RESUMEN
MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.
Asunto(s)
Aminopeptidasas , Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Reproducibilidad de los Resultados , Ensayos Analíticos de Alto Rendimiento/métodos , PéptidosRESUMEN
Optimization of a previously reported lead series of PI3Kδ inhibitors with a novel binding mode led to the identification of a clinical candidate compound 31 (GSK251). Removal of an embedded Ames-positive heteroaromatic amine by reversing a sulfonamide followed by locating an interaction with Trp760 led to a highly selective compound 9. Further optimization to avoid glutathione trapping, to enhance potency and selectivity, and to optimize an oral pharmacokinetic profile led to the discovery of compound 31 (GSK215) that had a low predicted daily dose (45 mg, b.i.d) and a rat toxicity profile suitable for further development.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Sulfonamidas/farmacología , Animales , Cristalografía por Rayos X , Femenino , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3/metabolismo , Unión Proteica , Ratas Wistar , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismoRESUMEN
α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
Asunto(s)
Diseño de Fármacos , Pliegue de Proteína , alfa 1-Antitripsina/metabolismo , Cristalización , Desarrollo de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Retículo Endoplásmico/metabolismo , Biblioteca de Genes , Hepatocitos/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , alfa 1-Antitripsina/genéticaRESUMEN
Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency.
Asunto(s)
Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina , Animales , Retículo Endoplásmico , Hepatocitos , Ratones , alfa 1-Antitripsina/genéticaRESUMEN
Integrins αvß5 and αvß3 are closely related, proangiogenic members of the wider RGD-binding integrin family. Due to their high sequence homology, the development of αvß5-selective compounds has remained elusive to synthetic and medicinal chemists. Herein, we describe a survey of SAR around a series of amide-containing 3-aryl-succinamic acid-based RGD mimetics. This resulted in the discovery of α,α,α-trifluorotolyl 12 which exhibits 800 × selectivity for αvß5versus αvß3 with a pyrrolidine amide linker that confers selectivity for αvß5 by positioning a key aryl ring in the SDL of αvß5 with good complementarity; binding in this mode is disfavoured in αvß3 due to clashes with key residues in the ß3-subunit. Compound 12 exhibits selective inhibition by a cell adhesion assay, high passive permeability and solubility which enables potential use of this inhibitor as an αvß5-selective in vitro tool compound.
Asunto(s)
Amidas/farmacología , Pirrolidinas/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Amidas/síntesis química , Amidas/metabolismo , Adhesión Celular/efectos de los fármacos , Humanos , Células K562 , Simulación del Acoplamiento Molecular , Estructura Molecular , Naftiridinas/síntesis química , Naftiridinas/metabolismo , Naftiridinas/farmacología , Unión Proteica , Pirrolidinas/síntesis química , Pirrolidinas/metabolismo , Receptores de Vitronectina/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A macrocyclization approach has been explored on a series of benzoxazine phosphoinositide 3-kinase δ inhibitors, resulting in compounds with improved potency, permeability, and in vivo clearance while maintaining good solubility. The thermodynamics of binding was explored via surface plasmon resonance, and the binding of lead macrocycle 19 was found to be almost exclusively entropically driven compared with progenitor 18, which demonstrated both enthalpic and entropic contributions. The pharmacokinetics of macrocycle 19 was also explored in vivo, where it showed reduced clearance when compared with the progenitor 18. This work adds to the growing body of evidence that macrocyclization could provide an alternative and complementary approach to the design of small-molecule inhibitors, with the potential to deliver differentiated properties.
RESUMEN
A late-stage functionalization of the aromatic ring in amino acid derivatives is described. The key step is a copper-catalysed diversification of a boronate ester by amination (Chan-Lam reaction) that can be carried out on a complex ß-aryl-ß-amino acid scaffold. This not only considerably extends the substrate scope of amination partners, but also delivers an array of potent and selective integrin inhibitors as potential treatment agents of idiopathic pulmonary fibrosis (IPF). This versatile chemical strategy, which is amenable to high-throughput-array protocols, allows the installation of pharmaceutically valuable heteroaromatic fragments at a late stage by direct coupling to NH heterocycles, leading to compounds with drug-like attributes. It thus constitutes a useful addition to the medicinal chemist's repertoire.
