Lyngbyapeptin A (1) is a linear modified tetrapeptide originally isolated from the marine cyanobacterium Moorena bouillonii in Papua New Guinea and Guam. In previous research, 1 did not show significant cytotoxicity but was not rigorously investigated due to insufficient material and the propensity of the (E)-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 1 from a Moorena collection. The first total synthesis of 1, of its keto analogue named 5-desmethyl-lyngbyapeptin A (2), and of acrylamide analogue 3 was also achieved by convergent liquid-phase peptide synthesis. Compounds 1-3 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 1, supported by secondary functional and binding assays. These receptors were also modulated by 2 and 3 but to a lesser extent, with 2- to 12-fold decrease in potency, demonstrating the role of the (E)-3-methoxy-2-butenoyl moiety in contributing to the GPCR modulating activity. The binding modes of 1 to the GPCR hits were further investigated using molecular modeling.