Synthetic short-chain peptide analogues of H1 relaxin lack affinity for the RXFP1 receptor and relaxin-like bioactivity. Clues to a better understanding of relaxin agonist design

Abstract

The peptide hormone relaxin (RLX), also available as clinical-grade recombinant protein (serelaxin), holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues common to all peptide drugs. In this study, by a computational modelling chemistry approach, we have synthesized and tested a set of low molecular weight peptides based on the putative receptor-binding domain of the B chain of human H1 RLX isoform, with the objective to obtain RLX analogues with improved pharmacokinetic features. Some of them were stabilized to induce the appropriate 3-D conformation by intra-chain tri-azolic staples, which should theoretically enhance their resistance to digestive enzymes making them suited for oral administration. Despite these favourable premises, none of these H1 peptides, either linear or stapled, revealed a sufficient affinity to the specific RLX receptor RXFP1. Moreover, none of them was endowed with any RLX-like biological effects in RXFP1-expressing THP-1 human monocytic cells and mouse NIH-3T3-derived myofibroblasts in in vitro culture, in terms of significantly relevant cAMP elevation and ERK1/2 phosphorylation, which represent two major signal transduction events downstream RXFP1 activation. This was at variance with authentic serelaxin, which induced a clear-cut, significant activation of both these classical RLX signaling pathways. Albeit negative, the results of this study offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues.

Further Developments towards a Minimal Potent Derivative of Human Relaxin-2

“…Unlike our previous work on the H3 relaxin B chain [ 29 ], the rigid HC staple dramatically reduced the binding affinity of B7-33 analogs. Our data confirm the previous findings from the Rovero group, where they showed triazole conformationally constrained B-chain analogs of H2 relaxin exhibit significantly reduced RXFP1 activity compared with non-stapled B7-33 [ 30 ]. These results suggest that B7-33 requires a certain degree of flexibility to adopt a high-affinity binding state for RXFP1/7BP.…”

“…The finding that Aib incorporation reduced helical propensity was unexpected, suggesting that sites of incorporation are important to determine the effect on the helicity of Aib residues. Further, the data presented here and elsewhere [ 30 ], that rigid stapling strategies are not suitable for single-chain H2 relaxin analogs, suggest that more flexible stapling strategies should be explored in the future.…”

Porcine Relaxin but Not Serelaxin Shows Residual Bioactivity after In Vitro Simulated Intestinal Digestion—Clues for the Development of New Relaxin Peptide Agonists Suitable for Oral Delivery

“…Additional studies are undoubtedly required to identify the amino-acidic sequence of this molecule, as such knowledge could be exploited for the development of synthetic relaxin agonists suitable for oral formulations. In this context, we have recently shown that human H1 RLX analogs can be stabilized by intra-chain tri-azolic staples in the appropriate 3-D conformation in order to further enhance their resistance to digestive enzymes [23]. Our forthcoming study will explore whether such chemical modification can also be applied to the pRLX component to obtain an effective and digestion-resistant RLX agonist.…”