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(A) Design of B7-33. Truncation of 6 residues from the N-terminus and elongation of 4 residues (KRSL) at the C-terminus of B1-29 (or truncation of 6 residues from B1-33) resulted in a soluble B-chain derivative of H2 relaxin, B7-33. The native H2 B-chain (B1-29) is insoluble (left vial) whereas the B7-33 is soluble (right vial) at concentration 4 mg mL⁻¹ H2O. (B) X-ray crystal structure of H2 relaxin showing the key RXFP1 binding residues (RB13, RB17 and IB20) in the B-chain
Source publication
Human gene-2 relaxin (H2 relaxin) is a pleiotropic hormone with powerful vasodilatory and anti-fibrotic properties which has led to its clinical evaluation and provisional FDA approval as a treatment for acute heart failure. The diverse effects of H2 relaxin are mediated via its cognate G protein coupled-receptor (GPCR), Relaxin Family Peptide Rece...
Citations
... 11 In addition, pathways activated by relaxin2 vary extremely be tween different types of native receptorexpressing cells, 8 as well as between nativeexpressing and RXFP1overexpressing cells: B(733) (B7), a researchonly singlechain derivative of human relaxin2, was shown to be a lowaffinity RXFP1 binder (B7, pK i [negative decadic logarithm of inhibition constant] 5.5; relaxin2, pK i 9.0) as well as a weak cAMP agonist in RXFP1over expressing HEKRXFP1 cells, but was fully active in human car diac fibroblasts as compared to the native peptide. 12 What does this have to do with the termination of LY3540378 development? The meticulous upengineering of halflife may have been achieved at the cost of essential signalling pathways -current relaxin2 mimetics may not really mimic the native peptide's complex and highly beneficial spectrum of effects. ...
On November 25, 2024, Eli Lilly and Company informed the public about the decision to terminate their phase-2 study, J3E-MC-EZDB with the relaxin-2 analogue, LY3540378, after data analysis of 40% of participants had indicated futility without immediate signs of patient risk. The study enrolled patients with heart failure with preserved ejection fraction within 2 weeks of an event of worsening heart failure with volume overload to receive 3 different doses of LY3540378 or placebo as weekly subcutaneous injection.In this Editorial, we compare LY3540378 and related long-acting relaxin-2 analogues with native relaxin-2. It is demonstrated that the modifications to native relaxin-2 to increase its half-life in circulation have been achieved at the cost of i) safety and ii) signalling pathways pivotal to the treatment of HFpEF. In terms of safety concerns, elevated immunogenicity attributed to the fusion to Fc or antibody fragments is forwarded, as well as impairment of therapy control in blood pressure-labile HFpEF patients due to overly prolonged administration intervals. With respect to signalling, we elaborate on the glucocorticoid-receptor and Wnt1 pathways that control anti-inflammatory and anti-arrhythmic therapy effects. It is highly unlikely that those pathways are activated by the long-acting relaxin-2 analogues. Using the Wn1 pathway, native relaxin-2 increases markedly the expression of the fast sodium channel, Nav1.5 in cardiomyocytes from aged rats, to ~200 % after 48 hours. In contrast, increasing doses of a single-chain analogue of relaxin-2, B(7-33) have no effect on Nav1.5.In summary, we make a case for the therapeutic use of full-length, native-structure human relaxin-2 in HF, especially in HFpEF. We need the full pleiotropy of the native peptide for a most complex clinical syndrome.
... Processing of the pro-RLN to RLN occurs in vivo through the endoproteolytic cleavage of the C-peptide. However, delivering the A and B-chain peptides separately often leads to reduced protein stability and assembly challenges 41 . Therefore, we retained the original mRNA sequence encoding the pro-RLN (Fig. 4a, Supplementary Data file 4). ...
