B7-33: a single-chain relaxin B-chain analog and functionally selective RXFP1 agonist

General Overview

B7-33 is a synthetic single-chain peptide derived from the B-chain of human relaxin-2 (H2 relaxin). It is the first described functionally selective (biased) agonist of the RXFP1 receptor, a G-protein-coupled receptor of the relaxin family, related to the LGR subfamily of receptors (leucine-rich repeat-containing GPCRs). Native relaxin-2 is a heterodimeric hormone with an insulin-like fold: the A- and B-chains are held together by three disulfide bridges (two interchain, one intrachain within the A-chain). B7-33 reproduces the hormone's key antifibrotic activity within a single short linear chain, with no disulfide bond at all. Unlike native relaxin-2, which activates both the cAMP- and ERK-dependent branches of RXFP1 signaling in parallel, B7-33 predominantly engages only one branch, so-called biased agonism. This material is intended exclusively as a laboratory research reagent (RUO); the chemistry, molecular mechanism, and published preclinical body of work are presented below without any indication for use in humans.

Origin and Design

B7-33 was engineered in the laboratory of Mohammed Akhter Hossain and Ross A. D. Bathgate (Florey Institute of Neuroscience and Mental Health, Melbourne) and first described in 2016 ^[1]. The motivation was twofold. First, recombinant relaxin-2 (serelaxin) is complex and expensive to manufacture: the two-chain structure, with the precise spatial arrangement of three disulfide bonds, requires either separate synthesis of the A- and B-chains followed by oxidative folding, or complex recombinant expression. Second, the pleiotropic signaling of the full-length hormone (parallel activation of the cAMP- and ERK-dependent cascades) is accompanied by effects that are partly undesirable for a narrowly antifibrotic application. In particular, relaxin is capable of stimulating proliferative processes. The authors asked whether the molecule could be reduced to a minimal single-chain fragment that retains the antifibrotic action without reproducing the receptor's full signaling repertoire. The name "B7-33" reflects the peptide's origin: the region of the relaxin-2 B-chain spanning approximately residues 7 to 33, i.e., the central "core" of the B-chain, excluding the terminal N- and C-terminal residues and excluding the A-chain entirely. This simplification was possible because the native B-chain contains a compact arginine motif (the "arginine cassette": residues Arg13, Arg17, and Ile/Val20) that is responsible for physical contact with the extracellular domain of RXFP1 and is retained within the 7–33 region even in the absence of the A-chain ^[8].

Structure and Physicochemistry

B7-33 is a short single-chain peptide approximately 27 residues in length, corresponding to the central portion of the relaxin-2 B-chain. The key design idea is the elimination of the A-chain and of both interchain disulfide bridges required in the native hormone for correct insulin-like folding and high-affinity receptor binding. This radically simplifies chemical synthesis compared with two-chain relaxin, but comes at a cost: the short linear molecule, lacking a disulfide scaffold, is structurally less rigid, retains an ordered conformation in solution less well, and undergoes proteolytic degradation in biological media more rapidly (see the Pharmacokinetics section). The molecular mass of B7-33 is correspondingly lower than that of heterodimeric relaxin-2. It is typically supplied as a lyophilized powder for research use.

Molecular Mechanism: Functional Selectivity at RXFP1

RXFP1 is the cognate receptor for relaxin-2. Its extracellular domain is built from an N-terminal LDLa module (which coordinates a Ca²⁺ ion, stabilized by three disulfide bonds of its own), connected by a flexible linker (~32 residues) to a domain of ten leucine-rich repeats (the LRR domain), which is in turn joined to the receptor's seven-transmembrane domain ^[7][8]. Binding of the native hormone occurs in two steps: first, the B-chain arginine cassette (Arg13, Arg17, Ile/Val20) makes high-affinity contact with acidic residues (Asp/Glu) on LRR4–8. This contact mediates binding but does not by itself activate the receptor; next, the LDLa module and the LDLa-LRR linker, adopting an ordered helical conformation under the hormone's influence, dock onto the transmembrane domain and trigger transduction ^[8]. The activated RXFP1 engages cAMP accumulation via Gαs and ERK1/2 phosphorylation via additional circuits in parallel ^[7].

Biased Agonism of B7-33

The defining property of B7-33 is preferential activation of pERK1/2 with substantial bypassing of the cAMP branch, to which part of the vascular and proliferative effects of full-length relaxin are attributed ^[1][3]. Because B7-33 lacks the A-chain, its contact with the LDLa module and the transmembrane domain is simplified relative to the two-chain hormone. This has been proposed as the reason for the shift in signaling balance toward ERK1/2. An important nuance: the potency of B7-33 depends on cellular context. In HEK293 cells overexpressing RXFP1, the peptide shows low affinity, whereas in fibroblasts with endogenous expression levels (human cardiac fibroblasts, rat renal myofibroblasts) it is practically equipotent with H2 relaxin ^[1][6]. The pERK1/2 axis is associated with suppression of fibroblast activation, reduced collagen deposition, and increased MMP-2 activity ^[1]. The antifibrotic action of B7-33 has been linked to heterodimerization of RXFP1 with the angiotensin II type 2 receptor (AT2R), that is, the effect is described as also depending on the interaction of RXFP1 with another GPCR ^[1]. The broader context of relaxin family signaling (RXFP1-RXFP4) is discussed in the review literature ^[7].

