RXFP2

On November 28, 2023, Tectonic Therapeutic, a biotech company dedicated to discovering novel GPCR-targeted therapies, announced the completion of dosing in the first patient enrolled for their Fc-relaxin fusion program targeting the RXFP1 receptor. Tectonic Therapeutic's TX000045 Fc-relaxin fusion protein, referred to as "TX45," is a potentially best-in-class formulation that, through protein engineering efforts, has overcome the biophysical limitations of human hormones, delivering optimal pharmacokinetics, target engagement, and developability. Following the completion of a dose-escalation safety study in healthy volunteers, the continued development of TX45 will focus on high unmet needs within cardiopulmonary indications. As an agonist for the relaxin-1 receptor (RXFP1), relaxin produces unique and diverse biological effects including pulmonary and systemic vasodilation, tissue remodeling/fibrosis reversal, and inflammation reduction. Owing to these characteristics, it has the potential for significant therapeutic effects in cardiopulmonary diseases. Additionally, it is also referred to as the "pregnancy hormone" as it is upregulated during pregnancy to help the cardiovascular system of expectant mothers meet the increasing demands of the developing fetus and to remodel tissues and musculoskeletal structures. Tectonic Therapeutics was co-founded in 2019 by Andrew Kruse and Tim Springer from Harvard Medical School, dedicated to transforming the discovery of antibodies and other biologics targeting GPCRs (G protein-coupled receptors). Building on the pioneering work from Dr. Kruse's laboratory, Tectonic Therapeutics' proprietary GEODe platform has overcome challenges previously encountered in identifying biologics that modulate GPCR signaling, thereby advancing the development of novel GPCR-targeted therapies. About Relaxin Receptors The Relaxin family peptide receptors (RXFP) consist of two members in the human body, RXFP1 and RXFP2, and are part of the leucine-rich repeat-containing G protein-coupled receptors (LGRs). LGRs are involved in various processes of reproductive and developmental biology. RXFP1 is the receptor for human relaxin-2 hormone and plays a crucial role during pregnancy. It is responsible for physiological changes, including increased cardiac output and remodeling of reproductive tissues to facilitate childbirth. RXFP1 signaling also regulates the physiological functions of many organs in both males and females, particularly the heart, lungs, liver, and kidneys. In these organs, the activation of RXFP1 by relaxin-2 leads to multiple cellular effects, including vasodilation, angiogenesis, anti-inflammatory responses, and remodeling of the extracellular matrix through collagen degradation. As a result, RXFP1 has emerged as a promising therapeutic target for the treatment of cardiovascular and fibrotic diseases. On April 20th, 2023, the team led by Andrew Kruse at Harvard University published a new mechanism by which the relaxin receptor RXFP1 transmits regulatory signals through an autoregulatory mechanism in the online edition of the journal Nature Chemical Biology. In the study, Professor Andrew C. Kruse's laboratory resolved the cryo-electron microscopy structure of the RXFP1 receptor in complex with the downstream remodeled miniGs protein trimer. The structure revealed that the extracellular loop 2 (ECL2) of RXFP1 occupies the GPCR orthogonal ligand-binding pocket in its active state. Subsequent structural and functional studies have determined that the conformation of ECL2 is regulated by a mechanism involving the receptor's leucine-rich repeats (LRRs) and the hinge region (a short segment between LRRs and the 7-transmembrane domains, or 7TMs). These studies suggest that the synergistic action of multiple receptor structural domains controls the transduction of RXFP1 signaling under the influence of its agonist, relaxin-2. Combining mutagenesis experiments and studies on second messenger activity at the cellular level, the authors validated the molecular mechanism by which ECL2 regulates receptor activation. Integrating multidimensional experimental validations such as molecular dynamics simulations, cryo-electron microscopy, and crosslinking