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

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily

Applying Molecular Networking for the Detection of Natural Sources and Analogues of the Selective Gq Protein Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the traditional Chinese medicine plant <i>Ardisia crenata</i>, is a potent Gq protein inhibitor and thus a valuable tool to study Gq-mediated signaling of G protein-coupled receptors. Two new FR analogues (<b>3</b> and <b>4</b>) were isolated from <i>A. crenata</i> together with the known analogues <b>1</b> and <b>2</b>. The structures of compounds <b>3</b> and <b>4</b> were established by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS² analysis. The latter approach led to the annotation of further FR analogues <b>5</b>–<b>9</b>. Comparative bioactivity tests of compounds <b>1</b>–<b>4</b> along with the parent molecule FR showed high-affinity binding to Gq proteins in the low nanomolar range (IC₅₀ = 2.3–16.8 nM) for all analogues as well as equipotent inhibition of Gq signaling, which gives important SAR insights into this valuable natural product. Additionally, FR was detected from leaves of five other <i>Ardisia</i> species, among them the non-nodulated leaves of <i>Ardisia lucida</i>, implying a much broader distribution of FR than originally anticipated