Preclinical Studies

Heart and Vasculature

In the 2016 study, B7-33 prevented or reduced organ fibrosis in three rodent models (MI in rats, isoprenaline-induced heart failure, and chronic allergic airway disease in mice) with potency comparable to H2 relaxin, and increased MMP-2 activity in human cardiac fibroblasts and rat renal myofibroblasts in vitro; unlike relaxin, it did not stimulate prostate tumor growth in vivo ^[1]. In an ischemia-reperfusion model (LAD ligation) in CD1 mice, B7-33 (0.25 mg/kg s.c. at the time of reperfusion, followed by twice daily dosing to day 7) reduced infarct size (approximately 22% versus 45% in controls), improved fractional shortening as early as 24 hours (approximately 29% versus 23%) and further by day 7, reduced LV fibrosis and markers of ER stress/inflammasome activation (GRP78, CHOP, ASC, TLR4) via an ERK1/2-dependent pathway; similar protection (50–100 nmol/L) was observed in cardiomyocytes under simulated ischemia-reoxygenation in vitro ^[3]. In an isoprenaline-induced cardiomyopathy model in 129sv mice, B7-33 (0.25 mg/kg/day, days 7–14 post-injury) was not inferior to relaxin-2 (0.5 mg/kg/day): both reduced LV fibrosis, inflammation, and cardiomyocyte hypertrophy, and restored vascular density and aortic contractility, whereas perindopril (1 mg/kg/day) lowered blood pressure and inflammation but not fibrosis or hypertrophy; the antifibrotic action of B7-33 set in more rapidly ^[9]. In isolated rat mesenteric arteries, equimolar doses of B7-33 reproduced the vasoprotective effects of serelaxin via endothelium-dependent hyperpolarization (EDH); in mouse arteries incubated with trophoblast-conditioned medium (a model of endothelial dysfunction in preeclampsia), B7-33 prevented impaired relaxation as effectively as serelaxin ^[2].

Skin Fibrosis, Scarring, and Biomaterials

In a subcutaneous polypropylene implant model in mice, coated with a PLGA polymer releasing B7-33 locally over 6 weeks, the coating produced a pronounced (~49%) reduction in fibrous capsule thickness compared with an uncoated control, an example of local antifibrotic application to mitigate the foreign-body response ^[10]. In a later study on human hypertrophic scar fibroblasts and normal dermal fibroblasts, B7-33 preserved high cell viability after 72-hour exposure and, at low (nanogram-range) concentrations, significantly reduced expression of the profibrotic markers TGF-β1, α-SMA, and COL1A1, more effectively than a reference antifibrotic comparator compound; an electrospun peptide-loaded wound-dressing format was proposed as a means of local delivery ^[11].

Maturity of the Evidence Base

The evidence base for B7-33 is exclusively preclinical: cell cultures, isolated vessels ex vivo, and rodent models (mice, rats). No clinical studies of B7-33 in humans have been published, and no approved indication or regimen for human use exists. It is important not to confuse B7-33 with serelaxin (recombinant two-chain relaxin-2). These are distinct molecules with different RXFP1 signaling profiles, and clinical data on serelaxin do not automatically transfer to B7-33. Serelaxin underwent a large-scale Phase III clinical trial program in acute heart failure (RELAX-AHF), but the confirmatory trial did not meet its primary endpoints, and the drug did not receive regulatory approval, illustrating how difficult it is to translate even a well-studied relaxin-family molecule into clinical benefit, while for the structurally and pharmacologically distinct B7-33 no such trajectory has been investigated at all. The evidence pathway is limited to: cells → isolated tissues → rodents, without progression to primates or humans. Any claims about a human "dose" of B7-33 have no support in the peer-reviewed literature.

Pharmacokinetics and Medicinal Chemistry

The main practical limitation of B7-33 is its short duration of action. The unmodified peptide shows low serum stability: the in vitro half-life is estimated at approximately 6 minutes, typical for a short linear peptide lacking a disulfide scaffold and protected termini, and thus readily accessible to proteases. Attachment of a lipid "anchor" via a PEG spacer (the construct AcK(PalmGlu)-PEG12-B7-33) enables reversible binding to serum albumin on the fatty-acid acylation principle also used for other peptide hormones; this extended the in vitro half-life to approximately 60 minutes without loss of activity at RXFP1 ^[5]. In parallel, efforts continue to identify a minimal potent relaxin-2 derivative through Aib substitutions and hydrocarbon "stapling" to artificially restore the helicity that the disulfide scaffold provides in the native hormone, an approach that has revealed a persistent problem with shorter analogs: weak affinity, especially in systems overexpressing RXFP1 ^[6]. Fundamentally new single-chain RXFP1 agonists (PEG spacer + lipidation) with subnanomolar activity and subcutaneous bioavailability are also being developed, characterized in cellular and translational in vivo models ^[4]. B7-33 itself should be regarded as a prototypical "minimal pharmacophore" rather than a final, pharmacokinetically optimized molecule.

