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. 2023 Apr 1;24(7):6616.
doi: 10.3390/ijms24076616.

A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity

Praveen Praveen  1 , Chao Wang  2 , Thomas N G Handley  1 , Hongkang Wu  1 , Chrishan S Samuel  2 , Ross A D Bathgate  3 , Mohammed Akhter Hossain  4
Affiliations

A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity

Praveen Praveen et al. Int J Mol Sci. .

Abstract

Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin's receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.

Keywords: B7-33; H2 relaxin; RXFP1; structure-activity relationship (SAR).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of fatty acid interacting with albumin. Lipid interacts non-covalently to albumin.
Figure 2
Figure 2
Primary structure and sequence of lipidated analogues. (A) Different lengths of fatty acids: (1) decanoic acid (C10), (2) myristic acid (C14), (3) palmitic acid (C16), (4) lysine palmitic acid (C16), and (5) lysine replaced with lysine palmitic acid at 9th position of B7-33. (B) Eu-H2 relaxin competition binding of lipidated analogues in HEK-7BP cells. The data are the result of n = 3–4 independent experiments and expressed as mean ± SEM.
Figure 3
Figure 3
Primary structure and sequence of lipidated analogues. (A) Primary structure of tagged B7-33 analogues: (6) Tag structure, (7) Tag-B7-33 with no spacer, (8) Tag-PEG4-B7-33 with PEG4 spacer, and (9) Tag-PEG6-B7-33 with PEG6 spacer. (B) Eu-H2 relaxin competition binding of apidated analogues in HEK-7BP cells. The data are the result of n = 3–4 independent experiments and expressed as mean ± SEM.
Figure 4
Figure 4
Primary structure and sequence of lipidated analogues. (A) Primary structure and sequence of lipidated analogues with different lengths of spacer used between the fatty acid and B7-33. (B) Eu-H2 relaxin competition binding of lipidated analogues in HEK-7BP cells. The data are the result of n = 3–4 independent experiments and expressed as mean ± SEM.
Figure 5
Figure 5
In vitro serum stability assay. B7-33 degrades within minutes, whereas AcK(PalmGlu)-PEG12-B7-33 is stable in serum over time. There were significant differences between the peptides at any time point (p-value = 0.0222, t-test). One-phase exponential decay and paired t-test analyses were performed with GraphPad Prism 8.4.3. The data are the results of n = 3 independent experiments.
Figure 6
Figure 6
The effects of B7-33 vs. L-B7-33 on myofibroblast differentiation in vitro. (A) Representative Western blots of α-SMA expression and corresponding GAPDH expression from unstimulated BJ3 human dermal fibroblasts, TGF-β1 (T)-stimulated BJ3 human dermal myofibroblasts and T + B7-33- or T + L-B7-33-treated myofibroblasts after 3 days (D3); and T + B7-33- or T + L-B7-33-treated myofibroblasts after 5 days (D5), 7 days (D7), 10 days (D10) or 14 days (D14). (B) Also shown are the mean ± SEM levels of a-SMA expression from each treatment group, corrected for GAPDH loading and expressed relative to the unstimulated cell control group, which was expressed as 1 in each case; from n = 6 separate experiments conducted in duplicate. Statistical analysis: p < 0.05, * p < 0.01 vs. untreated (D3) group; # p < 0.05 vs. TGF- b1 [T] (D3) group; + p < 0.05 vs. T+B7-33 or T+L-B7-33 (D3) group, as determined using a non-parametric Kruskal Wallis test and Dunn’s post-hoc analysis to allow for multiple comparisons between the groups.
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