RESUMO
Osteoanabolic agents are used as a first line treatment in patients at high fracture risk. The PTH receptor 1 (PTH1R) agonists teriparatide (TPTD) and abaloparatide (ABL) increase bone formation, bone mineral density (BMD), and bone strength by activating PTH receptors on osteoblasts. Romosozumab (ROMO), a humanized monoclonal antibody against sclerostin, dramatically but transiently stimulates bone formation and persistently reduces bone resorption. Osteoanabolic agents increase BMD and bone strength while being more effective than antiresorptives in reducing fracture risk in postmenopausal women. However, direct comparisons of the antifracture benefits of osteoanabolic therapies are limited. In a direct comparison of TPTD and ABL, the latter resulted in greater BMD increases at the hip. While no differences in vertebral or non-vertebral fracture risk were observed between the two drugs, ABL led to a greater reduction of major osteoporotic fractures. Adverse event profiles were similar between the two agents except for hypercalcemia, which occurred more often with TPTD. No direct comparisons of fracture risk reduction between ROMO and the PTH1R agonists exist. Individual studies have shown greater increases in BMD and bone strength with ROMO compared with TPTD in treatment-naive women and in women previously treated with bisphosphonates. Some safety aspects, such as a history of tumor precluding the use of PTH1R agonists, and a history of major cardiovascular events precluding the use of ROMO, should also be considered when choosing between these agents. Finally, convenience of administration, reimbursement by national health systems and length of clinical experience may influence patient choice.
Assuntos
Anticorpos Monoclonais , Conservadores da Densidade Óssea , Densidade Óssea , Receptor Tipo 1 de Hormônio Paratireóideo , Teriparatida , Humanos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Conservadores da Densidade Óssea/uso terapêutico , Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/efeitos dos fármacos , Teriparatida/uso terapêutico , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Feminino , Fraturas por Osteoporose/prevenção & controle , Proteína Relacionada ao Hormônio Paratireóideo/uso terapêutico , Osteoporose Pós-Menopausa/tratamento farmacológicoRESUMO
Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo , Receptores Acoplados a Proteínas G , Transdução de Sinais , Anticorpos de Domínio Único , Ligantes , Humanos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Células HEK293 , Transdução de Sinais/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Ligação Proteica , Animais , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
CONTEXT: Hypoparathyroidism is a rare disorder characterized by a deficiency in PTH resulting in hypocalcemia, hyperphosphatemia, and hypercalciuria. Eneboparatide is an investigational peptide agonist of the PTH1 receptor for the treatment of chronic hypoparathyroidism (HP). OBJECTIVE: To evaluate the efficacy, safety, and tolerability of eneboparatide in HP patients. DESIGN: Open-label, phase 2 study. PARTICIPANTS: Twenty-eight patients (21 women, 7 men), mean age (range): 58 years (28-72), with HP were enrolled into 2 consecutive cohorts (C1, n = 12 and C2, n = 16). INTERVENTION: Following an optimization period, daily subcutaneous injections of eneboparatide were administered for 3 months at a 20 µg/day (C1) or 10 µg/day (C2) starting dose. Conventional therapy was progressively removed, and eneboparatide could be titrated up to 60 µg (C1) or 80 µg (C2). MAIN OUTCOMES: Proportion of patients achieving independence from conventional therapy, albumin-adjusted serum calcium (ADsCa), 24-h urine calcium (uCa), serum bone turnover markers (serum carboxy-terminal telopeptide of type I collagen and procollagen 1 intact N-terminal propeptide), bone mineral density (BMD), and adverse events (AEs). RESULTS: After 3 months, ≥ 88% of patients achieved independence from conventional therapy while mean ADsCa was maintained within target range (7.8-9â mg/dL). Eneboparatide induced a rapid and sustained reduction of mean 24-hour uCa, even among patients with hypercalciuria. Bone turnover markers slightly increased, and BMD remained unchanged, consistent with progressive resumption of physiologic bone turnover. Eneboparatide was well tolerated with no serious AEs. CONCLUSION: Eneboparatide allowed independence from conventional therapy and maintenance of serum calcium within a target range while normalizing uCa excretion and producing a balanced resumption of bone turnover.