Handling, Reconstitution, and Storage

Practical details on handling lyophilized B7-33 (reconstitution with bacteriostatic water, storage of dry and reconstituted material) are covered in a separate reference: B7-33: Form, Reconstitution, and Storage. Briefly: the dry lyophilizate should be stored frozen, protected from moisture and light; the reconstituted solution should be kept chilled, aliquoted into small portions immediately after dissolution, and repeated freeze-thaw cycles should be avoided, since the short linear structure without a disulfide scaffold is more susceptible to aggregation and denaturation than two-chain relaxin. Excessively vigorous mixing (vortexing) of the reconstituted solution is not recommended. Preferred dissolution is by gentle inversion or slow pipetting. These recommendations relate solely to reagent stability under laboratory conditions, not to any application.

RUO Status and Absence of an Established Human Dose

B7-33 is supplied exclusively for laboratory research use (research use only). No established human dosing regimen exists: there are no clinical trials of B7-33, no human toxicological or pharmacokinetic data, no immunogenicity data, no regulatory approval, and no validated human protocol. The dosages used in rodent studies (for example, 0.25 mg/kg in mouse models) pertain solely to a specific research protocol in a specific animal species and are not subject to direct conversion to humans: cross-species pharmacokinetic extrapolation is not valid for a short-lived, unmodified peptide. This material is scientific reference content and contains no medical advice, dosing recommendations, or guidance for use in humans.

References

1. Hossain MA, Kocan M, Yao ST, Royce SG, et al. A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1. Chemical Science (2016). doi:10.1039/C5SC04754D
2. Marshall SA, O'Sullivan K, Ng HH, Bathgate RAD, Parry LJ, Hossain MA, Leo CH. B7-33 replicates the vasoprotective functions of human relaxin-2 (serelaxin). European Journal of Pharmacology (2017). doi:10.1016/j.ejphar.2017.05.005
3. Devarakonda T, Mauro AG, Guzman G, Hovsepian S, et al. B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction-Related Adverse Cardiac Remodeling in Mice. Journal of the American Heart Association (2020). doi:10.1161/JAHA.119.015748
4. Illiano S, Poirier B, Minoletti C, Pasquier O, et al. Characterization of a new potent and long-lasting single chain peptide agonist of RXFP1 in cells and in vivo translational models. Scientific Reports (2022). doi:10.1038/s41598-022-24716-2
5. Praveen P, Wang C, Handley TNG, Wu H, Samuel CS, Bathgate RAD, Hossain MA. A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity. International Journal of Molecular Sciences (2023). doi:10.3390/ijms24076616
6. Handley TNG, Praveen P, Tailhades J, Wu H, Bathgate RAD, Hossain MA. Further Developments towards a Minimal Potent Derivative of Human Relaxin-2. International Journal of Molecular Sciences (2023). doi:10.3390/ijms241612670
7. Bathgate RAD, Halls ML, van der Westhuizen ET, Callander GE, Kocan M, Summers RJ. Relaxin Family Peptides and Their Receptors. Physiological Reviews (2013). doi:10.1152/physrev.00001.2012
8. Sethi A, Bruell S, Patil N, Hossain MA, Scott DJ, Petrie EJ, Bathgate RAD, Gooley PR. The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1. Nature Communications (2016). doi:10.1038/ncomms11344
9. Alam F, Gaspari TA, Kemp-Harper BK, Low E, Aw A, Ferens D, Spizzo I, Jefferis AM, Praveen P, Widdop RE, Bathgate RAD, Hossain MA, Samuel CS. The single-chain relaxin mimetic, B7-33, maintains the cardioprotective effects of relaxin and more rapidly reduces left ventricular fibrosis compared to perindopril in an experimental model of cardiomyopathy. Biomedicine & Pharmacotherapy (2023). doi:10.1016/j.biopha.2023.114370
10. Welch NG, Mukherjee S, Hossain MA, Praveen P, Werkmeister JA, Wade JD, Bathgate RAD, Winkler DA, Thissen H. Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation. ACS Applied Materials & Interfaces (2019). doi:10.1021/acsami.9b17859
11. Tamburriello M, Benedetti L, Chiesa E, Pisani S, Genta I, Conti B, Ceccarelli G, Dorati R. B7-33 modulates fibrotic signalling in hypertrophic scar fibroblasts: An in vitro study supporting a novel therapeutic strategy. Journal of Drug Delivery Science and Technology (2026). doi:10.1016/j.jddst.2026.108327