RESUMO
BACKGROUND: Heart failure mortality remains high despite recent progress in pharmacological treatment. AZD3427 is a selective long-acting analog of relaxin, a vasodilatory hormone with antifibrotic effects. We assessed the safety, pharmacokinetics, and pharmacodynamics of AZD3427 in healthy volunteers and patients with heart failure on standard-of-care therapy. METHODS AND RESULTS: In this first-in-human, phase 1a/b, randomized, single-blind, placebo-controlled study, healthy volunteers were randomized 6:2 to receive a single dose of AZD3427 or placebo by subcutaneous injection in 5 mixed-ethnicity cohorts (5, 10, 30, 90, or 270 mg) and 1 Japanese-descent cohort (270 mg), or by intravenous injection in 1 cohort (15 mg). After confirming safety and tolerability in healthy volunteers, 3 cohorts of patients with heart failure and left ventricular ejection fraction ≤40% and 3 cohorts with ejection fraction ≥41% were randomized 6:2 to receive 5 weekly doses of AZD3427 (5, 15, or 45 mg) or placebo by subcutaneous injection. In total, 56 healthy volunteers and 48 patients with heart failure were randomized. AZD3427 was well tolerated at all doses. After subcutaneous administration, AZD3427 was absorbed slowly, and exposure was approximately linear across the dose range. In patients with heart failure, AZD3427 terminal half-life was 13 to 14 days and there were numerical increases in stroke volume and estimated glomerular filtration rate. No treatment-emergent antidrug antibodies were detected. CONCLUSIONS: AZD3427 had favorable safety and pharmacokinetic profiles. Hemodynamic changes in patients with heart failure were consistent with the anticipated effects of a relaxin analog. These findings support further development of AZD3427 as a novel long-term treatment for patients with heart failure. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04630067.
Assuntos
Insuficiência Cardíaca , Receptores Acoplados a Proteínas G , Humanos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Masculino , Feminino , Método Simples-Cego , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas G/agonistas , Adulto , Idoso , Resultado do Tratamento , Receptores de Peptídeos/agonistas , Volume Sistólico/efeitos dos fármacos , Injeções Subcutâneas , Função Ventricular Esquerda/efeitos dos fármacos , Adulto Jovem , Relaxina/farmacocinética , Relaxina/administração & dosagem , Relaxina/efeitos adversos , Relaxina/uso terapêutico , Relação Dose-Resposta a Droga , Injeções IntravenosasRESUMO
Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (â¼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (â¼3 nM) with reduced partial agonist activity, enhanced antagonist potency (â¼10 nM) and maximum agonist inhibition (â¼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.
Assuntos
Insulina , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animais , Humanos , Camundongos , Masculino , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/agonistas , Insulina/metabolismo , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Células HEK293 , Camundongos Endogâmicos C57BL , ProteínasRESUMO
INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.
Assuntos
Neuropeptídeos , Relaxina , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Relaxina/metabolismo , Patentes como Assunto , Insulina/metabolismo , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/metabolismoRESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Ratos , Suínos , Masculino , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/metabolismo , Porco Miniatura , Doenças Cardiovasculares/tratamento farmacológico , Nefropatias/tratamento farmacológico , Ratos Sprague-Dawley , Peptídeos/farmacologia , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Relaxina/farmacologia , Relaxina/administração & dosagem , Relaxina/farmacocinética , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismoRESUMO
Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.
Assuntos
Relaxina , Humanos , Masculino , Ratos , Animais , Relaxina/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistasRESUMO
We identified the anti-Mullerian hormone (also known as Müllerian inhibiting substance or MIS) as an inhibitory hormone that induces long-term contraception in mammals. The type II receptor to this hormone, AMHR2 (also known as MISR2), represents a promising druggable target for the modulation of female reproduction with a mechanism of action distinct from steroidal contraceptives. We designed an in vitro platform to screen and validate small molecules that can activate MISR2 signaling and suppress ovarian folliculogenesis. Using a bone morphogenesis protein (BMP)response element luciferase reporter cellbased assay, we screened 5,440 compounds from a repurposed drug library. Positive hits in this screen were tested for specificity and potency in luciferase doseresponse assays, and biological activity was tested in ex vivo Mullerian duct regression bioassays. Selected candidates were further evaluated in ex vivo follicle/ovary culture assays and in vivo in mice and rats. Here, we report that SP600125, CYC-116, gandotinib, and ruxolitinib can specifically inhibit primordial follicle activation and repress folliculogenesis by stimulating the MISR2 pathway.
