Cardiac contractility structure-activity relationship and ligand-receptor interactions; the discovery of unique and novel molecular switches in myosuppressin signaling.

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology.

Structure-activity relationships of FMRF-NH2 peptides demonstrate A role for the conserved C terminus and unique N-terminal extension in modulating cardiac contractility.

FMRF-NH2 peptides which contain a conserved, identical C-terminal tetrapeptide but unique N terminus modulate cardiac contractility; yet, little is known about the mechanisms involved in signaling. Here, the structure-activity relationships (SARs) of the Drosophila melanogaster FMRF-NH2 peptides, PDNFMRF-NH2, SDNFMRF-NH2, DPKQDFMRF-NH2, SPKQDFMRF-NH2, and TPAEDFMRF-NH2, which bind FMRFa-R, were investigated. The hypothesis tested was the C-terminal tetrapeptide FMRF-NH2, particularly F1, makes extensive, strong ligand-receptor contacts, yet the unique N terminus influences docking and activity. To test this hypothesis, docking, binding, and bioactivity of the C-terminal tetrapeptide and analogs, and the FMRF-NH2 peptides were compared. Results for FMRF-NH2 and analogs were consistent with the hypothesis; F1 made extensive, strong ligand-receptor contacts with FMRFa-R; Y → F (YMRF-NH2) retained binding, yet A → F (AMRF-NH2) did not. These findings reflected amino acid physicochemical properties; the bulky, aromatic residues F and Y formed strong pi-stacking and hydrophobic contacts to anchor the ligand, interactions which could not be maintained in diversity or number by the small, aliphatic A. The FMRF-NH2 peptides modulated heart rate in larva, pupa, and adult distinctly, representative of the contact sites influenced by their unique N-terminal structures. Based on physicochemical properties, the peptides each docked to FMRFa-R with one best pose, except FMRF-NH2 which docked with two equally favorable poses, consistent with the N terminus influencing docking to define specific ligand-receptor contacts. Furthermore, SDNAMRF-NH2 was designed and, despite lacking the aromatic properties of one F, it binds FMRFa-R and demonstrated a unique SAR, consistent with the N terminus influencing docking and conferring binding and activity; thus, supporting our hypothesis.