Accelerating drug lead discovery from nature by high-resolution bioactivity profiling combined with HPLC-HRMS-SPE-NMR

Efficient dereplication of known compounds as well as full structural identification of new compounds directly from crude plant extracts has become possible by hyphenation of analytical-scale high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR [1]. Cryoprobe technology even allows acquisition of direct-detected ¹³C NMR spectra of major constituents [2], and database-assisted NMR structure elucidation eases the otherwise time-consuming interpretation of the obtained NMR data [3]. However, HPLC-HRMS-SPE-NMR does only provide structural information of individual metabolites associated with the peaks in the HPLC chromatogram, and knowledge of their bioactivity has until now awaited preparative-scale isolation and subsequent pharmacological assaying. However, the recent combination of microplate-based high-resolution bioassays (yielding one or more biochromatogram(s)) and HPLC-HRMS-SPE-NMR [4 – 6], has allowed correlation of the biochromatogram(s) and the HPLC chromatogram – and thus allowed subsequent HPLC-HRMS-SPE-cryoNMR experiments towards the bioactive constituents only. A schematic workflow of the high-resolution bioassay/HPLC-HRMS-SPE-NMR technology (modified from reference 4) is shown in the figure below.

Some recent examples of the successful use of single, double and triple high-resolution bioactivity profiling in combination with HPLC-HRMS-SPE-cryoNMR will be presented. Emphasis will be on describing the processes involved in the high-resolution bioassay/HPLC-HRMS-SPE-cryoNMR technology – for e.g. α-glucosidase, α-amylase, and PTP1B – all key-enzymes in the management of type 2 diabetes.

Acknowledgements: Arife Önder is acknowledged for technical assistance in the mentioned work.

Keywords: High-resolution bioassay, microfractionation, biochromatogram, HPLC-HRMS-SPE-NMR.

References:

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