Assuntos
Anticoncepcionais , Reposicionamento de Medicamentos , Folículo Ovariano , Receptores de Peptídeos , Receptores de Fatores de Crescimento Transformadores beta , Bibliotecas de Moléculas Pequenas , Animais , Antracenos/química , Antracenos/farmacologia , Anticoncepcionais/química , Anticoncepcionais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Nitrilas/química , Nitrilas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Receptores de Peptídeos/agonistas , Receptores de Fatores de Crescimento Transformadores beta/agonistas , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazóis/química , Tiazóis/farmacologiaRESUMO
Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.
Assuntos
Ensaios de Triagem em Larga Escala , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The central relaxin-3/RXFP3 system plays important roles in stress responses, feeding, and motivation for reward. However, exploration of its therapeutic applications has been hampered by the lack of small molecule ligands and the cross-activation of RXFP1 in the brain and RXFP4 in the periphery. Herein, we report the first structure-activity relationship studies of a series of novel nonpeptide amidinohydrazone-based agonists, which were characterized by RXFP3 functional and radioligand binding assays. Several potent and efficacious RXFP3 agonists (e.g., 10d) were identified with EC50 values <10 nM. These compounds also had high potency at RXFP4 but no agonist activity at RXFP1, demonstrating > 100-fold selectivity for RXFP3/4 over RXFP1. In vitro ADME and pharmacokinetic assessments revealed that the amidinohydrazone derivatives may have limited brain permeability. Collectively, our findings provide the basis for further optimization of lead compounds to develop a suitable agonist to probe RXFP3 functions in the brain.
Assuntos
Hidrazonas/farmacologia , Indóis/química , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Modelos Moleculares , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
Relaxin family peptide receptors (RXFPs) are the potential therapeutic targets for neurological, cardiovascular, and metabolic indications. Among them, RXFP3 and RXFP4 (formerly known as GPR100 or GPCR142) are homologous class A G protein-coupled receptors with short N-terminal domain. Ligands of RXFP3 or RXFP4 are only limited to endogenous peptides and their analogues, and no natural product or synthetic agonists have been reported to date except for a scaffold of indole-containing derivatives as dual agonists of RXFP3 and RXFP4. In this study, a new scaffold of tricyclic derivatives represented by compound 7a was disclosed as a selective RXFP4 agonist after a high-throughput screening campaign against a diverse library of 52,000 synthetic and natural compounds. Two rounds of structural modification around this scaffold were performed focusing on three parts: 2-chlorophenyl group, 4-hydroxylphenyl group and its skeleton including cyclohexane-1,3-dione and 1,2,4-triazole group. Compound 14b with a new skeleton of 7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8(5H)-one was thus obtained. The enantiomers of 7a and 14b were also resolved with their 9-(S)-conformer favoring RXFP4 agonism. Compared with 7a, compound 9-(S)-14b exhibited 2.3-fold higher efficacy and better selectivity for RXFP4 (selective ratio of RXFP4 vs. RXFP3 for 9-(S)-14b and 7a were 26.9 and 13.9, respectively).
Assuntos
Cicloexanonas/farmacologia , Desenho de Fármacos , Pirimidinonas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Triazóis/farmacologia , Cicloexanonas/síntese química , Cicloexanonas/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Pirimidinonas/síntese química , Pirimidinonas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).
Assuntos
Lipopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Relaxina/análogos & derivados , Relaxina/farmacologia , Sequência de Aminoácidos , Animais , Doenças Cardiovasculares , Linhagem Celular Tumoral , Células HEK293 , Meia-Vida , Humanos , Lipopeptídeos/genética , Lipopeptídeos/farmacocinética , Masculino , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Subunidades Proteicas , Ratos Sprague-Dawley , Relaxina/genética , Relação Estrutura-AtividadeRESUMO
Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.