Assuntos
Hipoparatireoidismo , Receptor Tipo 1 de Hormônio Paratireóideo , Humanos , Feminino , Hipoparatireoidismo/tratamento farmacológico , Pessoa de Meia-Idade , Masculino , Adulto , Idoso , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Densidade Óssea/efeitos dos fármacos , Resultado do Tratamento , Cálcio/sangueRESUMO
Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.
Assuntos
Imidazolidinas , Receptores Acoplados a Proteínas G , Compostos de Espiro , Humanos , Arrestina/metabolismo , Sítios de Ligação , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Imidazolidinas/farmacologia , Ligantes , Peptídeos/farmacologia , Conformação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/classificação , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Desenho de FármacosRESUMO
LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.
Assuntos
Hipoparatireoidismo/tratamento farmacológico , Hormônio Paratireóideo/análogos & derivados , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Animais , Cálcio/sangue , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Metionina/metabolismo , Camundongos , Modelos Moleculares , Norleucina , Oxirredução , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Hormônio Paratireóideo/uso terapêutico , Ratos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
We have previously shown that the oral administration of the small molecule hPTHR1 agonist PCO371 and its lead compound, 1 (CH5447240) results in PTH-like calcemic and hypophostemic activity in thyroparathyroidectomized rats. However, 1 was converted to a reactive metabolite in a human liver microsome assay. In this article, we report on the modification path that led to an enhancement of PTHR1 agonistic activity and reduction in the formation of a reactive metabolite to result in a potent, selective, and orally active PTHR1 agonist 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (PCO371, 16c). This compound is currently being evaluated in a phase 1 clinical study for the treatment of hypoparathyroidism.
Assuntos
Imidazolidinas/administração & dosagem , Imidazolidinas/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Compostos de Espiro/administração & dosagem , Compostos de Espiro/metabolismo , Administração Oral , Animais , Feminino , Humanos , Hipoparatireoidismo/tratamento farmacológico , Hipoparatireoidismo/metabolismo , Imidazolidinas/química , Células LLC-PK1 , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/química , SuínosRESUMO
The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH2 and abaloparatide (a PTHrP derivative) with a single homologous ß-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/química , Sequência de Aminoácidos , AMP Cíclico/biossíntese , Células HEK293 , Humanos , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo-electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.
Assuntos
Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/química , Motivos de Aminoácidos , Microscopia Crioeletrônica , Humanos , Hormônio Paratireóideo/farmacologia , Hormônio Paratireóideo/fisiologia , Ligação Proteica , Domínios Proteicos , Receptor Tipo 1 de Hormônio Paratireóideo/ultraestruturaRESUMO
Aim: Parathyroid hormone-1 receptor (PTH1R) is a member of B G protein-coupled receptors. The agonistic activation of the PTH1R results in the production and secretion of osteoclast-stimulating cytokines while antagonists may be used to treat bone metastases, hypercalcemia, cachexia and hyperparathyroidism. Results: We built pharmacophore models and investigated the characteristics of PTH1R agonists and antagonists. The agonist model consists of three hydrophobic points, one hydrogen bond acceptor and one positive ionizable point. The antagonist model consists of one hydrogen bond donor and three hydrophobic points. Conclusion: The features of the two models are similar, but the hydrogen bond acceptor, which is the main difference between PTH1R agonists and antagonists, suggests it may be essential for the agonist.
