed publications. The work on two other extracts is still ongoing.
The first extract was an ethyl acetate extract from the aerial parts of A. argyi. From this extract, 18 compounds, including 5 new sesquiterpene lactones were isolated. A series of four isomeric sesquiterpene lactones, two diastereomeric seco-tanapartholides and two diastereomers of canin, showed activity at micromolar concentrations (IC50 values between 1.0 and 3.7 µM). Published data on the series of canin-diastereomers gave only unsatisfactory information on their identities and absolute configurations. Thus, their absolute configurations were determined independently using ECD and VCD. The relative configurations of seco-tanapartholides A and B were only known in part and interestingly, ECD gave almost enantiomeric spectra. Therefore, the absolute configuration was solved by VCD. Visual and computational methods were used for evaluation of the spectra. The knowledge of their absolute configurations can now be used as the basis for possible development as immunosuppressant lead compounds. These results are published in the Journal of Natural Products (Vol. 82, 2019).
Next, we investigated the mode of action of the isolated compounds and the ethyl acetate extract from A. argyi. To identify the affected transcription factors, reporter cell lines for AP-1, NFAT, and NF-κB were used. The target of the A. argyi extract and the tested sesquiterpene lactones was thereby found to be located upstream of NFAT and NF-κB. We further investigated the effects of both, the extract and the sesquiterpene lactones, on calcium signaling involved in the NFAT pathway. Although some of the compounds had an effect on calcium signaling, none of them, nor a combination of all, inhibited calcium influx into the cell or from the ER as effectively as the extract itself. These results were published in Frontiers in Pharmacology (Vol. 11, 2020).
The second extract we investigated in the course of this work was the MeOH extract from the roots of T. asiatica. Here, the alkaloid nitidine was identified as a highly active constituent with an IC50 value of 0.37 µM. Another, less active (IC50 6.72 µM), alkaloid was identified along with other inactive alkaloids, lignans and coumarins, some of them glycosylated. A combination of ECD, OR, GCMS analysis of hydrolyzed sugars, enantioselective HPLC analysis of hydrolyzed aglycones and NMR in the presence of the chiral shift reagent Eu(hbc)3 was used to identify the absolute configurations of these compounds. Three of the compounds were new natural products. The results are published in the Journal of Natural Products (Vol. 83, 2020).
From identifying several compounds with significant activity in inhibiting T cell proliferation, we found the CFSE assay to be useful for the identification of new lead compounds with relevant activities. As many of the natural products found did not have well described absolute configurations, we used a broad array of methods to fill this gap and publish the results for use by future researchers. The combination of ECD (as a sensitive method) with complementary techniques like VCD, chromatography of hydrolyzed compounds, or the use of shift reagents in NMR proved to be very effective. It enabled us to solve challenging cases like seco-tanapartholides A and B from A. argyi and the glycosylated coumarins from T. asiatica.
Advisors:Hamburger, Matthias and Smiesko, Martin and Opatz, Till
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Pharmazeutische Biologie (Hamburger)
UniBasel Contributors:Hamburger, Matthias and Smiesko, Martin
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13957
Thesis status:Complete
Number of Pages:ix, 225
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss139570
edoc DOI:
Last Modified:01 Mar 2021 15:27
Deposited On:14 Jan 2021 14:19

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