Combined Use of Extract Libraries and HPLC-Based Activity Profiling for Lead Discovery: Potential, Challenges, and Practical Considerations

Abstract

A 96-well format plant extract library and a tailored technology platform have been set up for the discovery of new natural product lead compounds. The considerable advantages of the library approach are discussed. Key considerations such as sample generation, logistics, and data management are addressed. The potential of a HPLC-based profiling approach combining off-line bioassays with in-line and off-line spectroscopy (including HR-ESIMS and microprobe NMR) for tracking bioactivity is demonstrated with a selection of examples encompassing different types of bioassay formats. The information generated by this approach with regards to hit prioritization and preliminary structure activity-relationships is discussed. Practical aspects, such as validation of profiling protocols, the amounts of extracts to be applied, and the re-dissolution of fractions are addressed. Such information is intended for scientists aiming at implementing library-based discovery platforms in their own laboratory.

Key words

extract library - database - microfractionation - HPLC-based activity profiling

• References

• 1 Newmann DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012; 75: 311-335
• 2 Potterat O, Hamburger M. Natural products in drug discovery – Concepts and approaches for tracking bioactivity. Curr Org Chem 2006; 10: 899-920
• 3 Camp D, Davis RA, Campitelli M, Ebdon J, Quinn RJ. Drug-like properties: guiding principles for design of natural product libraries. J Nat Prod 2012; 75: 72-81
• 4 Johnson TA, Sohn J, Inman WD, Estee SA, Loveridge ST, Vervoort HC, Tenney K, Liu J, Ang KH, Ratnam J, Bray WM, Gassner NC, Shen YY, Lokey RS, McKerrow JH, Boundy-Mills K, Nukanto A, Kanti A, Julistiono H, Kardono LBS, Bjeldanes LF, Crews P. Natural product libraries to accelerate the high-throughput discovery of therapeutic leads. J Nat Prod 2011; 74: 2545-2555
• 5 Bugni TS, Harper MK, McCulloch MWB, Reppart J, Ireland CM. Fractionated marine invertebrate extract libraries for drug discovery. Molecules 2008; 13: 1372-1383
• 6 Zitha-Bovens E, Maas P, Wife D, Tijhuis J, Hu QN, Kleinöder T, Gasteiger J. COMDECOM: Predicting the lifetime of screening compounds in DMSO solution. J Biomol Screen 2009; 14: 557-565
• 7 Hamburger M, Hostettmann K. Bioactivity in plants: The link between phytochemistry and medicine. Phytochemistry 1991; 30: 3864-3874
• 8 Potterat O, Hamburger M. Concepts and technologies for tracking bioactive compounds in natural product extracts: generation of libraries and hyphenation of analytical processes with bioassays. Nat Prod Rep 2013; 30: 546-564
• 9 Buckingham J. Dictionary of natural products on DVD. London: Chapman and Hall/CRC; 2014
• 10 Zaugg J, Baburin I, Sommer B, Kim HJ, Hering S, Hamburger M. HPLC-based activity profiling: Discovery of piperine as a positive GABA_A receptor modulator targeting a benzodiazepine-independent binding site. J Nat Prod 2010; 73: 185-191
• 11 Kim HJ, Baburin I, Khom S, Hering S, Hamburger M. HPLC-based activity profiling approach for the discovery of GABA_A receptor ligands using an automated two microelectrodes voltage clamp assay on Xenopus oocytes. Planta Med 2008; 74: 521-526
• 12 Adams M, Zimmermann S, Kaiser M, Brun R, Hamburger M. A protocol for HPLC-based activity profiling for natural products with activities against tropical parasites. Nat Prod Commun 2009; 4: 1377-1381
• 13 Bohni N, Cordero-Maldonado ML, Maes J, Siverio-Mota D, Marcourt L, Munck S, Kamuhabwa AR, Moshi MJ, Esguerra CV, de Witte PAM, Crawford AD, Wolfender JL. Integration of microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products. Plos One 2013; 8: e64006
• 14 Collin RA, Ng TB, Fong WP, Wan CC, Yeung HW. Removal of polyphenolic compounds from aqueous plant extracts using polyamide minicolumns. Biochem Molec Biol Inter 1998; 45: 791-796
• 15 Grabher P, Durieu E, Kouloura E, Halabaki M, Skaltsounis LA, Meijer L, Hamburger M, Potterat O. Library-based discovery of DYRK1A/CLK1 inhibitors from natural product extracts. Planta Med 2012; 78: 951-956
