RESUMO
L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4 contribute to its exquisite Group III functional agonist potency and selectivity.
Assuntos
Aminobutiratos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Aminobutiratos/química , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Ligação Proteica , Domínios Proteicos , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Antibodies at high concentrations often reveal unanticipated biophysical properties suboptimal for therapeutic development. The purpose of this work was to explore the use of point mutations based on crystal structure information to improve antibody physical properties such as viscosity and phase separation (LLPS) at high concentrations. An IgG4 monoclonal antibody (Mab4) that exhibited high viscosity and phase separation at high concentration was used as a model system. Guided by the crystal structure, four CDR point mutants were made to evaluate the role of hydrophobic and charge interactions on solution behavior. Surprisingly and unpredictably, two of the charge mutants, R33G and N35E, showed a reduction in viscosity and a lower propensity to form LLPS at high concentration compared to the wild-type (WT), while a third charge mutant S28K showed an increased propensity to form LLPS compared to the WT. A fourth mutant, F102H, had reduced hydrophobicity, but unchanged viscosity and phase separation behavior. We further evaluated the correlation of various biophysical measurements including second virial coefficient (A2), interaction parameter (kD), weight-average molecular weight (WAMW), and hydrodynamic diameters (DH), at relatively low protein concentration (4 to 15 mg/mL) to physical properties, such as viscosity and liquid-liquid phase separation (LLPS), at high concentration. Surprisingly, kD measured using dynamic light scattering (DLS) at low antibody concentration correlated better with viscosity and phase separation than did A2 for Mab4. Our results suggest that the high viscosity and phase separation observed at high concentration for Mab4 are mainly driven by charge and not hydrophobicity.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/imunologia , Extração Líquido-Líquido/métodos , Engenharia de Proteínas/métodos , Viscosidade , Substituição de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Cristalização , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Soluções Farmacêuticas , Conformação ProteicaRESUMO
The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.
Assuntos
Complexo Antígeno-Anticorpo , Fragmentos Fab das Imunoglobulinas , Humanos , Cristalização/métodos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/química , Complexo Antígeno-Anticorpo/química , Antígenos/química , Cristalografia por Raios X , Conformação ProteicaRESUMO
B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.
Assuntos
Receptores Imunológicos , Linfócitos T , Camundongos , Animais , Linfócitos T/metabolismo , Receptores Imunológicos/metabolismo , Anticorpos/metabolismoRESUMO
The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.
Assuntos
Proteínas Fúngicas/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Candida glabrata/química , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Membrana Nuclear/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/químicaRESUMO
Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Cadeias kappa de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica em Folha beta , CoelhosRESUMO
We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.
Assuntos
Anticorpos Biespecíficos , Doenças Autoimunes/tratamento farmacológico , Fator Ativador de Células B/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fator Ativador de Células B/imunologia , Feminino , Células HEK293 , Células HT29 , Humanos , Interleucina-17/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Phase II of the Protein Structure Initiative, funded by the NIH NIGMS (National Institute of General Medical Sciences), is a 5-year effort to determine thousands of protein structures. The New York SGX Research Center for Structural Genomics (NYSGXRC) is one of the four large-scale production centers tasked with determining 100-200 structures annually. Almost all protein production is carried out using the high throughput structural biology platform at SGX Pharmaceuticals (SGX), which supplies 120 or more ultrapure proteins per month for NYSGXRC crystallization and structure determination activities. Protocols for PCR, cloning, expression/solubility testing, fermentation, purification, and crystallization are described. General protocols and detailed experimental results for each target are updated weekly at the public PepcDB website (pepcdb.pdb.org/), and all NYSGXRC clones should be available in 2008 through the PlasmID resource operated by the Harvard Institute of Proteomics.
Assuntos
Proteínas/química , Proteínas/isolamento & purificação , Proteômica/métodos , Proteômica/organização & administração , Clonagem Molecular/métodos , Cristalografia por Raios X/métodos , Cidade de Nova Iorque , Reação em Cadeia da Polimerase/métodos , Proteínas/genéticaRESUMO
Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-abl/química , Trifosfato de Adenosina/química , Sítio Alostérico , Cinética , Cadeias de Markov , Ácido Mirístico/química , Conformação Proteica , TermodinâmicaRESUMO
( S)-3,4-Dicarboxyphenylglycine (DCPG) was first reported in 2001 as a potent orthosteric agonist with high subtype selectivity for the mGlu8 receptor, but the structural basis for its high selectivity is not well understood. We have solved a cocrystal structure of recombinant human mGlu8 amino terminal domain (ATD) protein bound to ( S)-DCPG, which possesses the largest lobe opening angle observed to date among known agonist-bound mGlu ATD crystal structures. The binding conformation of ( S)-DCPG observed in the crystal structure is significantly different from that in the homology model built from an l-glutamate-bound rat mGlu1 ATD crystal structure, which has a smaller lobe opening angle. This highlights the importance of considering various lobe opening angles when modeling mGlu ATD-ligand complex. New homology models of other mGlu receptors based on the ( S)-DCPG-bound mGlu8 ATD crystal structure were explored to rationalize ( S)-DCPG's high mGlu8 receptor subtype selectivity.
Assuntos
Benzoatos/química , Benzoatos/farmacologia , Glicina/análogos & derivados , Receptores de Glutamato Metabotrópico/agonistas , Sítios de Ligação , Desenho de Fármacos , Glicina/química , Glicina/farmacologia , Humanos , Ligantes , Modelos Moleculares , Domínios Proteicos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/síntese química , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Animais , Compostos Bicíclicos com Pontes/farmacocinética , Cristalografia por Raios X , AMP Cíclico/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacocinética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenciclidina/antagonistas & inibidores , Fenciclidina/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/uso terapêutico , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Feminino , Antagonistas do Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Tiofenos/química , Tiofenos/farmacocinética , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Humanos , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala41 , Glu44 , and His45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.
Assuntos
Anticorpos Neutralizantes/química , Especificidade de Anticorpos , Epirregulina/química , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Fator de Crescimento Transformador alfa , Animais , Humanos , Camundongos , Fator de Crescimento Transformador alfa/antagonistas & inibidores , Fator de Crescimento Transformador alfa/químicaRESUMO
A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies.
Assuntos
Anticorpos Biespecíficos/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Engenharia de Proteínas/métodos , Biologia Computacional/métodos , Cristalografia por Raios X , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Domínios Proteicos , Multimerização ProteicaRESUMO
As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure-activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand-protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharmacology. Further characterization of 19f in vivo demonstrated an antidepressant-like signature in the mouse forced-swim test (mFST) assay when brain levels of this compound exceeded the cellular mGlu2 IC50 value.
Assuntos
Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Descoberta de Drogas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Antidepressivos/síntese química , Antidepressivos/química , Encéfalo/efeitos dos fármacos , Cicloexanos/síntese química , Cicloexanos/química , Cicloexanos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/isolamento & purificação , Relação Estrutura-Atividade , NataçãoAssuntos
Benzoatos/metabolismo , Canais Iônicos/química , Porinas/química , Pseudomonas fluorescens/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Canais Iônicos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Porinas/metabolismo , Conformação Proteica , Homologia de Sequência de AminoácidosAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cromatografia em Gel , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.