Aims Relaxin-2, a well-known human hormone primarily associated with pregnancy, has shown promising cardiovascular benefits in both pre-clinical models and clinical trials. However, its therapeutic potential has been limited due to the short half-life and the short duration of treatment. To address this, we developed AZD3427, a novel long-acting relaxin-2 analogue, and assessed its efficacy during prolonged treatment in a large animal model with cardiac dysfunction.Methods and results Extensive protein engineering resulted in AZD3427, a novel fusion protein, which closely mimics the natural hormone's structure and consists of a single relaxin-2 and the Fc fragment of human IgG1 to extend its half-life. AZD3427 exhibits an improved pharmacokinetic profile, allowing for weekly or less frequent, subcutaneous dosing, and maintains the pharmacology profile of relaxin-2 with signalling via relaxin family peptide receptor 1 (RXFP1) in cell systems. The effects of chronic RXFP1 agonism with AZD3427 were investigated in a non-human primate (NHP) model with systolic dysfunction and metabolic syndrome. Administration of AZD3427 over a 21-week period led to significant improvements in cardiac function, as evidenced by increased ejection fraction, cardiac output, and stroke volume, as well as reduced systemic vascular resistance. Importantly, no adverse events related to treatments were observed and there were no concomitant changes in heart rate or blood pressure. During the 18-week washout period, the observed effects gradually disappeared.Conclusion Prolonged administration of AZD3427, a long-acting relaxin receptor RXFP1 agonist, resulted in remarkable improvement in cardiac function in a NHP model. Findings of this study are an important translational step to developing future therapies and support further clinical development of AZD3427 as a novel treatment for patients with heart failure.

The GDF15-GFRaL-RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15-GFRaL protein-protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.

Genetic variants in proteins can interfere with measurement of their circulating concentrations. Given the growing biomedical importance of GDF-15, we wished to establish whether a common histidine to aspartate variant present in position 6 of the mature GDF-15 protein (H202D variant) interfered with its measurement by two commonly used immunoassays. We first examined the detectability of recombinant monomers, homodimers and heterodimers of GDF-15 by assays and/or reagents used in two widely used immunoassays (Roche Elecsys GDF-15 and the R&D antibody combinations used in their Quantikine and DuoSet ELISAs). The Roche assay detected the H and D containing peptides similarly but the assays based on the R&D reagents consistently underreported concentrations of the D-containing variant peptide. Measurements of plasma concentrations of GDF-15 in genotyped human participants showed that the R&D reagents reported values in heterozygotes were ~25% lower, and in homozygotes, 50% lower than the Roche assay. We finally studied the activation of the GDF-15 receptor, GFRAL-Ret, in a cell based assay and found that the activities of the HH and DD containing GDF-15 peptide were indistinguishable. These results have implications for the interpretation of genetic epidemiological studies which have used the R&D reagents to measure GDF-15, and for the emerging clinical use of GDF-15 as a diagnostic and prognostic biomarker. We provide correction equations, which may be of utility for the analysis of data generated with the R&D reagents where the genotype of the participants is known.