RESUMEN
Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.
Asunto(s)
Receptor Muscarínico M1/química , Receptor Muscarínico M4/química , Acetilcolina/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Enfermedad de Alzheimer , Cristalización , Cristalografía por Rayos X , Agonismo Inverso de Drogas , Humanos , Modelos Moleculares , Ácidos Nicotínicos/metabolismo , Ácidos Nicotínicos/farmacología , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/metabolismo , Esquizofrenia , Electricidad Estática , Especificidad por Sustrato , Propiedades de Superficie , Tiofenos/metabolismo , Tiofenos/farmacología , Bromuro de Tiotropio/farmacologíaRESUMEN
The observation that cholinergic deafferentation of circuits projecting from forebrain basal nuclei to frontal and hippocampal circuits occurs in Alzheimer's disease has led to drug-targeting of muscarinic M1 receptors to alleviate cognitive symptoms. The high homology within the acetylcholine binding domain of this family however has made receptor-selective ligand development challenging. This work presents the synthesis scheme, pharmacokinetic and structure-activity-relationship study findings for M1-selective ligand, LY593093. Pharmacologically the compound acts as an orthosteric ligand. The homology modeling work presented however will illustrate that compound binding spans from the acetylcholine pocket to the extracellular loops of the receptor, a common allosteric vestibule for the muscarinic protein family. Altogether LY593093 represents a growing class of multi-topic ligands which interact with the receptors in both the ortho- and allosteric binding sites, but which exert their activation mechanism as an orthosteric ligand.
Asunto(s)
Amidas/química , Amidas/farmacología , Diseño de Fármacos , Receptor Muscarínico M1/agonistas , Amidas/síntesis química , Animales , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
Structure-based computational methods have been widely used in exploring protein-ligand interactions, including predicting the binding ligands of a given protein based on their structural complementarity. Compared to other protein and ligand representations, the advantages of a surface representation include reduced sensitivity to subtle changes in the pocket and ligand conformation and fast search speed. Here we developed a novel method named PL-PatchSurfer (Protein-Ligand PatchSurfer). PL-PatchSurfer represents the protein binding pocket and the ligand molecular surface as a combination of segmented surface patches. Each patch is characterized by its geometrical shape and the electrostatic potential, which are represented using the 3D Zernike descriptor (3DZD). We first tested PL-PatchSurfer on binding ligand prediction and found it outperformed the pocket-similarity based ligand prediction program. We then optimized the search algorithm of PL-PatchSurfer using the PDBbind dataset. Finally, we explored the utility of applying PL-PatchSurfer to a larger and more diverse dataset and showed that PL-PatchSurfer was able to provide a high early enrichment for most of the targets. To the best of our knowledge, PL-PatchSurfer is the first surface patch-based method that treats ligand complementarity at protein binding sites. We believe that using a surface patch approach to better understand protein-ligand interactions has the potential to significantly enhance the design of new ligands for a wide array of drug-targets.
Asunto(s)
Algoritmos , Simulación del Acoplamiento Molecular/métodos , Proteínas/química , Sitios de Unión , Ligandos , Unión Proteica , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.
Asunto(s)
Compuestos de Anilina/síntesis química , Modelos Moleculares , Sulfonamidas/síntesis química , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/química , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Espectroscopía de Resonancia Magnética , Unión Proteica , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/química , Proteína bcl-X/químicaRESUMEN
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.
Asunto(s)
Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Descubrimiento de Drogas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Antidepresivos/síntesis química , Antidepresivos/química , Encéfalo/efectos de los fármacos , Ciclohexanos/síntesis química , Ciclohexanos/química , Ciclohexanos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Ratones Endogámicos , Modelos Moleculares , Estructura Molecular , Actividad Motora/efectos de los fármacos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/aislamiento & purificación , Relación Estructura-Actividad , NataciónRESUMEN
As part of our ongoing interest in identifying novel agonists acting at metabotropic glutamate (mGlu) 2/3 receptors, we have explored the effect of structural modifications of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), a potent and pharmacologically balanced mGlu2/3 receptor agonist. Incorporation of relatively small substituents (e.g., F, O) at the C4 position of this molecule resulted in additional highly potent mGlu2/3 agonists that demonstrate excellent selectivity over the other mGlu receptor subtypes, while addition of larger C4-substituents (e.g., SPh) led to a loss of agonist potency and/or the appearance of weak mGlu2/3 receptor antagonist activity. Further characterization of the α-fluoro-substituted analogue (LY459477) in vivo revealed that this molecule possesses good oral bioavailability in rats and effectively suppresses phencyclidine-evoked locomotor activity at doses that do not impair neuromuscular coordination. This molecule therefore represents a valuable new addition to the arsenal of pharmacological tools competent to investigate mGlu2/3 receptor function both in vitro and in vivo.