mass spectrometry, the authors proposed a new model for the auto-inhibition regulation mechanism of the relaxin receptor RXFP1. Drug Development Targeting the Relaxin Receptor  According to statistics from the Synapse Database, there are a total of 11 new drug development pipelines worldwide focusing on relaxin receptors. Among them, recombinant relaxin-2 Serelaxin was approved in Russia in 2015 for the treatment of fibrosis, heart failure, and portal hypertension. Serelaxin (RLX030, Novartis) is a recombinant form of human relaxin-2, a naturally occurring peptide. It is a hormone produced during pregnancy, which softens the cervix and the pubic symphysis (the joint at the pubic bone) and facilitates the birth process by allowing these areas to expand. It is a heterodimeric protein secreted by the corpus luteum and placenta during pregnancy. According to a clinical study published in The Lancet in 2013, treatment with Serelaxin for acute heart failure can relieve dyspnea and improve other clinical outcomes, but it does not affect readmission for treatment. Additionally, the tolerance and safety of Serelaxin treatment are good. In the U.S. market, Serelaxin (RLX030), developed by Novartis, was granted the designation of a "Breakthrough Therapy" by the FDA in 2013. However, in 2014, both the U.S. FDA and European regulatory authorities rejected the marketing application for RLX030, stating that the initial, smaller scale Phase III RELAX-AHF study did not prove the drug's ability to provide short-term relief of dyspnea within 24 hours, and further research was required to demonstrate its efficacy. In 2017, a global trial involving 6,600 patients over a period of 3.5 years, the RELAX-AHF-2 trial, evaluated the treatment effects of Serelaxin in addition to standard therapy, but it failed to meet its primary endpoints of reducing cardiovascular death by day 180, or worsening heart failure by day 5. Novartis claims that Serelaxin is a recombinant form of human relaxin-2 hormone, which is produced at higher levels in pregnant women, and the company believes it could help meet the increased demands on the cardiovascular system during pregnancy. The recombinant polypeptide AZD-3427 and the small molecule compound AZD-5462, developed by AstraZeneca, are currently in Phase 2 and Phase 1 clinical trials, respectively. Recently, AstraZeneca has initiated patient enrollment for the Phase 2b clinical trial of AZD-3427. The indications for this trial are heart failure and pulmonary arterial hypertension caused by left heart disease.

Relaxin is a pleiotropic hormone that mediates systemic hemodynamics and renal adaptive changes during pregnancy. Relaxin has also been shown to have anti-fibrotic properties and beneficial effects on heart failure, such as acute decompensated heart failure (ADHF). Heart failure has a high incidence and mortality rate. Its characteristics include complex tissue remodeling, including myocardial cell death and interstitial fibrosis increase. Relaxin activates many signal cascade reactions, which are beneficial in the conditions of ischemia reperfusion and heart failure.  As shown in Figure 1, these signaling pathways include the PI3K pathway and nitric oxide signaling pathway. Relaxin Family Peptide Receptor 1 (RXFP1) is the receptor of relaxin-2, and relaxin-2 is an important regulator of reproduction and cardiovascular physiology. RXFP1 is a well-validated target, a pharmacologically important member of the GPCR family 1c, whose activation by relaxin hormone is associated with hemodynamics, anti-fibrosis, and anti-inflammatory properties. Activation of RXFP1 can reduce vascular resistance and potential lung pressure, thereby increasing cardiac output, enhancing perfusion of important central vascular beds in the kidney, lung, and liver. Figure1 RXFP1-Mediated Signaling PathwayCurrently, there is only one marketable agonist targeting RXFP1, which is a recombinant natural hormone analogue named serelaxin, developed by Novartis. The agonists in the clinical stage include a Relaxin analogue AZD3427 (Phase II), a small molecule AZD5462 (Phase I), and an mRNA-type agonist mRNA-0184 (Phase I) developed by Moderna Therapeutics, which has been highly recognized for its COVID-19 vaccine. Also, there is a pre-clinical stage peptide agonist CGEN-25010 targeting RXFP1 and RXFP2 (no progress).  