Assuntos
Desenho de Fármacos , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
Fractures typically heal via endochondral and intramembranous bone formation, which together form a callus that achieves union and biomechanical recovery. PTHrP, a PTH receptor agonist, plays an important physiological role in fracture healing as an endogenous stimulator of endochondral and intramembranous bone formation. Abaloparatide, a novel systemically-administered osteoanabolic PTH receptor agonist that reduces fracture risk in women with postmenopausal osteoporosis, has 76% homology to PTHrP, suggesting it may have potential to improve fracture healing. To test this hypothesis, ninety-six 12-week-old male rats underwent unilateral internally-stabilized closed mid-diaphyseal femoral fractures and were treated starting the next day with daily s.c. saline (Vehicle) or abaloparatide at 5 or 20 µg/kg/d for 4 or 6 weeks (16 rats/group/time point). Histomorphometry and histology analyses indicated that fracture calluses from the abaloparatide groups exhibited significantly greater total area, higher fluorescence scores indicating more newly-formed bone, and higher fracture bridging scores versus Vehicle controls. Callus bridging score best correlated with callus cartilage score (r = 0.64) and fluorescence score (r = 0.67) at week 4, and callus area correlated with cartilage score (r = 0.60) and fluorescence score (r = 0.89) at Week 6. By micro-CT, calluses from one or both abaloparatide groups had greater bone volume, bone volume fraction, bone mineral content, bone mineral density, and cross-sectional area at both time points versus Vehicle controls. Destructive bending tests indicated greater callus maximum load and stiffness in one or both abaloparatide groups at both time points versus Vehicle controls. These results provide preliminary preclinical evidence for improved fracture healing with systemically-administered abaloparatide. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
Assuntos
Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/uso terapêutico , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Animais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Masculino , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share â¼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single ß-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The ß-scan series allowed us to identify two homologs, each containing two αâß replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both ß2- and ß3-residues, which differ in terms of side-chain location.
Assuntos
Hormônio Paratireóideo/química , Peptídeos/síntese química , Peptídeos/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 2 de Hormônio Paratireóideo/agonistas , Sequência de Aminoácidos , Células HEK293 , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
During the course of derivatization of HTS hit 4a, we have identified a novel small-molecule hPTHR1 agonist, 1-(3,5-dimethyl-4-(2-((2-((1 R,4 R)-4-methylcyclohexyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1-methylurea (CH5447240, 14l). Compound 14l exhibited a potent in vitro hPTHR1 agonist effect with EC20 of 3.0 µM and EC50 of 12 µM and showed excellent physicochemical properties, such as high solubility in fasted state simulated intestinal fluid and good metabolic stability in human liver microsomes. Importantly, 14l showed 55% oral bioavailability and a significantly elevated serum calcium level in hypocalcemic model rats.
Assuntos
Descoberta de Drogas , Hipoparatireoidismo/tratamento farmacológico , Compostos de Metilureia/uso terapêutico , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacocinética , Sulfonas/uso terapêutico , Ureia/farmacologia , Ureia/farmacocinética , Administração Oral , Disponibilidade Biológica , Linhagem Celular , Humanos , Compostos de Metilureia/administração & dosagem , Compostos de Metilureia/farmacologia , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/uso terapêutico , Sulfonas/administração & dosagem , Sulfonas/farmacologia , Ureia/administração & dosagem , Ureia/uso terapêuticoRESUMO
