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2.
Cell Discov ; 10(1): 18, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38346960

RESUMO

Class B1 G protein-coupled receptors (GPCRs) are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist-receptor-G protein complex. However, intracellular signal transducers can also activate the receptor independent of extracellular stimuli, suggesting an intrinsic role of G proteins in this process. Here, we report cryo-electron microscopy structures of the human GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) in complex with Gs proteins without the presence of cognate ligands. These ligand-free complexes share a similar intracellular architecture to those bound by endogenous peptides, in which, the Gs protein alone directly opens the intracellular binding cavity and rewires the extracellular orthosteric pocket to stabilize the receptor in a state unseen before. While the peptide-binding site is partially occupied by the inward folded transmembrane helix 6 (TM6)-extracellular loop 3 (ECL3) juncture of GIPR or a segment of GCGR ECL2, the extracellular portion of GLP-1R adopts a conformation close to the active state. Our findings offer valuable insights into the distinct activation mechanisms of these three important receptors. It is possible that in the absence of a ligand, the intracellular half of transmembrane domain is mobilized with the help of Gs protein, which in turn rearranges the extracellular half to form a transitional conformation, facilitating the entry of the peptide N-terminus.

3.
Proc Natl Acad Sci U S A ; 120(33): e2303696120, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37549266

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR), two members of class B1 G protein-coupled receptors, play important roles in glucose homeostasis and energy metabolism. They share a high degree of sequence homology but have different functionalities. Unimolecular dual agonists of both receptors developed recently displayed better clinical efficacies than that of monotherapy. To study the underlying molecular mechanisms, we determined high-resolution cryo-electron microscopy structures of GLP-1R or GCGR in complex with heterotrimeric Gs protein and three GLP-1R/GCGR dual agonists including peptide 15, MEDI0382 (cotadutide) and SAR425899 with variable activating profiles at GLP-1R versus GCGR. Compared with related structures reported previously and supported by our published pharmacological data, key residues responsible for ligand recognition and dual agonism were identified. Analyses of peptide conformational features revealed a difference in side chain orientations within the first three residues, indicating that distinct engagements in the deep binding pocket are required to achieve receptor selectivity. The middle region recognizes extracellular loop 1 (ECL1), ECL2, and the top of transmembrane helix 1 (TM1) resulting in specific conformational changes of both ligand and receptor, especially the dual agonists reshaped ECL1 conformation of GLP-1R relative to that of GCGR, suggesting an important role of ECL1 interaction in executing dual agonism. Structural investigation of lipid modification showed a better interaction between lipid moiety of MEDI0382 and TM1-TM2 cleft, in line with its increased potency at GCGR than SAR425899. Together, the results provide insightful information for the design and development of improved therapeutics targeting these two receptors simultaneously.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Receptores de Glucagon , Microscopia Crioeletrônica , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Ligantes , Lipídeos , Peptídeos/química , Receptores de Glucagon/agonistas
4.
Cell Discov ; 9(1): 81, 2023 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-37524700

RESUMO

Members of the melanocortin receptor (MCR) family that recognize different melanocortin peptides mediate a broad spectrum of cellular processes including energy homeostasis, inflammation and skin pigmentation through five MCR subtypes (MC1R-MC5R). The structural basis of subtype selectivity of the endogenous agonist γ-MSH and non-selectivity of agonist α-MSH remains elusive, as the two agonists are highly similar with a conserved HFRW motif. Here, we report three cryo-electron microscopy structures of MC3R-Gs in complex with γ-MSH and MC5R-Gs in the presence of α-MSH or a potent synthetic agonist PG-901. The structures reveal that α-MSH and γ-MSH adopt a "U-shape" conformation, penetrate into the wide-open orthosteric pocket and form massive common contacts with MCRs via the HFRW motif. The C-terminus of γ-MSH occupies an MC3R-specific complementary binding groove likely conferring subtype selectivity, whereas that of α-MSH distances itself from the receptor with neglectable contacts. PG-901 achieves the same potency as α-MSH with a shorter length by rebalancing the recognition site and mimicking the intra-peptide salt bridge in α-MSH by cyclization. Solid density confirmed the calcium ion binding in MC3R and MC5R, and the distinct modulation effects of divalent ions were demonstrated. Our results provide insights into ligand recognition and subtype selectivity among MCRs, and expand the knowledge of signal transduction among MCR family members.