The Phase III clinical trial for heart failure treatment with serelaxin failed because it did not meet the primary endpoint, leaving it no choice but to be marketed in Russia. Besides, the half-life of serelaxin is only 10 minutes, therefore, it has to be administrated via intravenous injection. Both the Relaxin analogue AZD3427 and mRNA-0184 also require injection. For the sake of enhancing patients' compliance, the development of small molecule agonists has a broad market.  Targeting RXFP1 with small molecules poses significant challenges due to the lack of direct protein structure information and the poor drugability of the small molecules obtained from early high-throughput screenings. In 2013, the National Institutes of Health in the United States screened over 350,000 compounds through high-throughput methods and discovered the compound ML290. Both AstraZeneca (AZ) and Bristol-Myers Squibb (BMS) optimized based on ML290. Eventually, in June 2022, AZD5462 became the first orally taken small molecule RXFP1 agonist entering the clinical stage. Figure2 Structure of AZD5462The biennial EFMC-ISMC is a significant conference in the field of medicinal chemistry and drug discovery. At the EFMC-ISMC conference in September 2022, AstraZeneca disclosed the structural information of AZD5462. As shown in Figure 2, the structure of AZD5462 contains an interesting cyclohexane ring reduced to an ethyl group and is connected to a cis-cyclohexanecarboxylic acid fragment. AZD5462 is currently in Phase I clinical trials, where its safety, tolerability, and pharmacokinetics are being evaluated in Japanese healthy volunteers with both single and multiple escalating doses. Preclinical studies on monkey models with heart failure reduced ejection fraction (HFrEF) have shown that after 8 weeks of administration, cardiac systolic function was improved, without any significant increase in mean arterial blood pressure or heart rate. AZD5462 exhibits high selectivity for RXFP1, it shows no activity in the seven closest GPCRs (greater than 10µM), and does not interact with targets in the CEREP Safety panel. Figure 3 Discovery of AZD5462 - Introduction of Phenyl Group Bioisosteric ReplacementBased on the SAR information disclosed at the conference, it's simple to speculate on the discovery process of AZD5462, which is a case of traditional medicinal chemistry modification. The structure of ML290 is shown by the arrow in Figure 3, and its high LogD7.4 is likely to present toxicity risks. The benzene ring of the para-Aminobenzoic fragment was initially replaced with a non-aromatic fragment, such as in Rac-4 and Rac-5, but both experienced a decrease in activity levels and an increased rate of clearance.  As shown in Figure 4, the key modification involves the incorporation of a carboxyl group. This may have been done to reduce the LogD7.4 or improve the drug's tissue distribution. Indeed, from the subsequently provided Vss, it can be inferred that the volume of distribution was reduced. A comparison between Rac-12 and Rac-13 indicates that having 4 carbon atoms in between the carboxyl group and the oxygen atom is highly beneficial for increasing activity, with improvements of over ten times compared to Rac-11. Further attaching a cyclohexane carboxylic group and saturating the double bond in the non-aromatic ring of Rac-11 resulted in compound 14, which displayed a 10,000-fold increase in activity compared to Rac-11. However, the LogD7.4 of compound 14 remains high at 4.9. Figure 4 Discovery of AZD5462 - Introduction of Cyclohexanecarboxylic AcidFurther, replacing the benzene ring on the far right with non-aromatic fragments to reduce lipophilicity, decrease LogD7.4, and introducing tert-butyl, cyclobutyl, cyclohexyl, etc., produced the compounds 15, AZD5462, 16, 17, 18 as shown in Figure 5. Although the activity decreased, other properties were balanced. For example, LogD7.4 is within a reasonable range and the clearance rate will not be too fast. The predicted human administration dose is also within 100mg/70kg/d (except for compound 16, where the predicted dose reaches 