RESUMEN
Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool within drug discovery over the last two decades. While traditionally being used by medicinal chemists for small molecule structure elucidation, it can also be a valuable tool for the identification of small molecules that bind to drug targets, for the characterization of target-ligand interactions and for hit-to-lead optimization. Here, we describe how NMR spectroscopy is integrated into the Pfizer drug discovery pipeline and how we utilize this approach to identify and validate initial hits and generate leads.
Asunto(s)
Descubrimiento de Drogas/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Ligandos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Bibliotecas de Moléculas Pequeñas , Soluciones/químicaRESUMEN
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (ß and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1ß1γ1, α1ß2γ1, α1ß2γ3, α2ß1γ1, α2ß2γ1 and α2ß2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were â¼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with ß1-containing heterotrimers, ß2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Adenosina Monofosfato/química , Regulación Alostérica , Compuestos de Bifenilo , Activación Enzimática , Activadores de Enzimas/química , Pruebas de Enzimas , Humanos , Isoenzimas/química , Cinética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Pironas/química , Proteínas Recombinantes/química , Tiofenos/químicaRESUMEN
The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.
Asunto(s)
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/química , Acetilgalactosamina/análogos & derivados , Humanos , Ligandos , Estructura Molecular , EstereoisomerismoRESUMEN
Fragment Based Drug Discovery (FBDD) continues to advance as an efficient and alternative screening paradigm for the identification and optimization of novel chemical matter. To enable FBDD across a wide range of pharmaceutical targets, a fragment screening library is required to be chemically diverse and synthetically expandable to enable critical decision making for chemical follow-up and assessing new target druggability. In this manuscript, the Pfizer fragment library design strategy which utilized multiple and orthogonal metrics to incorporate structure, pharmacophore and pharmacological space diversity is described. Appropriate measures of molecular complexity were also employed to maximize the probability of detection of fragment hits using a variety of biophysical and biochemical screening methods. In addition, structural integrity, purity, solubility, fragment and analog availability as well as cost were important considerations in the selection process. Preliminary analysis of primary screening results for 13 targets using NMR Saturation Transfer Difference (STD) indicates the identification of uM-mM hits and the uniqueness of hits at weak binding affinities for these targets.
Asunto(s)
Descubrimiento de Drogas , Fragmentos de Péptidos/química , Proteínas/química , Sitios de Unión , Técnicas Químicas Combinatorias/métodos , Cristalografía por Rayos X , Industria Farmacéutica , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Biblioteca de Péptidos , Conformación ProteicaRESUMEN
The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.
RESUMEN
Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu â Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Hemoglobina A/efectos de los fármacos , Hemoglobina Falciforme/efectos de los fármacos , Quinolinas/farmacología , Quinolinas/uso terapéutico , Animales , Eritrocitos/metabolismo , Ratones , Oxígeno/metabolismo , Quinolinas/químicaRESUMEN
Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.
RESUMEN
The identification of small molecule modulators of biological processes mediated via protein-protein interactions has generally proved to be a challenging endeavor. In the case of the thrombopoietin receptor (TPOr), however, a number of small molecule types have been reported to display biological activity similar to that of the agonist protein TPO. Through a detailed analysis of structure-activity relationships, X-ray crystal structures, NMR coupling constants, nuclear Overhauser effects, and computational data, we have determined the agonism-inducing conformation of one series of small molecule TPOr agonists. The relationship of this agonism-inducing conformation to that of other series of TPO receptor agonists is discussed.
Asunto(s)
Benzamidas/farmacología , Pirimidinas/farmacología , Receptores de Trombopoyetina/agonistas , Trombopoyetina , Animales , Benzamidas/química , Línea Celular , Simulación por Computador , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Pirimidinas/química , Receptores de Trombopoyetina/química , Relación Estructura-Actividad , Trombopoyetina/química , Trombopoyetina/metabolismoRESUMEN
Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Medición de Intercambio de Deuterio/métodos , Activadores de Enzimas/química , Resonancia por Plasmón de Superficie/métodos , Proteínas Quinasas Activadas por AMP/aislamiento & purificación , Animales , Sitios de Unión , Cristalografía por Rayos X , Medición de Intercambio de Deuterio/instrumentación , Activación Enzimática , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Cinética , Ligandos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.
