RESUMEN
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria.
RESUMEN
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
Asunto(s)
Antibacterianos/clasificación , Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Biocatálisis/efectos de los fármacos , Productos Biológicos/clasificación , Productos Biológicos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/enzimología , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/patogenicidad , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/patogenicidad , Lisina/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/química , Porinas , Unión Proteica , Dominios Proteicos , Serina Endopeptidasas , Especificidad por SustratoRESUMEN
UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Animales , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Expresión Génica , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Selección Genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , VirulenciaRESUMEN
A series of 4-azaindole-containing p21-activated kinase-1 (PAK1) inhibitors was prepared with the goal of improving physicochemical properties relative to an indole starting point. Indole 1 represented an attractive, non-basic scaffold with good PAK1 affinity and cellular potency but was compromised by high lipophilicity (clogD=4.4). Azaindole 5 was designed as an indole surrogate with the goal of lowering logD and resulted in equipotent PAK1 inhibition with a 2-fold improvement in cellular potency over 1. Structure-activity relationship studies around 5 identified additional 4-azaindole analogs with superior PAK1 biochemical activity (Ki <10nM) and up to 24-fold selectivity for group I over group II PAKs. Compounds from this series showed enhanced permeability, improved aqueous solubility, and lower plasma protein binding over indole 1. The improvement in physicochemical properties translated to a 20-fold decrease in unbound clearance in mouse PK studies for azaindole 5 relative to indole 1.
Asunto(s)
Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Perros , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismoRESUMEN
p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Piridonas , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismoRESUMEN
Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.
RESUMEN
The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirazoles/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Humanos , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacocinética , Pirazoles/farmacocinética , Ratas , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismoRESUMEN
Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/química , Histonas/química , Acetilación , Benzamidas/química , Unión Competitiva , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Piridinas/química , Transcripción Genética , VorinostatRESUMEN
Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.
Asunto(s)
Caspasa 6/metabolismo , Inhibidores de Caspasas/química , Inhibidores de Caspasas/farmacología , Secuencia de Aminoácidos , Caspasa 6/química , Inhibidores de Caspasas/análisis , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Especificidad por Sustrato/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.
Asunto(s)
Bioensayo/métodos , Caspasa 6/metabolismo , Células/enzimología , Pruebas de Enzimas/métodos , Lamina Tipo A/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Inhibidores de Caspasas , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacología , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Identifying chemical lead matter by high-throughput screening (HTS) has been a common practice in early stage drug discovery. Evolution of small-molecule library composition to include more drug-like molecules with desirable physical chemical properties combined with improving assay technologies has vastly enhanced the capability of HTS. However, HTS campaigns can still be plagued by false positives arising from nonspecific inhibitors. The generation of assay-ready plates has permitted an incremental advancement to the speed and efficiency of HTS but has the potential to enhance the occurrence of nonspecific inhibitors. A subtle change in the order of reagent addition to the assay-ready plates can greatly alleviate false-positive inhibition. Our case studies with six different kinase and protease targets reveal that this type of inhibition affects targets regardless of enzyme class and is unpredictable based on protein construct or inhibitor chemical scaffold. These case studies support a model where a diversity set of compounds should be tested first for hit rates as a function of order of addition, carrier protein, and relevant mechanistic studies prior to launch of the HTS campaign.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Péptido Hidrolasas/química , Proteínas Quinasas/química , Animales , Caspasa 6/química , Bovinos , Simulación por Computador , Evaluación Preclínica de Medicamentos , Reacciones Falso Positivas , Humanos , Modelos Teóricos , Péptido Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Albúmina Sérica/química , Bibliotecas de Moléculas Pequeñas/análisis , gammaglobulinas/químicaRESUMEN
Reducing production of amyloid-ß (Aß) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer's disease. However, small-molecule inhibitors of the ß-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aß production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aß peptide concentrations. Anti-BACE1 also reduces central nervous system Aß concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer's disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/deficiencia , Ácido Aspártico Endopeptidasas/metabolismo , Bioensayo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cristalografía por Rayos X , Endocitosis/efectos de los fármacos , Humanos , Macaca fascicularis , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos , Resultado del TratamientoRESUMEN
The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
The design, synthesis, and SAR of a series of substituted spirohydantoins are described. Optimization of an in-house screening hit gave compounds that exhibited potent binding affinity and functional activity at MCH-R1.