Assuntos
Queratinócitos/metabolismo , Cininas/metabolismo , Pele , Calicreínas Teciduais/metabolismo , Cicatrização/fisiologia , Homeostase , Humanos , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Pele/imunologia , Pele/metabolismoRESUMO
Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células CHO , Cricetulus , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
The neuropeptide relaxin-3 is expressed by the pontine nucleus incertus. Relaxin-3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the relaxin-family peptide 3 receptor (RXFP3). Despite the presence of RXFP3 in the nucleus of the solitary tract (NTS), the ability of RXFP3 to modulate NTS-mediated cardiorespiratory functions has not been explored. Therefore, we examined the effects of bilateral microinjections of the selective agonist, RXFP3-A2 (40⯵M, 100â¯nL/side), into the NTS in perfused working-heart-brainstem-preparations from rats (nâ¯=â¯6), while recording phrenic, vagal, and thoracic sympathetic chain activity (PNA, VNA, t-SCA) and heart rate (HR). RXFP3-A2 significantly increased respiratory rate and shortened post-inspiratory VNA. RXFP3-A2 in the NTS also significantly enhanced arterial chemoreceptor reflex (a-CR)-mediated tachypnea. However, RXFP3-A2 had no significant effect on HR and t-SCA at baseline or during the a-CR. These data represent the first evidence that RXFP3 activation in the NTS can selectively modulate respiration at baseline and during reflex behaviour.
Assuntos
Células Quimiorreceptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Taxa Respiratória/fisiologia , Núcleo Solitário/metabolismo , Animais , Células Quimiorreceptoras/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Microinjeções/métodos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Taxa Respiratória/efeitos dos fármacos , Núcleo Solitário/efeitos dos fármacosRESUMO
Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.
Assuntos
Peptídeos/síntese química , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Peptídeos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Dicroísmo Circular , Cricetulus , Humanos , Insulina/química , Ligantes , Ligação Proteica , Proteínas/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Relaxina/química , TransfecçãoRESUMO
BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
Assuntos
Rim/patologia , Miocárdio/patologia , Miofibroblastos/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Peptídeos/fisiologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Benzimidazóis/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Células Cultivadas , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Proteínas Recombinantes , Relaxina/fisiologia , Tetrazóis/uso terapêuticoRESUMO
Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a Gi/o -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.
Assuntos
Ansiedade/metabolismo , Comportamento Animal/fisiologia , Neurônios GABAérgicos/metabolismo , Hipocampo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Comportamento Social , Animais , Comportamento Animal/efeitos dos fármacos , Neurônios GABAérgicos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistasRESUMO
The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Bibliotecas de Moléculas Pequenas/química , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Descoberta de Drogas , Ligantes , Peptídeos/química , Peptídeos/farmacologia , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Relaxina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
There are seven human relaxin family peptides that have two chains (A and B) and three disulfide bonds. The target receptors for four of these peptides are known as relaxin family peptide receptors, RXFP1-RXFP4. Detailed structure-activity relationship (SAR) studies of relaxin family peptides have been reported over the years and have led to the design of new analogs with agonistic and antagonistic properties. This review briefly summarizes the SAR of human relaxin 2 (H2 relaxin) and human relaxin 3 (H3 relaxin) leading to the design and development of single-B-chain only agonists, B7-33 and peptide 5. The physiological functions of these new peptides agonists in cellular and animal models are also described.
Assuntos
Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Sequência de Aminoácidos , Animais , Humanos , Peptídeos/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Relaxina/química , Relaxina/farmacologia , Relação Estrutura-AtividadeRESUMO
In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the Gi/o-protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Peptídeos/administração & dosagem , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Reconhecimento Psicológico/efeitos dos fármacos , Comportamento Social , Ácido gama-Aminobutírico/metabolismo , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/enzimologia , Animais , Neurônios GABAérgicos/enzimologia , Infusões Intraventriculares , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Ocitocina/metabolismo , Fosforilação , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293â¯cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.