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The 5'-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 β 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the current study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant RUTA GRAVEOLENS L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the IN SILICO prediction of putative biological targets, i. e., target fishing. Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB (2)). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory IN VITRO activity on AChE (IC (50) 34.7 muM). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC (50) values of 11.98 muM and 3.19 muM, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB (2) ligand was obtained, i. e., rutamarin. Interestingly, in experimental studies only this compound showed a selective activity to the CB (2) receptor ( Ki of 7.4 muM) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. GRAVEOLENS on three different proteins has shown promise as an IN SILICO tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

We developed an immunoassay with antibodies against retrorsine to detect the closely related senecionine, the main alkaloid in various Asteraceae. Sensitivity is about 23 pg (68 fmol) for senecionine. Cross reactivity of monocrotalin, retrorsine N-oxide, senkirkine (< 0.1%), and seneciphylline (3.6-34.5%) was resolved. The alkaloid content in leaves of Petasites hybridus (Asteraceae) was determined to be 3.86 ppm, calculated as senecionine, which was far less than in rhizomes (104.8 ppm). Additionally, we proved specificity by taking immunograms from different parts of Petasites hybridus. The test is a suitable tool for assessing the toxic potential of medicinal plants containing senecionine.