5.
Proc Natl Acad Sci U S A ; 119(20): e2200155119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35561211

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.


Assuntos
Ciclobutanos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptidomiméticos , Microscopia Crioeletrônica , Ciclobutanos/química , Ciclobutanos/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Humanos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Domínios Proteicos
6.
Nat Commun ; 13(1): 1057, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217653

RESUMO

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dual agonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their multiplexed pharmacological actions over monoagonists such as semaglutide, we determine cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR. The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the near-atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility of tirzepatide and peptide 20.


Assuntos
Diabetes Mellitus Tipo 2 , Receptores de Glucagon , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/uso terapêutico , Humanos , Peptídeos/química , Receptores Acoplados a Proteínas G
7.
Trends Pharmacol Sci ; 43(4): 321-334, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35078643

RESUMO

Class B1 G protein-coupled receptors (GPCRs) play important roles in human physiology and disease pathology. Using cryo-electron microscopy (cryo-EM) and X-ray crystallography, the 3D structures of all 15 members of this receptor subfamily have been determined in recent years at the near-atomic level. Although they share many structural commonalities, they show distinct features in terms of ligand recognition and receptor activation. In-depth structural analyses have yielded valuable insights into the N termini of both peptide hormones and cognate receptors, the outward movement of transmembrane helix 6 (TM6), the allosteric modulation sites located in the transmembrane domain (TMD), and the constitutive signaling bias mediated by receptor splice variants. These provide new directions for the design of better therapeutic agents, thereby making these targets more druggable.


Assuntos
Receptores Acoplados a Proteínas G , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Ligantes , Domínios Proteicos , Receptores Acoplados a Proteínas G/química
8.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34599099

RESUMO

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.


Assuntos
Processamento Alternativo/genética , Variação Genética/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Células PC-3 , Células Sf9 , Transdução de Sinais/genética , beta-Arrestinas/genética
9.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34353904

RESUMO

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.


Assuntos
Receptor Tipo 2 de Hormônio Paratireóideo/química , Receptor Tipo 2 de Hormônio Paratireóideo/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutação , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Conformação Proteica , Receptor Tipo 2 de Hormônio Paratireóideo/genética
11.
Nat Commun ; 12(1): 3763, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145245

RESUMO

The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer insights into the structural basis of ago-allosterism at GLP-1R and may aid the design of better therapeutics.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Animais , Sítios de Ligação/fisiologia , Células CHO , Linhagem Celular , Cricetulus , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativação Enzimática/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos Semelhantes ao Glucagon/farmacologia , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Células Sf9 , Spodoptera
12.
Cell Res ; 30(12): 1098-1108, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33239759

RESUMO

Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn's disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 2/química , Receptor do Peptídeo Semelhante ao Glucagon 2/ultraestrutura , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Homologia Estrutural de Proteína
13.
Nat Commun ; 11(1): 5205, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060564

RESUMO

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/química , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Nanismo Hipofisário/genética , Proteínas de Ligação ao GTP , Hormônio Liberador de Hormônio do Crescimento/deficiência , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Transdução de Sinais
14.
Int J Biol Macromol ; 107(Pt A): 855-864, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28935541