Metabolic dysfunction-associated steatohepatitis (MASH) poses challenges for targeted delivery and retention of therapeutic proteins due to excess extracellular matrix (ECM). Here we present a new approach to treat MASH, termed “Fibrosis overexpression and retention (FORT)”. In this strategy, we design (1) retinoid-derivative lipid nanoparticle (LNP) to enable enhanced mRNA overexpression in fibrotic regions, and (2) mRNA modifications which facilitate anchoring of therapeutic proteins in ECM. LNPs containing carboxyl-retinoids, rather than alcohol- or ester-retinoids, effectively deliver mRNA with over 10-fold enhancement of protein expression in fibrotic livers. The carboxyl-retinoid rearrangement on the LNP surface improves protein binding and membrane fusion. Therapeutic proteins are then engineered with an endogenous collagen-binding domain. These fusion proteins exhibit increased retention in fibrotic lesions and reduced systemic toxicity. In vivo, fibrosis-targeting LNPs encoding fusion proteins demonstrate superior therapeutic efficacy in three clinically relevant male-animal MASH models. This approach holds promise in fibrotic diseases unsuited for protein injection.
... Different options to extend the pharmacokinetic half-life of relaxin-2 include PEGylation, fatty acid (Muppidi et al. 2019) Fc fusion (e.g., AZD3427), or fusion with the serum albumin binding VHH domain (albVHH) (LY3540378) (Verdino et al., 2023). In parallel, small molecule agonists of RXFP1 were identified (Sabnis, 2022), and pioneering studies showed that a single-chain peptide derived from the relaxin B chain (B7-33) maintains activity as a functional agonist for RXFP1, providing an important step towards simplification of the relaxin structure (Hossain et al., 2016). ...
Background
The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half‐life of serelaxin. Optimization of fatty acid‐acetylated single‐chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half‐life.
Experimental Approach
Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo‐allergic reactions and CD4+ T‐cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs.
Key Results
In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half‐life. The in vitro assays with LAD2 cells and CD4+ T‐cells showed that R2R01 had low potential for pseudo‐allergic and immunogenic reactions, respectively.
Conclusion and implications
R2R01 is a potent RXFP1 agonist with an extended half‐life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.
... However, delivering the A and B-chain peptides separately often leads to reduced protein stability and assembly challenges. 37 Therefore, we retained the original mRNA sequence encoding the pro-RLN (Fig. 4a). As the B-chain's two receptor binding sites are crucial for RLN function, we added the CBD peptides adjacent to the A-chain and close to the C terminus. ...
Fibrotic diseases, like non-alcoholic steatohepatitis (NASH), pose challenges for targeted delivery and retention of therapeutic proteins due to increased extracellular matrix (ECM) deposition. Here we present a new approach to treat fibrotic diseases, termed “Fibrosis overexpression and retention (FORT)”. In this two-step strategy, we design 1) a retinoid derivative lipid nanoparticle (LNP) to enable specific mRNA overexpression in hepatic stellate cells, and 2) mRNA modifications which facilitate anchoring of therapeutic proteins in the fibrotic ECM. LNPs containing carboxyl retinoid derivatives, as opposed to alcohol or ester retinoid derivatives, effectively delivered mRNA, resulting in more than 10- fold enhancement of protein expression within the fibrotic liver. The carboxyl retinoid rearrangement on the LNP surface improved protein binding, sprouting, and membrane fusion. Therapeutic relaxin fusion proteins were then engineered with an endogenous collagen-binding domain. These fusion proteins exhibited increased retention in fibrotic lesions and reduced systemic side effects. In vivo, fibrosis-targeting LNPs encoding for mRNA fusion proteins demonstrated superior therapeutic efficacy in three clinically relevant NASH mouse models. This approach holds promise in chronic fibrotic diseases that are unsuited for direct injections of recombinant proteins.
... It also cross-reacts with RXFP2, a native receptor for the related peptide INSL3 [12], with currently unknown consequences. H2 relaxin's strong activation of cAMP signaling may also induce side effects, including tumor growth, in long-term use [13]. Excessive cAMP activation in the ischemic heart could also potentially increase heart rate and myocardial workload [14], further exacerbating the contractile demand on myocytes [15]. ...
... Sci. 2023, 24, 12670 2 of 9 potent single-B-chain analog of H2 relaxin, B7-33 ( Figure 1C) [13]. B7-33 consists of residues 7-29 of the B chain of H2 relaxin ( Figure 1B,C), with the addition of a KRSL sequence from positions 30-33 of the B1-33 isoform of H2 relaxin, which dramatically improves solubility over B7-29 [13]. ...