304mg/70kg/d). However, in compounds 17 and 18, the R fragment has been changed from F to CN, reducing the predicted human dose but was not selected. The author speculates this might have been based on considerations of safety. Figure5 Discovery and Medicinal Optimization of AZD5462As shown in Figure 6, the various data for AZD5462 is reorganized. Due to the fact that the indications targeted by the RXFP1 agonist are chronic diseases, safety is undoubtedly the most important. The properties of AZD5462 are balanced, and in the evaluation of the most critical safety parameters, there is no risk of CYP450 enzyme inhibition, no TDI risk (CPY3A4/5), no hERG toxicity risk, and no Ames toxicity risk. In the Caco-2 test, although the efflux reached 3.6 and there was a certain degree of excretion, good absorption compensated for the risk of excretion. Although there is a certain degree of inhibition on the transporters OATP1B1 and OATP1B3, the impact of the drug concentration in the blood under specific dosing should still be considered. In addition, the LogD7.4 of AZD5462 was successfully lowered to 3.2 due to the de-aromatization of two aromatic rings and the introduction of carboxylic acid fragments. The target selectivity in the CEREP Safety panel is also more than 1000 times. Vss is within a reasonable range and will not lead to excessively widespread tissue distribution and affect the efficacy of the drug. The bioavailability in rats reached 58%, and although the bioavailability in crab-eating monkeys was only 12%, the predicted bioavailability in human bodies reached 70% based on the differences in the metabolic stability of AZD5462 in hepatocytes of different species. Taking into account the above benefit-risk ratio brought about, AZD5462 finally entered phase I clinical trials to evaluate its potential for treating cardiovascular disorders. Figure6 Key Evaluation Data of AZD5462In Patsnap Patent Analytics Database, RXFP1 has been searched, where it is mainly Astrazeneca and Bristol-Myers Squibb that are laying out the targeting point RXFP1. There are no patents disclosed by domestic companies following it yet.  Astrazeneca has applied for a patent WO2022122773A1, which contains 624 embodiments, including AZD5462. Bristol-Myers Squibb disclosed four patents related to RXFP1 agonists on May 4, 2023. The first one is WO2023077041A1, which only has 19 embodiments. The second one is WO2023077040A1, featuring 377 embodiments, with a small part of the compounds achieving nanomolar level. The third patent, WO2023077070A1, includes 188 embodiments, with the majority of the compounds achieving nanomolar activity. Structurally, it is further optimized based on the second patent structure. The fourth one is WO2023076626A1, with 976 embodiments. The structure extends the third one, just replacing the pyridine with a phenyl ring. Activities are mostly at the nanomolar level. It has been a decade since the structural reporting of ML290 by the National Institutes of Health in the United States. When Astrazeneca and Bristol-Myers Squibb entered the field of small molecule RXFP1 agonists, they took ML290 as the lead compound and optimized its medicinal properties according to the traditional medicinal chemical modification concepts. In 2022, the CryoEM structure of the active state RFXP1 was published on bioRxiv, which will stimulate the discovery of more new structural types of RXFP1 small molecule agonists. As AZD5462 enters clinical evaluation for safety and efficacy, the passion for research and development in this field has been reignited. We look forward to more positive results and hope that this will bring good news for patients with cardiovascular diseases. Reference 1.Ram W. Sabnis. Novel RXFP1Modulators for Treating Heart Failure. ACS Med. Chem. Lett. 2022, 13, 1213−1214. 2.Maik Gollasch et al; RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Gαi2-Protein/PI3Kß/γ/Nitric Oxide-Coupled Pathway. doi: 10.3389/fphys.2018.01234. 3.https://drughunter.com/first-disclosures-from-esmc-ifmc-nice-2022/. 4.Pamela Brown et al; Recent trends in medicinal chemistry and enabling technologies. Highlights from the Society for Medicines Research Conference. Drugs of the Future 2023, 48(3): 211-219.