Development of a pharmacophoric deconvolution method for the targeted discovery of bioactive natural products

A new method combining 2D-NMR techniques and bioactivity assay was developed with the aim of accelerating the discovery process of new bioactive natural products. Based on differential analysis of 2D-NMR spectra (DANS), the method allows identifying molecular fingerprints related to biological activity (pharmacophoric fingerprint) within a complex natural mixture. This method combined with the HPLC-DAD-MS-SPE-NMR hyphenated technique leads to the reliable structural identification of the activity-bearing compounds.

The proof of concept was made with an algal extract (Bifurcaria bifurcata). Subtraction of active fractions 3 (98% of inhibition at 100 µg/mL) and 4 (97% of inhibition at 100 µg/mL) allowed to obtain the pharmacophoric fingerprint and led to the identification of eleganolone as active compound. Compared to the traditional “bioguided fractionation” method, it was three times faster (3 weeks/3 months) and needed about 400 times less starting raw material (0.5 mg/200 mg) than the traditional method [1].

Fig. 1: Workflow of the pharmacophoric deconvolution method. A biologically active extract is fractioned by MPLC to give five fractions (step 1). Each fraction is biologically evaluated and analysed by NMR spectroscopy (step 2). Differential analyses of NMR data allow the access to fingerprint of active molecule(s) (step 3). The molecules contained in the active fraction are separated and analyzed by HPLC-DAD-MS-SPE-NMR (step 4). The active molecule is identified by comparison between the pharmacophoric fingerprint obtained in the step 3 and ¹H-¹H COSY spectra of each molecule obtained in the step 4, as well as by complementary data acquired in this step (¹H, ¹H-¹H NOESY, ¹H-¹³C HSQC).

Acknowledgements: We are grateful to Labex Medalis and Région Alsace for a fellowship (L. Margueritte).

Keywords: Pharmacophoric deconvolution, fingerprint, DANS, HPLC-DAD-MS-SPE-NMR.

References:

[1] Galle JB, Attioua B, Kaiser M, Rusig AM, Lobstein A, Vonthron-Senecheau C. Eleganolone, a diterpene from the french marine alga Bifurcaria bifurcata inhibits growth of the human pathogens Trypanosoma brucei and Plasmodium falciparum. Mar Drugs 2013; 11: 599 – 610