Asunto(s)
Calmodulina/química , Indoles/química , Oximas/química , Pirazoles/química , Pirimidinas/química , Riluzol/química , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/química , Regulación Alostérica , Secuencias de Aminoácidos , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Sitios de Unión , Calmodulina/genética , Calmodulina/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Indoles/metabolismo , Modelos Moleculares , Oximas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Pirazoles/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riluzol/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismoRESUMEN
Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.
Asunto(s)
Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Activadores de Enzimas/síntesis química , Activadores de Enzimas/farmacología , Indoles/síntesis química , Indoles/farmacología , Animales , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacocinética , Humanos , Indoles/farmacocinética , Absorción Intestinal , Riñón/efectos de los fármacos , Riñón/enzimología , Masculino , Modelos Moleculares , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.
Asunto(s)
Diseño de Fármacos , Fructoquinasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Cristalografía por Rayos X , Fructoquinasas/química , Fructoquinasas/metabolismo , Humanos , Masculino , Simulación del Acoplamiento Molecular , Piridinas/química , Piridinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a ß-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Interleucina-17/antagonistas & inhibidores , Péptidos/metabolismo , Receptores de Interleucina-17/metabolismo , Sustitución de Aminoácidos , Células Cultivadas , Cristalografía por Rayos X , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Interleucina-17/química , Tamizaje Masivo , Modelos Moleculares , Mutagénesis , Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Unión Proteica , Conformación ProteicaRESUMEN
Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.
Asunto(s)
Interleucina-17/antagonistas & inhibidores , Interleucina-17/química , Péptidos Cíclicos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Algoritmos , Sitios de Unión , Células Cultivadas , Diseño de Fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Simulación de Dinámica Molecular , Péptidos Cíclicos/química , Unión Proteica , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Activadores de Enzimas/química , Indoles/química , Administración Oral , Adsorción , Animales , Cristalografía por Rayos X , Perros , Activadores de Enzimas/síntesis química , Activadores de Enzimas/farmacocinética , Activadores de Enzimas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Indazoles/síntesis química , Indazoles/química , Indazoles/farmacología , Indoles/síntesis química , Indoles/farmacocinética , Indoles/farmacología , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal ß-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.
Asunto(s)
Péptidos Cíclicos/química , Receptores de Glucagón/agonistas , Animales , Células CHO , Dicroismo Circular , Cricetulus , AMP Cíclico/biosíntesis , Receptor del Péptido 1 Similar al Glucagón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/farmacología , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.
Asunto(s)
Péptidos/metabolismo , Proproteína Convertasas/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Colesterol/metabolismo , Factor de Crecimiento Epidérmico/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis , Péptidos/síntesis química , Péptidos/química , Proproteína Convertasa 9 , Proproteína Convertasas/química , Proproteína Convertasas/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1ß1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2ß1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/fisiología , Activación Enzimática/fisiología , Modelos Moleculares , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Monofosfato/metabolismo , Sitio Alostérico/genética , Compuestos de Bifenilo , Sistemas de Liberación de Medicamentos , Humanos , Cinética , Ligandos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Pironas/metabolismo , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Tiofenos/metabolismoRESUMEN
ß-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimer's disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aß(X-40) levels at a free drug exposure equivalent to the whole cell IC(50) (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aß lowering versus that observed in wild-type (WT) mouse at an equivalent dose.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Compuestos Aza/síntesis química , Encéfalo/metabolismo , Compuestos de Espiro/síntesis química , Sulfonamidas/síntesis química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/química , Compuestos Aza/farmacocinética , Compuestos Aza/farmacología , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Femenino , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Permeabilidad , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , TransfecciónRESUMEN
The aspartyl protease ß-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aß-peptide, a major component of plaques in the brains of Alzheimer's disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizer's proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.