• 16 Schramm A, Jähne EA, Baburin I, Hering S, Hamburger M. Natural products as potential hERG channel inhibitors – Outcomes from a screening of widely used herbal medicines and edible plants. Planta Med 2014; , submitted
• 17 Khom S, Strommer B, Schöffmann A, Hintersteiner J, Baburin I, Erker T, Schwarz T, Schwarzer C, Zaugg J, Hamburger M, Hering S. GABA_A receptor modulation by piperine and a non-TRPV1 activating derivative. Biochem Pharmacol 2013; 85: 1827-1836
• 18 Li Y, Plitzko I, Zaugg J, Hering S, Hamburger M. HPLC-based activity profiling for GABA_A receptor modulators: A new dihydroisocoumarin from Haloxylon scoparium . J Nat Prod 2010; 73: 768-770
• 19 Zaugg J, Ebrahimi SN, Smiesko M, Baburin I, Hering S, Hamburger M. Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations. Phytochemistry 2011; 72: 2385-2395
• 20 Zaugg J, Khom S, Eigenmann D, Baburin I, Hamburger M, Hering S. Identification and characterization of GABA_A receptor modulatory diterpenes from Biota orientalis that decreases locomotor activity in mice. J Nat Prod 2011; 74: 1764-1772
• 21 Zaugg J, Eickmeier E, Rueda DC, Hering S, Hamburger M. HPLC-based activity profiling of Angelica pubescens roots for new positive GABA_A receptor modulators in Xenopus oocytes. Fitoterapia 2011; 82: 434-440
• 22 Zaugg J, Eickmeier E, Ebrahimi SN, Baburin I, Hering S, Hamburger M. Positive GABA_A receptor modulators from Acorus calamus and structural analysis of (+)-dioxosarcoguaiacol by 1D and 2D NMR and molecular modeling. J Nat Prod 2011; 74: 1437-1443
• 23 Yang X, Baburin I, Plitzko I, Hering S, Hamburger M. HPLC-based activity profiling for GABA_A receptor modulators from the traditional Chinese herbal drug Kushen (Sophora flavescens root). Mol Divers 2011; 15: 361-372
• 24 Kim HJ, Baburin I, Zaugg J, Ebrahimi SN, Hering S, Hamburger M. HPLC-based activity profiling – Discovery of sanggenons as GABA_A receptor modulators in the traditional Chinese drug Sang bai pi (Morus alba root bark). Planta Med 2012; 78: 440-447
• 25 Rueda DC, Zaugg J, Quitschau M, Reich E, Hering S, Hamburger M. Discovery of GABA_A receptor modulator aristolactone in a commercial sample of the Chinese herbal drug „Chaihu“ (Bupleurum chinense roots) unravels adulteration by nephrotoxic Aristolochia manshuriensis roots. Planta Med 2012; 78: 207-210
• 26 Schramm A, Ebrahimi SN, Raith M, Zaugg J, Rueda DC, Hering S, Hamburger M. Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators. Phytochemistry 2013; 96: 318-329
• 27 Rueda DC, Schöffmann A, De Mieri M, Raith M, Jähne E, Hering S, Hamburger M. Identification of dihydrostilbenes in Pholidota chinensis as a new scaffold for GABA_A receptor modulators. Bioorg Med Chem 2014; 22: 1276-1284
• 28 Rueda DC, De Mieri N, Hering S, Hamburger M. HPLC-based activity profiling for GABA_A receptor modulators in Adenocarpus cincinnatus . J Nat Prod 2014; 77: 640-649
• 29 Zimmermann S, Kaiser M, Brun R, Hamburger M, Adams M. Cynaropicrin: The first plant natural product with in vivo activity against Trypanosoma brucei . Planta Med 2012; 78: 553-556
• 30 Julianti T, Hata Y, Zimmermann S, Kaiser M, Hamburger M, Adams M. Antitrypanosomal sesquiterpene lactones from Saussurea costus . Fitoterapia 2011; 82: 955-959
• 31 Adams M, Gschwind S, Zimmermann S, Kaiser M, Hamburger M. Renaissance remedies: Antiplasmodial triterpenoids from Alisma plantago-aquatica L. (Alismataceae). J Ethnopharmacol 2011; 135: 43-47
• 32 Zimmermann S, Thomi S, Kaiser M, Hamburger M, Adams M. Screening and HPLC-based activity profiling for new antiprotozoal leads from European plants. Sci Pharm 2012; 80: 205-213
• 33 Adams M, Christen M, Plitzko I, Zimmermann S, Brun R, Kaiser M, Hamburger M. Antiplasmodial lanostanes from the Ganoderma lucidum mushroom. J Nat Prod 2010; 73: 897-900
• 34 Adams M, Plitzko I, Kaiser M, Brun R, Hamburger M. HPLC-profiling for antiplasmodial compounds – 3-Methoxycarpachromene from Pistachia atlantica . Phytochem Lett 2009; 2: 159-162