RESUMO

In this study, we present a novel cross-linked unimolecular micelle based on chitosan. For controlling drug delivery via oral administration, emodin (EMO) encapsulated micelles were loaded into sodium alginate hydrogel matrix to construct the pH-sensitive hydrogel/micelle composites. The optimized formulation of micelle that consists of 8.06% CaCl2, 1.71% chitosan and 26.52% ß-GP was obtained by the combination of Box-Behnken experimental design and response surface methodology. The morphological analysis showed that the micelles exhibited a smaller diameter of about 80nm in aqueous solution, but dilated to 100-200nm in hydrogel owing to the formation of polyelectrolyte complexes. The physical characteristics in simulated digestive fluids were investigated, demonstrating that the ratio of hydrogel to micelle distinctly affected swelling, degradation and in vitro drug release behaviors. The hydrogel/micelle (1:1) exhibited a sustained-release profile, while hydrogel/micelle (3:1) exhibited a colon-specific profile. Their corresponding release mechanisms revealed that the release of drug from these two formulations followed a complex process, in which several mechanisms were involved or occurred simultaneously. These results demonstrated that the pH-sensitive hydrogel/micelle composites constructed with biocompatible materials can be a promising sustained-release or site-specific drug delivery system for instable or hydrophobic drugs.


Assuntos
Materiais Biocompatíveis/química , Quitosana/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Alginatos/química , Alginatos/uso terapêutico , Materiais Biocompatíveis/uso terapêutico , Quitosana/uso terapêutico , Ácido Glucurônico/química , Ácido Glucurônico/uso terapêutico , Ácidos Hexurônicos/química , Ácidos Hexurônicos/uso terapêutico , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapêutico , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Micelas
15.
Drug Dev Ind Pharm ; 43(4): 558-573, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27935330

RESUMO

OBJECTIVE: This work aimed to develop an alternative sustained-release thermosensitive praziquantel-loaded nanoemulsion (PZQ-NE) hydrogel for better schistosomiasis treatment. SIGNIFICANCE: PZQ-NE-dispersed chitosan/glycerol 2-phosphate disodium/HPMC (NE/CS/ß-GP/HMPC) hydrogel was successfully prepared to improve bioavailability of PZQ. METHODS: Solubility tests and pseudo-ternary phase diagrams were applied to screen optimal oils, surfactants and co-surfactants of NE. The hydrogels were characterized for gelling time, surface exudates, rheological properties and in vitro drug release. Formulation optimization of NE/CS/ß-GP/HMPC hydrogel was conducted by Box-Behnken experimental design combined with response surface methodology. In vitro cytotoxicity of hydrogel was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. The sustained-release property of PZQ in NE and optimized hydrogel was evaluated by pharmacokinetic study in rabbits. RESULTS: The formulation of PZQ-NE consisted of mass ratio of 12.5% capryol 90 containing PZQ (160 mg/g), 40% cremophor RH 40/tween 20 and transcutol HP (S/CoS = 2:1), 47.5% deionized water. PZQ releasing from NE/CS/ß-GP/HMPC hydrogels was best fitted to Higuchi model and governed by diffusion. Rheological investigation evidenced the themosensitive gelation of different hydrogel systems and their gel-like character at 37 °C. The optimized hydrogel formulation consisted of HPMC solution (103.69 mg/g), 3.03% (w/v) chitosan and 14.1% (w/v) ß-GP showed no cytotoxicity when the addition of NE was no more than 100 mg/g. Pharmacokinetic parameters indicated that NE/CS/ß-GP/HMPC hydrogel can significantly slow down drug elimination, prolong mean residence time and improve bioavailability of PZQ. CONCLUSIONS: NE/CS/ß-GP/HMPC hydrogel possessed sustained-release property and could be an alternative antischistosomal drug delivery system with improved therapeutic effect.


Assuntos
Preparações de Ação Retardada/química , Emulsões/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Nanopartículas/química , Praziquantel/química , Animais , Disponibilidade Biológica , Química Farmacêutica/métodos , Quitosana/química , Preparações de Ação Retardada/metabolismo , Feminino , Glicerofosfatos/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Masculino , Praziquantel/metabolismo , Coelhos , Reologia , Solubilidade , Soluções/química , Tensoativos/química , Temperatura
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