Asunto(s)
Fármacos Antiobesidad/síntesis química , Hidantoínas/síntesis química , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Compuestos de Espiro/síntesis química , Fármacos Antiobesidad/farmacología , Diseño de Fármacos , Humanos , Hidantoínas/farmacología , Cinética , Unión Proteica , Compuestos de Espiro/farmacología , Relación Estructura-ActividadRESUMEN
A series of thienopyrimidinone bis-aminopyrrolidine ureas were designed, synthesized, and evaluated for their ability to bind melanin-concentrating hormone receptor-1. These compounds exhibit potent binding affinity (K(i)=3 nM) and good in vitro metabolic stability.
Asunto(s)
Química Farmacéutica/métodos , Pirrolidinas/química , Receptores de Somatostatina/antagonistas & inhibidores , Urea/análogos & derivados , Urea/química , Animales , Inhibidores del Citocromo P-450 CYP2D6 , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Cinética , Ratones , Modelos Químicos , Conformación Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.
Asunto(s)
Receptores LHRH/metabolismo , Conteo por Cintilación/métodos , Unión Competitiva/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análisis , Hormona Liberadora de Gonadotropina/metabolismo , Cinética , Ligandos , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.
Asunto(s)
Espectroscopía de Resonancia Magnética , Péptidos/farmacología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/química , Aminoácidos , Sitios de Unión , Humanos , Fragmentos de Péptidos/farmacología , Péptidos/química , Unión Proteica , Conformación ProteicaRESUMEN
The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.
Asunto(s)
Piridazinas/química , Piridazinas/farmacocinética , Receptores de Somatostatina/antagonistas & inhibidores , Receptores de Somatostatina/metabolismo , Somatostatina/antagonistas & inhibidores , Aminoácidos/química , Animales , Calcio/metabolismo , Agonistas de los Canales de Calcio/química , Agonistas de los Canales de Calcio/farmacocinética , Canales de Calcio/metabolismo , Células Cultivadas , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Neuronas/efectos de los fármacos , Ratas , Receptores de Somatostatina/químicaRESUMEN
Numerous nonpeptide ligands have been developed for the human gonadotropin-releasing hormone (GnRH) receptor as potential agents for treatment of disorders of the reproductive-endocrine axis. While the equilibrium binding of these ligands has been studied in detail, little is known of the kinetics of their receptor interaction. In this study we evaluated the kinetic structure-activity relationships (SAR) of uracil-series antagonists by measuring their association and dissociation rate constants. These constants were measured directly using a novel radioligand, [3H] NBI 42902, and indirectly for unlabeled ligands. Receptor association and dissociation of [3H] NBI 42902 was monophasic, with an association rate constant of 93+/-10 microM(-1) min(-1) and a dissociation rate constant of 0.16+/-0.02 h(-1) (t(1/2) of 4.3 h). Four unlabeled compounds were tested with varying substituents at the 2-position of the benzyl group at position 1 of the uracil (-F, -SO(CH3), -SO2(CH3) and -CF3). The nature of the substituent did not appreciably affect the association rate constant but varied the dissociation rate constant >50-fold (t(1/2) ranging from 52 min for -SO(CH3) to >43 h for -CF3). This SAR was poorly resolved in standard competition assays due to lack of equilibration. The functional consequences of the varying dissociation rate were investigated by measuring antagonism of GnRH-stimulated [3H] inositol phosphates accumulation. Slowly dissociating ligands displayed insurmountable antagonism (decrease of the GnRH E(max)) while antagonism by more rapidly dissociating ligands was surmountable (without effect on the GnRH E(max)). Therefore, evaluating the receptor binding kinetics of nonpeptide antagonists revealed SAR, not evident in standard competition assays, that defined at least in part the mode of functional antagonism by the ligands. These findings are of importance for the future definition of nonpeptide ligand SAR and for the identification of potentially useful slowly dissociating antagonists for the GnRH receptor.
Asunto(s)
Relación Estructura-Actividad Cuantitativa , Receptores LHRH/antagonistas & inhibidores , Uracilo/farmacología , Unión Competitiva/efectos de los fármacos , Humanos , Cinética , Ligandos , Estructura Molecular , Ensayo de Unión Radioligante/métodos , Receptores LHRH/metabolismo , Timina/análogos & derivados , Timina/metabolismo , Tritio , Uracilo/química , Uracilo/metabolismoRESUMEN
The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.