Native metabolomics identifies the rivulariapeptolide family of protease inhibitors

Abstract

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides

“…18 The top CSI:FingerID results did not contain endolides, but the CANOPUS annotations "valine and derivatives" hinted at a valine moiety, while "pyrrolidinecarboxamides" and "azacyclic compounds" hinted at a proline moiety in the new compound (Figure S6) (as reported before for other proline-containing natural products). 18,33 Altogether, the CANOPUS and SIRIUS results supported the MassQLannotated molecular networking predicted amino acid composition NMefAla, Pro, Leu, and Val for m/z 461.2754.…”

Marine indole alkaloid diversity and bioactivity. What do we know and what are we missing?

“…Examples include NP Analyst, an open access online platform that combines metabolomics data (from most commonly used open-source MS platforms) with bioactivity data, 60 and approaches that combine native metabolomics with native and non-targeted LCMS data for the detection of metabolite protein binding. 61 While these platforms represent innovative steps forward for the future exploration of NP biological activity, both resources will only be as useful as the diversity of the deposited biological data (NP Analyst) and the diversity of protein targets available for interrogation (native metabolomics approach). Further, purely MS-based metabolomics approaches have inherent limitations including the over-representation of un-isolatable NPs resulting from the inherently high sensitivity of MS, problems associated with the ionizability of some NP structures (decreased MS signal intensities), variation of results obtained across instruments and platforms, and difficulties associated with examining complex NP fragments obtained in the gas phase.…”

Computational Approaches to Enzyme Inhibition by Marine Natural Products in the Search for New Drugs

“…An additional hydrogen bond relative to lyngbyastatin 7 has been proposed as the element responsible for its enhanced inhibitory potency [ 183 ]. More recently, yet another family of new Ahp-cyclodepsipeptides, the rivulariapeptolides, with nanomolar potency as serine protease inhibitors, was identified from an environmental cyanobacteria community using a scalable, bioactivity-focused, native metabolomics approach [ 184 ].…”