... 2023, 24, 12670 2 of 9 potent single-B-chain analog of H2 relaxin, B7-33 ( Figure 1C) [13]. B7-33 consists of residues 7-29 of the B chain of H2 relaxin ( Figure 1B,C), with the addition of a KRSL sequence from positions 30-33 of the B1-33 isoform of H2 relaxin, which dramatically improves solubility over B7-29 [13]. B7-33, being a single-chain peptide, is far easier and cheaper to produce than the parent peptide, making it an excellent scaffold for modification with the aim of improving its pharmacological properties as an anti-fibrotic therapeutic [13] and a component of anti-fibrotic device coating [16]. ...
Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.
... Similarly, another phase III trial exploring serelaxin in scleroderma had demonstrated limited efficacy [130]. Researchers have also directed their efforts towards the development of novel relaxin analogous and agonists, including ML290 and B7-33, which have exhibited promising outcomes in reducing fibrosis in heart and kidney of rodent models [131]. While preclinical in vivo models provide compelling evidence for potential therapeutic application of serelaxin and relaxin mimetics in fibrotic diseases, the underlying mechanisms of these findings remain unclear. ...
Fibrosis is a prevalent and detrimental condition associated with various diseases with a high impact on global morbidity and mortality rates. Despite its diverse causes and affected organs, common underlying mechanisms drive the development and progression of the disease. These mechanisms include an exaggerated inflammatory response, excessive activation of fibroblasts, and abnormal tissue remodeling following severe or repetitive tissue injury. Although significant advancements have been achieved to enhance our understanding of fibrosis, there is still a gap between identifying potential antifibrotic targets and successfully translating them into effective clinical interventions. Novel approaches that target specific cellular and molecular processes involved in fibrosis hold promise for reducing the pathological consequences of the disease. Understanding the pathogenesis and clinical implications of fibrotic diseases is crucial for developing effective therapeutic strategies and improving patient outcomes. In this review, we introduce the concept of fibrosis, discuss the mechanisms by which it arises, and explore existing and emerging therapeutic approaches in development.
... [17] Some easily synthesized single chain H2 relaxin variants have been reported, but their relative cAMP activity was reduced. [18,19] Herein, we report that H2 relaxin analogues incorporating a pre-made diaminodiacid (DADA) bridge (Scheme 1) [20][21][22][23] in place of the A24 Cys -B23 Cys disulfide can be easily and rapidly synthesized by a single-shot solid-phase synthesis (SPPS) [24][25][26][27][28][29][30] and expedient one-step folding. This new synthesis would enable the production of a myriad of derivatives with optimized potency, selectivity, and stability; a strategy that also proved applicable to the synthesis of the disulfide analogues of other ISPs such as insulin and H3 relaxin. ...
... native H2 relaxin, H2-1 b, H2-1 b-B28 Ala , H2-1 b-B28 Val , H2-1 b-B28 Leu and H2-1 b-B28 Ile , defined as EC 50 (RXFP2)/EC 50 -(RXFP1), were 18,41,120,194,196, and 220, respectively (Figure 3d). These results are consistent with the previous finding that the B28 Trp to B28 Ala mutation can improve the selectivity and established for the first time that the B28 Trp to B28 Ile mutation improves the selectivity the most (220fold). ...
Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next‐generation drugs. Separate synthesis of multiple peptide fragments and tedious chain‐to‐chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre‐made diaminodiacid bridge at A‐B chain terminal disulfide can be easily and rapidly synthesized by a single‐shot automated solid‐phase synthesis and expedient one‐step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.
... The combination of recombinant technologies and post translational chemical modifications resulted in the discovery of long-lasting double chain relaxin analogues 11 . The recent identification of a single chain peptide derived from the relaxin B chain (B7-33) as a functional agonist of RXFP1 was an important step towards simplification of the relaxin structure 12 . We have recently identified new short single B chain modified peptides that are highly potent RXFP1 agonists 13 . ...
... The differences in the engagement of some signaling pathway could be cell type dependent or due to the use of primary cells at very early passages. Since new RXFP1 agonists (B7-33, ML290) described so far are biased agonists 12,14 impedance experiments were performed to assess the full phenotypic response to relaxin and SA10SC-RLX in EA.hy926 or EA.hy926_RXFP1 cells. Neither relaxin nor SA10SC-RLX induced a response in wild type EA.hy926 cells. ...
Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.