Abaloparatide, a novel PTH1 receptor agonist, increased bone formation in osteopenic ovariectomized cynomolgus monkeys while increasing cortical and trabecular bone mass. Abaloparatide increased bone strength and maintained or enhanced bone mass-strength relationships, indicating preserved or improved bone quality. INTRODUCTION: Abaloparatide is a selective PTH1R activator that is approved for the treatment of postmenopausal osteoporosis. The effects of 16 months of abaloparatide administration on bone formation, resorption, density, and strength were assessed in adult ovariectomized (OVX) cynomolgus monkeys (cynos). METHODS: Sixty-five 9-18-year-old female cynos underwent OVX surgery, and 15 similar cynos underwent sham surgery. After a 9-month period without treatments, OVX cynos were allocated to four groups that received 16 months of daily s.c. injections with either vehicle (n = 17) or abaloparatide (0.2, 1, or 5 µg/kg/day; n = 16/dose level), while Sham controls received s.c. vehicle (n = 15). Bone densitometry (DXA, pQCT, micro-CT), qualitative bone histology, serum calcium, bone turnover markers, bone histomorphometry, and bone strength were among the key measures assessed. RESULTS: At the end of the 9-month post-surgical bone depletion period, just prior to the treatment phase, the OVX groups exhibited increased bone turnover markers and decreased bone mass compared with sham controls. Abaloparatide administration to OVX cynos led to increased bone formation parameters, including serum P1NP and endocortical bone formation rate. Abaloparatide administration did not influence serum calcium levels, bone resorption markers, cortical porosity, or eroded surfaces. Abaloparatide increased bone mass at the whole body, lumbar spine, tibial diaphysis, femoral neck, and femoral trochanter. Abaloparatide administration was associated with greater lumbar vertebral strength, and had no adverse effects on bone mass-strength relationships for the vertebrae, femoral neck, femoral diaphysis, or humeral cortical beams. CONCLUSIONS: Abaloparatide administration was associated with increases in bone formation, bone mass and bone strength, and with maintenance of bone quality in OVX cynos, without increases in serum calcium or bone resorption parameters.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Reabsorção Óssea/prevenção & controle , Osteogênese/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/uso terapêutico , Absorciometria de Fóton/métodos , Animais , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/fisiopatologia , Feminino , Vértebras Lombares/fisiopatologia , Macaca fascicularis , Osteogênese/fisiologia , Ovariectomia , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Microtomografia por Raio-X/métodosRESUMO
The parathyroid hormone receptor 1 (PTHR1) is a member of the B-family of GPCRs; these receptors are activated by long polypeptide hormones and constitute targets of drug development efforts. Parathyroid hormone (PTH, 84 residues) and PTH-related protein (PTHrP, 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1-34), is used clinically to treat osteoporosis. Conventional peptides in the 20-40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1-ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with ß-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α â ß replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis, in cases remaining active after extended exposure to simulated gastric fluid. The strategy we have employed suggests a path toward identifying protease-resistant agonists of other B-family GPCRs.
Assuntos
Proteólise/efeitos dos fármacos , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Aminoácidos/química , Aminoácidos/farmacologia , Humanos , Ligantes , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Inibidores de Proteases/síntese químicaRESUMO
The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.
Assuntos
Modelos Moleculares , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Glucagon/agonistas , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/química , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro , Homologia Estrutural de ProteínaRESUMO
Parathyroid hormone (PTH) is essential for calcium homeostasis and its action is mediated by the PTH type 1 receptor (PTHR1), a class B G-protein-coupled receptor. Hypoparathyroidism and osteoporosis can be treated with PTH injections; however, no orally effective PTH analogue is available. Here we show that PCO371 is a novel, orally active small molecule that acts as a full agonist of PTHR1. PCO371 does not affect the PTH type 2 receptor (PTHR2), and analysis using PTHR1-PTHR2 chimeric receptors indicated that Proline 415 of PTHR1 is critical for PCO371-mediated PTHR1 activation. Oral administration of PCO371 to osteopenic rats provokes a significant increase in bone turnover with limited increase in bone mass. In hypocalcemic rats, PCO371 restores serum calcium levels without increasing urinary calcium, and with stronger and longer-lasting effects than PTH injections. These results strongly suggest that PCO371 can provide a new treatment option for PTH-related disorders, including hypoparathyroidism.