Asunto(s)
Aminoácidos/química , Cobre/química , Integrinas/antagonistas & inhibidores , Aminación , Catálisis , Integrinas/químicaRESUMEN
Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound 19. Selectivity profiling of compound 19 showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound 19 was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound 41, which showed improved toxicological outcomes at similar exposure levels to compound 19.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacocinética , Animales , Unión Competitiva , Disponibilidad Biológica , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Isoenzimas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3/toxicidad , Ratas , Relación Estructura-ActividadRESUMEN
Phosphoinositide-3-kinase δ (PI3Kδ) is a critical regulator of cell growth and transformation and has been explored as a therapeutic target for a range of diseases. Through the exploration of the thienopyrimidine scaffold, we have identified a ligand-efficient methylation that leads to remarkable selectivity for PI3Kδ over the closely related isoforms. Interrogation through the Free-Wilson analysis highlights the innate selectivity the thienopyrimidine scaffold has for PI3Kδ and provides a predictive model for the activity against the PI3K isoforms.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/química , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Relación Estructura-Actividad , Aminas/química , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Metilación , Nitrógeno/química , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Pirimidinas/química , Albúmina Sérica Humana/metabolismoRESUMEN
The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvß3 and αvß5 significantly change the biological activities against αvß6 and αvß8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).
Asunto(s)
Fibrosis Pulmonar Idiopática/patología , Integrina alfaV/química , Fenilbutiratos/química , Administración Oral , Animales , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Integrina alfaV/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/metabolismo , Integrinas/antagonistas & inhibidores , Integrinas/metabolismo , Conformación Molecular , Simulación del Acoplamiento Molecular , Fenilbutiratos/farmacocinética , Fenilbutiratos/uso terapéutico , Estructura Terciaria de Proteína , Ratas , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/metabolismo , Relación Estructura-ActividadRESUMEN
A quaternary ammonium betaine 7 is described which shows exceptional potency and selectivity (1.4 to >3 logs) for the αvß6 integrin receptor over the other αv integrins as determined in cell adhesion assays. 7 is prepared by remarkably stereoselective methylation, the origins of which are discussed. The chemical, biological, physicochemical, and pharmacokinetic properties of 7 and its docking into αvß6 are described along with related analogues.
Asunto(s)
Betaína/farmacología , Integrinas/antagonistas & inhibidores , Pirrolidinas/química , Compuestos de Amonio Cuaternario/farmacología , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Betaína/química , Betaína/farmacocinética , Células Cultivadas , Cristalografía por Rayos X , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Integrinas/química , Integrinas/metabolismo , Metilación , Modelos Químicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacocinética , Ratas , EstereoisomerismoRESUMEN
Up to 45 % of deaths in developed nations can be attributed to chronic fibroproliferative diseases, highlighting the need for effective therapies. The RGD (Arg-Gly-Asp) integrin αvß1 was recently investigated for its role in fibrotic disease, and thus warrants therapeutic targeting. Herein we describe the identification of non-RGD hit small-molecule αvß1 inhibitors. We show that αvß1 activity is embedded in a range of published α4ß1 (VLA-4) ligands; we also demonstrate how a non-RGD integrin inhibitor (of α4ß1 in this case) was converted into a potent non-zwitterionic RGD integrin inhibitor (of αvß1 in this case). We designed urea ligands with excellent selectivity over α4ß1 and the other αv integrins (αvß3, αvß5, αvß6, αvß8). Inâ silico docking models and density functional theory (DFT) calculations aided the discovery of the lead urea series.
Asunto(s)
Fenilalanina/análogos & derivados , Receptores de Vitronectina/antagonistas & inhibidores , Urea/análogos & derivados , Animales , Sitios de Unión , Diseño de Fármacos , Estabilidad de Medicamentos , Humanos , Ligandos , Hígado/metabolismo , Masculino , Fenilalanina/síntesis química , Fenilalanina/metabolismo , Ratas Sprague-Dawley , Receptores de Vitronectina/química , Receptores de Vitronectina/metabolismo , Urea/síntesis química , Urea/metabolismoRESUMEN
A deconstruction of previously reported phosphoinositide 3-kinase δ (PI3Kδ) inhibitors and subsequent regrowth led to the identification of a privileged fragment for PI3Kδ, which was exploited to deliver a potent, efficient, and selective lead series with a novel binding mode observed in the PI3Kδ crystal structure.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Relación Estructura-Actividad , Administración por Inhalación , Animales , Fosfatidilinositol 3-Quinasa Clase Ia/química , Cristalografía por Rayos X , Perros , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Enlace de Hidrógeno , Isoquinolinas/química , