Identification of a pepducin acting as S1P₃ receptor antagonist

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

Pepducins and Other Lipidated Peptides as Mechanistic Probes and Therapeutics

“…Among them, S1P 3 has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 is a pepducin agonist derived from the i2 intracellular loop, which mimics the effects of S1P by specifically activating S1P 3 [58]. KRX-725 derivatives with deletions of two amino acids from both N- and C-termini have the ability to inhibit both KRX-725-induced vasorelaxation and fibroblast proliferation [58].…”

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis

“…We have already identified a pepducin (compound KRX-725-II in Table 1) acting as an S1P 3 receptor antagonist [10]. Here, we have synthesized a series of new derivatives to optimize the activity and selectivity profile toward S1P 3 .…”

“…As expected, KRX-725-II significant inhibited KRX-725-induced relaxation (Figure 1A), as well as KRX-722-induced vasorelaxation (Figure 1B), demonstrating a limited selectivity. The Ala-scan, previously conducted on KRX-725, suggested its high tolerance to amino acid substitutions [10], and directed the research towards more selective S1P3 antagonists by chemical modification of the native sequence. Firstly, we focused our attention on Tyr, in position 4, and synthesized a new series of compounds to verify if: (1) the presence of Tyr is determinant in the interaction with S1P 3 ; (2) the introduction of molecular constraints could address the selectivity towards one specific receptor.…”

“…Moreover, an increasing amount of data indicate how these cellpenetrating lipopeptides can access receptor conformations not available to orthosteric ligands. Using this approach, we have synthesized a pepducin-based S1P 3 antagonist, namely KRX-725-II (Myristoyl-GRPYDAN-NH2) [10,11]. Here, to overcome the poor selectivity between S1P 1 and S1P 3 , due to the high structural homology between the two receptors [12], we have synthesized several derivatives of KRX-725-II.…”

Utilization of Biased G Protein-Coupled Receptor Signaling towards Development of Safer and Personalized Therapeutics

“…Apart from transmembrane region and surrounding medium, the intracellular region of GPCRs, to which effectors couple, have also been targeted for more than a decade by means of pepducins and RNA aptamers. As to pepducins, which are cell-penetrating peptides, in particular, KRX-725 targets Spingosine-1-phosphate receptor subtype 3 (S1P3) to induce fibroblast proliferation and vasorelaxation by mimicking the effect of sphingosine-1-phosphate [91]. In addition, a study conducted by Carr et al has indicated that β-arrestin-biased pepducin that targets β 2 AR can be used to prevent congestive heart failure [92].…”