• 35 Slusarczyk S, Zimmermann S, Kaiser M, Matkowski A, Hamburger M, Adams M. Antiplasmodial and antitrypanosomal activity of tanshinone-type diterpenoids from Salvia miltiorrhiza . Planta Med 2011; 77: 1594-1596
• 36 Moradi-Afrapoli F, Ebrahimi SN, Smiesko M, Raith M, Zimmermann S, Brun R, Hamburger M. Bisabololoxide derivatives from Artemisia persica, and determination of their absolute configurations by ECD. Phytochemistry 2013; 85: 143-152
• 37 Ebrahimi SN, Zimmermann S, Zaugg J, Smiesko M, Brun R, Hamburger M. Abietane diterpenoids from Salvia sahendica – antiprotozoal activity and determination of their absolute configurations. Planta Med 2013; 79: 150-156
• 38 Hata Y, Raith M, Ebrahimi SN, Zimmermann S, Mokoka T, Naidoo D, Fouche G, Maharaj V, Kaiser M, Brun R, Hamburger M. Antiprotozoal isoflavan quinones from Abrus precatorius ssp. africanus . Planta Med 2013; 79: 492-498
• 39 Mokoka TA, Xolani KP, Zimmermann S, Hata Y, Moodley N, Adams M, Kaiser M, Maharaj V, Koorbanally NA, Hamburger M, Brun R, Fouche G. Antiprotozoal Screening of 60 South African plants, and the identification of the antitrypanosomal eudesmanolides schkurin 1 and 2. Planta Med 2013; 79: 1380-1384
• 40 Hata Y, Ebrahimi SN, De Mieri M, Zimmermann S, Mokoka T, Naidoo D, Fouche G, Maharaj GV, Kaiser M, Brun R, Potterat O, Hamburger M. Antitrypanosomal isoflavan quinones from Abrus precatorius . Fitoterapia 2014; 93: 81-87
• 41 Vidal V, Potterat O, Louvel S, Hamy F, Mojarrab M, Sanglier JJ, Klimkait T, Hamburger M. Library-based discovery and characterization of daphnane diterpenes as potent and selective HIV inhibitors in Daphne gnidium . J Nat Prod 2012; 75: 414-419
• 42 Schramm A, Baburin I, Herring S, Hamburger M. hERG channel inhibitors in extracts of Coptidis rhizome. Planta Med 2012; 77: 692-697
• 43 Danz H, Stoyanova S, Wippich P, Brattström A, Hamburger M. Identification and Isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria . Planta Med 2001; 67: 411-416
• 44 Oberthür C, Jäggi U, Hamburger M. HPLC-based activity profiling of a lipophilic Isatis tinctoria leaf extract for 5-lipoxygenase inhibitory activity. Fitoterapia 2005; 76: 324-332
• 45 Dittmann K, Riese U, Hamburger M. HPLC-based bioactivity profiling of plant extracts – development of an assay for the identification of monoamine oxidase A inhibitors using human recombinant MAO A. Phytochemistry 2004; 65: 2885-2891
• 46 Dittmann K, Gerhäuser C, Hamburger M. HPLC-based activity profiling of Salvia miltiorrhiza for MAO A and iNOS inhibitory activities. Planta Med 2004; 70: 909-913
• 47 Lin Y, Schiavo S, Orjala J, Vouros P, Kautz R. Microscale LC-MS-NMR Platform applied to the identification of active cyanobacterial metabolites. Anal Chem 2008; 80: 8045-8054
• 48 Glauser G, Gindro K, Fringeli J, De Joffrey JP, Rudaz S, Wolfender JL. Differential analysis of mycoalexins in confrontation zones of grapevine fungal pathogens by ultrahigh pressure liquid chromatography/time-of-flight mass spectrometry and capillary nuclear magnetic resonance. J Agric Food Chem 2009; 57: 1127-1134
• 49 Gassner NC, Tamble CM, Bock JE, Cotton N, White KN, Tenney K, St Onge RP, Proctor MJ, Giaever G, Nislow C, Davis RW, Crews P, Holman TR, Lokey RS. Accelerating the discovery of biologically active small molecules using a high-throughput yeast halo assay. J Nat Prod 2007; 70: 383-390
• 50 Van Elswijk DA, Irth H. Analytical tools for the detection and characterization of biologically active compounds from Nature. Phytochem Rev 2003; 1: 427-439
• 51 Kool J, Giera M, Irth H, Niessen WMA. Advances in mass spectrometry-based post column bioaffinity profiling of mixtures. Anal Bioanal Chem 2011; 399: 2655-2668
• 52 Giera M, Heus F, Janssen L, Kool J, Lingeman H, Irth H. Microfractionation revisited: A 1536 well high resolution screening assay. Anal Chem 2009; 81: 5460-5466
• 53 Kool J, Heus F, De Kloe G, Lingeman H, Smit AB, Leurs R, Edink E, De Esch IJP, Irth H, Niessen WMA. High-resolution bioactivity profiling of mixtures toward the acetylcholine binding protein using a nanofractionation spotter technology. J Biomol Screen 2011; 16: 917-924