Assuntos
Hipoparatireoidismo/tratamento farmacológico , Imidazolidinas/síntese química , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Compostos de Espiro/síntese química , Animais , Cães , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazolidinas/farmacologia , Masculino , Estrutura Molecular , Mutação , Glândulas Paratireoides/efeitos dos fármacos , Glândulas Paratireoides/cirurgia , Ratos , Compostos de Espiro/farmacologiaRESUMO
Ligands possessing different physico-chemical structures productively interact with G protein-coupled receptors generating distinct downstream signaling events due to their abilities to activate/select idiosyncratic receptor entities ('receptorsomes') from the full spectrum of potential receptor partners. We have employed multiple novel informatic approaches to identify and characterize the in vivo transcriptomic signature of an arrestin-signaling biased ligand, [D-Trp(12),Tyr(34)]-bPTH(7-34), acting at the parathyroid hormone type 1 receptor (PTH1R), across six different murine tissues after chronic drug exposure. We are able to demonstrate that [D-Trp(12),Tyr(34)]-bPTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated, in an arrestin signaling-dependent manner, across more tissues than that of the pluripotent endogenous PTH1R ligand, hPTH(1-34). This arrestin-focused response signature is strongly linked with the transcriptional regulation of cell growth and development. Our informatic deconvolution of a conserved arrestin-dependent transcriptomic signature from wild type mice demonstrates a conceptual framework within which the in vivo outcomes of biased receptor signaling may be further investigated or predicted.
Assuntos
Redes Reguladoras de Genes/fisiologia , Informática/métodos , Hormônio Paratireóideo/farmacologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Animais , Bovinos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
Biased G protein-coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in cellula efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [d-Trp(12), Tyr(34)]bovine PTH(7-34) in six different murine tissues after chronic drug exposure. We find that [d-Trp(12), Tyr(34)]bovine PTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, human PTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized.
Assuntos
Arrestinas/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Transcriptoma , Animais , Arrestinas/genética , Bovinos , Biologia Computacional , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Especificidade da Espécie , Transcrição GênicaRESUMO
With an increasing number of new chemical entities entering clinical studies, and an increasing share of the market, peptides and peptidomimetics constitute one of the most promising classes of therapeutics. The success of synthetic peptides as therapeutics relies on the lead optimization step in which the lead candidates are modified to improve drug-like properties of peptides related to potency, pharmacokinetics, solubility, and stability, among others. Peptidomimetics based on the N-terminal stretch of the first 11 amino acids of the PTH have been investigated as potential lead compounds for the treatment of osteoporosis. On the basis of a peptide reported in the literature, referred to here as the Parent Peptide (H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2), we conducted systematic SAR analyses to investigate the effects of altering peptide hydrophobicity on PTH receptor functional potency as measured by the cAMP (cyclic adenosine monophosphate) accumulation and ß-arrestin recruitment assays. Among hydrophobic residues, we found that the Val2 position shows the least flexibility in terms of the SAR studies, whereas the Leu7 position appeared to be most flexible. Through circular dichroism and nuclear magnetic resonance spectroscopy studies, we were able to establish that changes in hydrophobic residues significantly change the extent of peptide helicity and that the helical character correlates well with receptor agonist activity. Here, we report several novel PTH 1-11 peptidomimetics that show comparable or enhanced potency to stimulate Gs-signaling over ß-arrestin recruitment as compared with such properties of PTH 1-34 and the Parent Peptide.
Assuntos
Sondas Moleculares/farmacologia , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Relação Estrutura-AtividadeRESUMO
Parathyroid hormone (PTH) is an anabolic agent that mediates bone formation through activation of the Gα(s)-, Gα(q)- and ß-arrestin-coupled parathyroid hormone receptor type 1 (PTH1R). Pharmacological evidence based on the effect of PTH(7-34), a PTH derivative that is said to preferentially activate ß-arrestin signaling through PTH1R, suggests that PTH1R-activated ß-arrestin signaling mediates anabolic effects on bone. Here, we performed a thorough evaluation of PTH(7-34) signaling behaviour using quantitative assays for ß-arrestin recruitment, Gα(s)- and Gα(q)-signaling. We found that PTH(7-34) inhibited PTH-induced cAMP accumulation, but was unable to induce ß-arrestin recruitment, PTH1R internalization and ERK1/2 phosphorylation in HEK293, CHO and U2OS cells. Thus, the ß-arrestin bias of PTH(7-34) is not apparent in every cell type examined, suggesting that correlating in vivo effects of PTH(7-34) to in vitro pharmacology should be done with caution.