Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists

Abstract

Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α'-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.

Keywords: RXFP3; helical stapling; relaxin-3; α-aminoisobutyric acid.

Figure 1

Three-dimensional structure of relaxin-3. The A-chain and B-chain are shown in grey and dark orange respectively. The structure is braced by an intra-chain disulfide in the A-chain and two inter-chain disulfide bonds, shown in yellow. Residues in blue are important for binding to RXFP3, whereas the green residues are required to activate the receptor.

Scheme 1

Stabilisation strategies for single-chain relaxin-3 agonists. (A) stapling using dicarba strategies. (B) stapling using thioether strategies.

Figure 2

Comparison of secondary Hα chemical shift of Aib containing and stapled relaxin-3 analogues. Secondary Hα chemical shifts are presented as the recorded chemical shift subtracted by the random coil chemical shift for each amino acid. An extended region of negative values indicates an α-helical structure. Analogues were compared to H3 relaxin B-chain of the native peptide or Ac-R3B10-27 [13/17 HC] (HC) and R3B1-22R. (A) Comparison between 1 (Ac-R3 B10-27 13, 17 Aib), 2 (Ac-R3 B10-27 13, 14, 17 Aib) and 7 (R3 B10-27 13, 17, 22 Aib), whereas (B) shows comparison of 3 (R3 B10-27 13, 17, 18 Aib), 5 (R3B 10-27 13, 17, 21 Aib) and 9 (R3 B10-27 13, 17, 18, 21 Aib). Cysteine-based stapled analogues 14 (R3 B10-27 13, 17 DCA) and 16 (R3 B10-27 13, 17 DBX) are compared in (C). * thiol stapling positions. Random coil shifts for Aib and Pa are not available. Oxidised Cys random coil value was for calculation of secondary Hα chemical shift at stapled sites of analogues 14 and 16.

Figure 3

Binding affinity and potency of relaxin-3 agonist analogues at RXFP3. (A,C) illustrate the binding of the Aib and stapled analogues, respectively. (B,D) show the potency of Aib containing and stapled analogues, respectively. Data are shown as mean ± SEM of triplicate points from a minimum of three independent experiments. HC represents Ac-R3B10-27 [13/17 HC].

Figure 4

Model of analogue 4 bound to RXFP3. The predicted helical structure allows the same key interactions between arginine residues in relaxin-3 and acidic residues in RXFP3, as well as hydrophobic contacts [22]. Residues in analogue 4 are labelled with three-letter amino acid codes and residue numbers based on the relaxin-3 sequence, and residues in RXFP3 are labelled with single letter amino acid codes and residue numbers.