... RLX binds with high affinity to the LRRs and low affinity to the linker enabling the LDLa-linker domain to form a conformation leading to G-protein-dependent signal transduction pathways, which differ depending on the target cell types (Halls et al., 2007;Sethi et al., 2021). The most common signaling mechanisms downstream of RXFP1 include the elevation of cAMP, the increased phosphorylation of extracellular-regulated protein kinases (ERK) 1/2 (Hossain et al., 2016) and the up-regulation of nitric oxide production (Baccari & Bani 2008). This unique mode of interaction of RLX with its receptor has represented a challenge for the generation of effective RXFP1 agonists. ...
... This unique mode of interaction of RLX with its receptor has represented a challenge for the generation of effective RXFP1 agonists. To date, three different conceptual pathways have been explored, namely: peptide and non-peptide RXFP1 agonists designed on receptor complementarity (Shemesh et al., 2009;Xiao et al., 2013;Agoulnik et al., 2017), low molecular weight RLX analogues designed on the functional B chain domains (Hossain et al., 2016;Marshall et al., 2017;Mallart et al., 2021) and semi-synthetic doublechain RLX analogues modified to extend their half-life (Muppidi et al., 2019). Another approach focuses on H1 RLX, whose gene Rln1 is only present in primates as a likely ortholog of the RLX-encoding gene Rln2 (Hansell et al., 1991). ...
... Materials-Serelaxin (batch B917056/1/1, prepared by Boehringer-Ingelheim Inc.) was kindly donated by the Relaxin RRCA Foundation, Florence, Italy. Short-chain peptide analogues of H1 RLX (Table 1) and the known RXFP1 agonist B7-33 (Hossain et al., 2016) were synthesized as previously described (D'Ercole et al., 2020;Nuti et al., 2010). Full analytical characterization (HPLC and MS) of the synthetic peptides is available as Supplementary Material (Table 1). ...
The peptide hormone relaxin (RLX), also available as clinical-grade recombinant protein (serelaxin), holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues common to all peptide drugs. In this study, by a computational modelling chemistry approach, we have synthesized and tested a set of low molecular weight peptides based on the putative receptor-binding domain of the B chain of human H1 RLX isoform, with the objective to obtain RLX analogues with improved pharmacokinetic features. Some of them were stabilized to induce the appropriate 3-D conformation by intra-chain tri-azolic staples, which should theoretically enhance their resistance to digestive enzymes making them suited for oral administration. Despite these favourable premises, none of these H1 peptides, either linear or stapled, revealed a sufficient affinity to the specific RLX receptor RXFP1. Moreover, none of them was endowed with any RLX-like biological effects in RXFP1-expressing THP-1 human monocytic cells and mouse NIH-3T3-derived myofibroblasts in in vitro culture, in terms of significantly relevant cAMP elevation and ERK1/2 phosphorylation, which represent two major signal transduction events downstream RXFP1 activation. This was at variance with authentic serelaxin, which induced a clear-cut, significant activation of both these classical RLX signaling pathways. Albeit negative, the results of this study offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues.
... The combination of recombinant technologies and post translational chemical modi cations resulted in the discovery of long-lasting double chain relaxin analogues (Muppidi et al. 2019). The recent identi cation of a single chain peptide derived from relaxin B chain (B7-33) as a functional agonist of RXFP1 was an important step towards simpli cation of the relaxin structure (Hossain et al. 2016). We have recently identi ed new short single B chain modi ed peptides that are highly potent RXFP1 agonists (Mallart et al. 2021). ...
... The differences in the engagement of some signaling pathway could be cell type dependent or due to use of primary cells at very early passages. Since new RXFP1 agonists (B7-33, ML290) described so far are biased agonists (Hossain et al. 2016, Kocan et al. 2017 and discrepancy between signaling have been observed with these compounds, impedance experiments were performed to assess the full phenotypic response to relaxin and SA10SC-RLX in endothelial cells (EA.hy926) expressing or not human recombinant RXFP1. Neither relaxin nor SA10SC-RLX induced a response in wild type EA.hy926 cells. ...
Despite beneficial effects in acute heart failure, recombinant relaxin-2 full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life.
SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with study of different RXFP1 mutant were used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration and this, without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported with relaxin in humans.
In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.
