Biosynthesis, Chemical Synthesis, and Pharmacological Evaluation of Lyngbyapeptin A as a GPCR Antagonist of Motilin, Cannabinoid, and Amylin Receptors

Lyngbyapeptin A (<b>1</b>) is a linear modified tetrapeptide originally isolated from the marine cyanobacterium <i>Moorena bouillonii</i> in Papua New Guinea and Guam. In previous research, <b>1</b> did not show significant cytotoxicity but was not rigorously investigated due to insufficient material and the propensity of the (<i>E</i>)-3-methoxy-2-butenoyl moiety to undergo conversion into a ketone, preventing further biological testing. In this study, we report the identification and characterization of the biosynthetic gene cluster (BGC) of <b>1</b> from a <i>Moorena</i> collection. The first total synthesis of <b>1</b>, of its keto analogue named 5-desmethyl-lyngbyapeptin A (<b>2</b>), and of acrylamide analogue <b>3</b> was also achieved by convergent liquid-phase peptide synthesis. Compounds <b>1</b>-<b>3</b> were subjected to functional GPCR target-based β-arrestin screens to identify their activity profiles. Four GPCRs, including amylin receptor 2 (CALCR-RAMP2), motilin receptor (MLNR), and cannabinoid receptors CNR1 and CNR2, were antagonized by <b>1</b>, supported by secondary functional and binding assays. These receptors were also modulated by <b>2</b> and <b>3</b> but to a lesser extent, with 2- to 12-fold decrease in potency, demonstrating the role of the (<i>E</i>)-3-methoxy-2-butenoyl moiety in contributing to the GPCR modulating activity. The binding modes of <b>1</b> to the GPCR hits were further investigated using molecular modeling.