Johannesen, Ane

Johansen, Kenneth

Jäger, Anna

Kjellerup, Lasse

Nyberg, Nils

Stærk, Dan

Vinther, Anne-Marie

Wubshet, Sileshi Gizachew

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetIdentification of Fungal H+-ATPase Inhibitors by Microfractionation and HPLC-HRMS-SPE-NMRWubshet, Sileshi Gizachew; Johansen, Kenneth; Johannesen, Ane; Nyberg, Nils; Jäger,Anna; Kjellerup, Lasse; Vinther, Anne-Marie; Stærk, DanPublication date:2014Citation for published version (APA):Wubshet, S. G., Johansen, K., Johannesen, A., Nyberg, N., Jäger, A., Kjellerup, L., ... Stærk, D. (2014).Identification of Fungal H+-ATPase Inhibitors by Microfractionation and HPLC-HRMS-SPE-NMR. Poster sessionpræsenteret ved 35th Danish NMR Meeting, Kobæk Strand, Danmark.Download date: 03. Feb. 2020Identification of Fungal H+-ATPase Inhibitors by Microfractionation and HPLC-HRMS-SPE-NMR   Background - Fungal Fight    Sileshi Wubshet1*, Kenneth Kongstad1, Ane Johannesen1, Nils Nyberg1, Anna K. Jäger1, Lasse Kjellerup2, Anne-Marie Winther2, Dan Stærk1  1Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark 2PCOVERY, Thorvaldsensvej 57, DK-1871 Frederiksberg C, Denmark *E-mail: sgw@sund.ku.dk FA C U LT Y  O F  H E A LT H  A N D  M E D I C A L  S C I E N C E S   U N I V E R S I T Y  O F  C O P E N H A G E N    A large number of fungal proteins have been proposed as potential targets for novel antifungal agents,1 however, current available antifungal agents are primarily targeting the intracellular membrane biosynthesis1 and thus need to enter the fungus to act. In our search for novel and more efficient antifungal compounds, we are focusing on the plasma membrane (PM) H+-ATPase enzyme as target.  Plants are exposed to a wide array of phytopathogenic fungi in their natural habitat, and have been forced to develop antifungal metabolites in order to survive.2,3 Hence, as previously suggested by Monk and coworkers,4 it is reasonable to assume that some plants have the PM H+-APTase enzyme as target for the antifungal metabolites. However, plant extracts are very complex mixtures, and the traditional bioassay-guided fractionation used for identification of individual bioactive components are very time-consuming and suffers from inherently low resolution during the fractionation process. To circumvent this, we have developed a bioanalytical platform that combines high-resolution microplate bioassays5  with HPLC-SPE-NMR.6 In the present work, we report crude extract screening of 48 plant-extracts for fungal PM H+-ATPase inhibitors - followed by high-resolution bioassay and HPLC-SPE-NMR analysis for identification of individual active constituents.   Crystal structure of the H+-ATPase in plants.     The setup - HR screening/HPLC-SPE-NMR  Empowered by the separation capabilities of analytical HPLC, high-resolution bioassay-guided HPLC-SPE-NMR has been shown to provide a platform that can substitute the traditional bio-guided fractionation with considerable reduction of workload and improved efficiency.7 In this approach, chromatographically separated fractions will be subjected to a given bio-activity test to afford the so-called biochromatogram. The resulting biochromatogram will then be used as a tool for targeted analysis of active constituents. Such biochromatogram can be acquired through micro-fractionation followed by offline assay.              HR screening/HPLC-SPE-NMR     HPLC-HRMS-SPE-NMR    Results - Crude extract screening § From 48 plants to 20 plants Extracts were tested in three different concentrations and those showing inhibition higher than 95% for all concentrations or a concentration-dependent activity profile were selected for semi-high-resolution screening.          No Species Family Plant part Percent inhibition 7.5 mg/mL  15 mg/mL  30 mg/mL 1 Alafia barteri Apocynaceae Leaves + twigs 27 11 33 19 33 27 2 Alchornea cordifolia Euphorbiaceae 99 98 98 98 97 97 6 Baissea leonensis Apocynaceae Leaves + twigs 99 99 99 99 99 100 7 Baissea leonensis Apocynaceae Fruit 98 98 98 98 97 97 11 Caloncoba gilgiana Achariaceae Leaves 99 99 98 97 99 97 12 Croton longiracemosus Euphorbiaceae Branches -8 -13 3 9 19 7 13 Croton longiracemosus Euphorbiaceae Root bark 20 12 27 19 34 18 14 Croton membranaceus Euphorbiaceae Leaves + twigs 40 18 40 28 53 41 15 Dovyalis macrocalyx Salicaceae Stem -4 -13 21 1 35 18 16 Euadenia eminens Capparaceae Aerial parts 33 15 43 25 49 32 17 Euadenia eminens Capparaceae Root 2 -47 4 -36 13 -7 18 Gymnema sylvestre Apocynaceae Aerial parts 34 19 38 26 59 46 19 Haplocoelum foliolosum Sapindaceae Aerial parts 99 99 100 99 100 100 20 Hubertia ambavilla Asteraceae Leaves + twigs 27 29 56 58 84 86 21 Hubertia tomentosum Asteraceae Leaves 36 19 50 31 75 71 22 Hypericum scabrum Hypericaceae Whole plant 59 51 96 75 97 96 23 Lophira alata Achnaceae Leaves 41 38 60 56 96 90 24 Momordica charantia Curcurbitaceae Aerial parts 31 27 33 35 42 39 25 Mussaenda tristigmatica Rubiaceae Leaves + twigs 99 98 99 98 99 98 47 Sphenocentrum jollyanum Menispermaceae Leaves + twigs 39 24 55 36 76 68 48 Sauvagesia erecta Ochnaceae Whole plant 98 98  99 98  97 98 Results – Semi-HR-screening § From 20 plants to 2 plants The 20 samples selected for semi-high-resolution screening (assay resolution: 2.66 data points per min) were assayed for their ability to inhibit the PM H+-ATPase.          