Zimmermann, Stefanie. Screening, identification, structure-activity, and mode of action studies with new antitrypanosomal leads of plant and fungal origin. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10482
This thesis describes the search of new natural products (NPs) from nature. Over the last seven years we collected 724 plants and 64 fungi. The material was subsequently extracted and tested in vitro against T. b. rhodesiense, Plasmodium falciparum (the causative agent of malaria), Leishmania donovani (leishmaniasis), and T. cruzi (Chagas disease) to find potential hits. From the total 2151 extracts, 17.9% showed activity of more than 50% at 4.81 µg/mL test concentration against at least one parasite, and 3.4% showed potency of more than 50% at 0.81 µg/mL test concentration, respectively. Overall the plant extracts had six times higher "hit-rates" (15.3%) than the fungi extracts (2.6%), both resulting in high potencies against T. b. rhodesiense and P. falciparum. Yet, with up to 5 millions fungi, which outnumber higher plants by 16:1, the kingdom remains a relatively poorly studied source.
One of the antitrypanosomal hits was a dichloromethane (DCM) extract of the cornflower Centaurea salmantica with a growth inhibition of 61% tested at 4.81 µg/mL against T. b. rhodesiense. HPLC-based activity profiling led to the identification of the sesquiterpene lactone (STL) cynaropicrin (CYN), which was the first plant NP to show in vivo efficacy in T. b. rhodesiense infected mice, treated i.p. at 10 mg/kg/b.i.d. for four consecutive days. Despite of more than 10'000 known STLs is a better understanding of the structural features, which contribute to activity, expedient. The established structure-activity relationship (SAR) study included 18 natural STLs and demonstrated that antitrypanosomal and cytotoxic effect depended on their a,ß-unsaturated enone moieties. Many bioactivities of STLs have been attributed to a nucleophilic Michael-addition of these functional motifs to biological thiols. Considering that trypanosomes depend on their unique trypanothione-based redox system to deal with oxidative stress and to maintain a reducing intracellular milieu and that CYN contains reactive exocyclic a,ß-unsaturated methylenes, we anticipated that the mechanism of action depended on a direct interference with glutathione (GSH) and trypanothione (T(SH)2) in the cells. After 5 min. of CYN's exposure to trypanosomes, the intracellular thiol pool was completely depleted and a GS-CYN-monoadduct as well as a T(S-CYN)2-bisadduct were formed. This led to apoptosis of the trypanosomes over 40 min. linked to phenotype transformations from the typical slender to a stumpy-like form. Additionally, ornithine quantification studies by tandem mass spectroscopy (MS/MS) showed that ornithine decarboxylase (ODC) is a potential secondary target for CYN.
To improve CYN's pharmacokinetic (PK) profile the a,ß-unsaturated exocyclic double bond at the lactone was masked to create an amine prodrug with increased aqueous solubility and reduced unspecific binding to biological thiols. Through subsequent bioactivation the prodrug would be converted back to CYN and it would display a higher concentration on the target side. The lead optimization did not reward any better antitrypanosomal in vivo efficacy after oral application, but the prodrug had an improved in vivo cytotoxic profile. Further PK studies with other orally applied STL amino derivatives are needed to demonstrate if the use of amino STLs as prodrugs is a reasonable approach to improve STLs suitability as antitrypanosomal drug.
|Advisors:||Hamburger, Matthias Otto|
|Committee Members:||Brun, Reto and Seebeck, Thomas|
|Faculties and Departments:||05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Pharmazeutische Biologie (Hamburger)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||131 S.|
|Last Modified:||30 Jun 2016 10:53|
|Deposited On:||28 Aug 2013 13:01|
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