B7-33: Research Guide & Chemical Profile

B7-33 is a synthetic single-chain peptide analog of human relaxin-2 that functions as a biased agonist at the relaxin family peptide receptor 1 (RXFP1). Developed at the Florey Institute of Neuroscience and Mental Health, B7-33 was designed to retain the anti-fibrotic and vasodilatory properties of native relaxin while eliminating the requirement for the complex two-chain A-B disulfide-bonded structure that makes recombinant relaxin-2 expensive and difficult to manufacture. B7-33 selectively activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway downstream of RXFP1 while showing reduced activation of the cAMP/PKA pathway compared to native relaxin-2.

Native human relaxin-2 (H2 relaxin) is a 6-kDa heterodimeric peptide consisting of an A chain (24 amino acids) and a B chain (29 amino acids) linked by two inter-chain disulfide bonds plus one intra-chain disulfide bond in the A chain. This structural complexity presents significant manufacturing challenges. B7-33 circumvents this by using only a modified B chain of relaxin-3 — specifically residues B7 through B33 — as a single-chain peptide of approximately 3 kDa.

The B7-33 sequence retains the critical Arg-B13, Arg-B17, and Ile-B20 residues that form the receptor-binding cassette for RXFP1. Structure-activity studies demonstrated that these three residues, presented within the alpha-helical context of the B chain, are necessary and sufficient for RXFP1 engagement. The truncation removes the N-terminal B1-B6 segment, which contributes to full cAMP activation but is dispensable for ERK1/2 signaling.

B7-33 exemplifies the concept of biased agonism — the ability of a ligand to preferentially activate certain signaling pathways over others through the same receptor. At RXFP1, native relaxin-2 activates multiple pathways: cAMP accumulation via Gs coupling, ERK1/2 phosphorylation, and nitric oxide production via nNOS/eNOS. B7-33 activates ERK1/2 phosphorylation to approximately 80% of the native relaxin-2 response but generates less than 15% of the cAMP signal in HEK293 cells stably expressing RXFP1.

This signaling bias is pharmacologically significant because the anti-fibrotic effects of relaxin are primarily mediated through ERK1/2-dependent pathways (including downstream activation of the nNOS-NO-cGMP cascade), whereas some cardiovascular effects such as chronotropy appear to be cAMP-dependent. The biased profile of B7-33 may therefore offer a more targeted anti-fibrotic effect.

In a mouse model of isoproterenol-induced cardiac fibrosis, B7-33 administered via subcutaneous osmotic minipump (0.5 mg/kg/day for 14 days) significantly reduced left ventricular collagen deposition as measured by picrosirius red staining. Collagen volume fraction decreased by approximately 40% compared to vehicle-treated fibrotic controls, an effect comparable to equimolar doses of recombinant human relaxin-2 (serelaxin). Echocardiographic assessment showed preserved left ventricular function and reduced diastolic stiffness in B7-33-treated animals.

Gene expression analysis of cardiac tissue revealed that B7-33 suppressed collagen type I alpha 1 (Col1a1) and collagen type III alpha 1 (Col3a1) mRNA expression while upregulating matrix metalloproteinase-2 (MMP-2) and MMP-9 — shifting the collagen balance toward degradation. Additionally, B7-33 reduced transforming growth factor beta 1 (TGF-beta1) expression and attenuated Smad2/3 phosphorylation in cardiac fibroblasts, consistent with anti-fibrotic activity at the transcriptional level.

The anti-fibrotic mechanism of B7-33 involves ERK1/2-dependent activation of neuronal nitric oxide synthase (nNOS), leading to increased NO production and downstream activation of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). This nNOS-NO-cGMP-PKG signaling axis directly inhibits myofibroblast differentiation by preventing the nuclear translocation of Smad proteins and by promoting myofibroblast apoptosis.

In primary human cardiac fibroblast cultures, B7-33 at 100 nM inhibited TGF-beta1-induced alpha smooth muscle actin expression (a myofibroblast marker) by 55-60% as measured by Western blot and immunofluorescence. The effect was abolished by the NOS inhibitor L-NAME and by the ERK1/2 inhibitor U0126, confirming the proposed signaling pathway.

Beyond cardiac fibrosis, B7-33 has been investigated for metabolic effects. In high-fat diet-fed mice, B7-33 treatment improved glucose tolerance and reduced hepatic steatosis. The metabolic effects are attributed to RXFP1-mediated enhancement of insulin sensitivity in hepatocytes and reduced hepatic inflammation, though the precise molecular mechanisms remain under investigation.

Vascular studies have shown that B7-33 induces endothelium-dependent vasodilation in isolated mesenteric arteries, with an efficacy approximately 60% of that achieved by native relaxin-2. The reduced vasodilatory potency compared to native relaxin may be advantageous in a therapeutic context, as excessive vasodilation was a dose-limiting effect observed in clinical trials of serelaxin for acute heart failure.

Serelaxin (recombinant human relaxin-2) reached Phase 3 clinical trials for acute heart failure before the RELAX-AHF-2 trial failed to meet its primary endpoint. B7-33 represents a next-generation approach with potential advantages: single-chain structure enables simpler synthesis and lower manufacturing cost, biased signaling may reduce off-target cardiovascular effects, and the smaller molecular size may improve tissue penetration. However, B7-33 remains in preclinical development and has not yet entered human clinical trials.

Research-grade B7-33 is characterized by HPLC purity of 95% or higher, confirmed by reversed-phase C18 chromatography. Mass spectrometry should confirm the expected molecular ion consistent with the B7-33 sequence. Given the importance of the alpha-helical conformation for bioactivity, circular dichroism spectroscopy is recommended as a supplementary quality control measure. Certificates of Analysis should include amino acid analysis, mass spectrometry data, and endotoxin testing.

• Hossain MA et al. (2016). A single-chain derivative of the relaxin hormone is a functionally selective agonist of RXFP1. Chem Sci, 7(6):3805-3819.
• Kocan M et al. (2017). ML290 and B7-33 receptor pharmacology. Br J Pharmacol, 174(10):1032-1047.
• Samuel CS et al. (2017). Anti-fibrotic actions of relaxin. Br J Pharmacol, 174(10):962-976.
• Bathgate RAD et al. (2013). Relaxin family peptides and their receptors. Physiol Rev, 93(1):405-480.
• Teerlink JR et al. (2013). Serelaxin in acute heart failure (RELAX-AHF). Lancet, 381(9860):29-39.
• Hossain MA et al. (2019). B7-33 and single-chain relaxin mimetics. Front Chem, 7:110.
• Mookerjee I et al. (2009). Relaxin inhibits renal myofibroblast differentiation. FASEB J, 23(4):1219-1229.

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