Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1

Abstract

The anti-fibrotic, vasodilatory, and pro-angiogenic therapeutic properties of recombinant relaxin peptide hormone have been investigated in several diseases and recent clinical trial data has shown benefit in treating acute heart failure. However, the remodeling capacity of these peptide hormones is difficult to study in chronic settings due to their short half-life and the need for intravenous administration. Here we present the first small-molecule series of human relaxin receptor 1 (RXFP1) agonists. These molecules display similar efficacy as the natural hormone in several functional assays. Mutagenesis studies indicate that the small molecules activate relaxin receptor through an allosteric site. These compounds have excellent physical and in vivo pharmacokinetic properties to support further investigation of relaxin biology and animal efficacy studies of the therapeutic benefits of RXFP1 activation.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…Substitution of eight A-or B-chain residues with alanine does not affect receptor signaling (Bullesbach and Schwabe, 2005a). Disruption of the intra-A-chain disulfide bond (C10S/C15S; or C10del/C15del) produces peptides that retain RXFP2 Xiao et al (2013) binding but do not activate cAMP signaling (Table 2). The INSL3 B-chain alone is also a RXFP2 antagonist, although it only has low affinity for the receptor (Del Borgo et al, 2006;Shabanpoor et al, 2007).…”

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

“…However, all mutants, except P565A, also showed significant perturbation of activation by the small molecule activator ML290. Importantly, this compound activates RXFP1 by an allosteric mechanism, potentially involving EL3, which does not require the LDLa module (31) and was used as a control for potential disruption in EL structure. Because ML290 activity and H2 relaxin binding are not significantly different for P565A, but H2 relaxin induced signaling is perturbed, this indicates that this residue may be directly involved in the interaction with the LDLa module.…”

“…Because our mutations of Gly 561 and Val 562 showed no change to signaling, suggesting that residues on the N-terminal side of Cys 563 site are not a part of the binding site, we also mutated Pro 565 , the next residue C-terminal of Phe 564 to further investigate H2 relaxin interactions with that region of EL2. Because the F564A mutation showed poor expression in transient transfections and to aid in characterization of new mutations, HEK-293T cells stably expressing mutant receptors were prepared and characterized for H2 relaxin binding and signaling in response to H2 relaxin and the small molecule allosteric modulator of RXFP1, ML290 (31).…”

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists

“…30 In addition, ML290 adopts the same intramolecular hydrogen-bonding conformation in the binding pocket as is observed in the crystal structure of the isolated small molecule and by solution NMR. 9 …”