References 1.  Odds, F. C.; Brown, A. J. P.; Gow, N. A. R. Trends Microbiol. 2003, 11, 272-279.  2.  Bell, A. A. Ann. Rev. Plant Physiol. 1981, 32, 21-81.  3.  Maor, R.; Shirasu, K. Curr. Opin. Microbiol. 2005, 8, 399-404.  4.  Monk, B. C.; Perlin, D. S. Crit. Rev. Microbiol. 1994, 20, 209-223.  5.  Shi, S.-Y.; Zhou, H.-H.; Zhang, Y.-P.; Jiang, X.-Y.; Chen, X.-Q.; Huang, K.-L. TRAC – Trend. Anal. Chem. 2009, 28, 865-877. 6.  Stærk, D.; Lambert, M.; Jaroszewski, J.W. HPLC-NMR techniques for plant extract analysis. In Medicinal Plant Biotechnology. From Basic Research to Applications. Kayser, O.; Quax, W., Eds.; Viley-VCH, Weinheim, Germany, 2006, pp. 29-48. 7.  Wubshet, S.G.; Nyberg, N.T.; Tejesvi, M.V.; Pirttilä, A.M.; Kajula, M.; Mattila, S.; Staerk, D. J. Chromatogr., A 2013, 1302, 34-39.      Acknowledgment The project FF is granted by the Danish Research Council for Strategic research – Food and Health. HPLC equipment used for high-resolution bioassay profiles was obtained via a grant from The Carlsberg Foundation. The 600 MHz HPLC-HRMS-SPE-NMR system used in this work was acquired through a grant from “Apotekerfonden af 1991”, The Carlsberg Foundation, and the Danish Agency for Science, Technology and Innovation via the National Research Infrastructure funds.  Concluding remarks §  Thorough investigation of 48 plant extracts for fungal PM H+-ATPase inhibitors led to identification of two active metabolites. i.e., Chebulagic acid (1) and  Tellimagrandin II (2). §  Systematic combination of crude extract screening, high-resolution screening and HPLC-HRMS-SPE-NMR analysis allowed optimized workflow. §  High-resolution PM H+ ATPase inhibition assay allows subsequent HPLC-SPE-NMR analysis to be targeted bioactive constituents only §  Cryogenic probe detection (1.7 mm) allowed characterization of metabolites (with high PM H+-ATPase inhibition) direct from analytical-scale HPLC of crude extract     Results - HR-screening § From 2 pants to 2 compounds The two plants (Haplocoelum foliolosum and Sauvagesia erecta) showing distinct chromatographic peaks correlated with PM H+-ATPase inhibition in semi-HR screening were subjected to high-resolution screening (assay resolution: 5.33 data points per min).               From H. foliosum two peaks (peak 1 and 2) were correlated with > 80% inhibition of the PM H+-ATPase. However, despite the noticeable inhibition in both crude extract screening and semi-high resolution assay, S. erecta did not show any peak correlated to a defined inhibition profile in high-resolution assay. This can be attributed to the possible loss of aggregate activities of multiple constituents due to lower residual complexity of the tested compounds in the HR screening compared to both semi-HR and crude extract screening.  Results - HPLC-HRMS-SPE-NMR Detailed analysis of HRMS and NMR data acquired via HPLC-HRMS-SPE-NMR, led to identification of the two active metabolites as Chebulagic acid (1) and  Tellimagrandin II (2).     Results - IC50 and MIC The two HITs (Chebulagic acid (1) and Tellimagrandin II (2)) were purified by preparative scale HPLC and subjected to IC50 determination in PM H+-ATPase as well as Na+,K+-ATPase inhibition assay.                 Moreover, minimum inhibitory concentrations of the two compounds were assessed against growth of two fungal strains (Candida albicans and Saccharomyces cerevisiae).            10 plants: No significant inhibition from individual constituents 8 plants: Inhibition correlated to tannins (large polyphenols with inherent ability of precipitating proteins) 2 plants: inhibition profiles correlated to distinct peaks      1 2 Compound IC50 [µM] Pma1  NaK  Chebulagic acid  12 0,5 Tellimagrandin II 10 0,8 H. foliolosum 

Identification of Fungal H<sup>+</sup>-ATPase Inhibitors by Microfractionation and HPLC-HRMS-SPE-NMR

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Identification of Fungal H<sup>+</sup>-ATPase Inhibitors by Microfractionation and HPLC-HRMS-SPE-NMR

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