Developing small peptidomimetics to target RXFP1 for the treatment of acute heart failure

Florey patented H2 relaxin was recently granted a “breakthrough therapy” designation by the FDA for the treatment of acute heart failure. 

The indications are that H2 relaxin will enter the clinic soon by next couple of years. It is likely that the beneficial effects of H2 relaxin were also related to its anti-fibrotic effects, which we have shown occurs rapidly and independently of the cause of disease. This project aims to improve this current drug and develop the next generation molecule.

In order to maximize its future translational potential, we will address the three following issues:

Large size and complex structure: The size (53 amino acids) and complex structure (two chains, linked by three disulfide bonds; Fig. 1) of H2 relaxin represent a considerable challenge for its synthesis; limiting modifications of the peptide to optimize its efficacy and stability. 

Cross-reactivity with other receptors: H2 relaxin exerts its biological actions through its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1; initially discovered as LGR7). However, it also activates RXFP2, the native receptor for the related insulin-like peptide 3, INSL3; opening the possibility of potential side-effects through RXFP2-mediated physiological processes. 

Short half-life in blood: Like insulin, H2 relaxin has a very short half-life. Hence, when injected into patients, H2 relaxin will lose half its activity within 10 minutes because it is degraded by blood enzymes and cleared by the kidney and liver.

Thus, there is an urgent need to develop simpler H2 relaxin analogues that are easier to prepare and modify, have high selectivity for RXFP1 and retain their activity for an extended therapeutic time-frame in patients with acute heart failure.  A considerable effort has been made to reduce “H2 relaxin” into developing a single chain analogue with RXFP1-specific agonist activity. As the B-chain of H2 relaxin seems to possess most, if not all, of the amino acids responsible for RXFP1 binding and activation, it was probable that a B-chain-only analogue would be active at RXFP1. Preliminary single-chain analogues (such as native B-chain, B1-29) were found to be insoluble (Fig. 1B) and inactive over the years, but, very recently, in an exciting finding we have identified an N-terminus-truncated and C-terminus-extended B-chain analogue (B7-33) (Fig. 1A), which was found to be soluble in water (Fig 1B), binds to RXFP1 (data now shown) and acts as an agonist in over-expressing RXFP1 cells, albeit with less potency compared with native H2-relaxin (Fig 1C). However, unlike “H2 relaxin”, “B7-33 is selective at RXFP1 over RXFP2 (data not shown), and demonstrates similar activity to H2 relaxin in cells natively expressing RXFP1 in vitro (in rat renal myofibrobalst cells, Fig. 1D) and in models of diseases in vivo (Isoproterenol ISO-induced heart fibrosis model Fig. 1E; Ovalbumin OVA-induced lung fibrosis model Fig. 1F, G). The “B7-33” peptide provides us with a great starting point for further development as small peptidomimetic agonists of RXFP1. The peptide "B7-33", however, is less potent compared with native H2 relaxin. This proposal focuses on the utility of this lead compound as the basis for its further modification to improve its potency as well as in vivo stability. We will improve the structures, stability and potency of single chain "B7-33" analogue by using rational and structure-based drug design, point mutations with natural, non-natural, isometric amino acids, dicarba stapling, combinatorial library and lipidation.

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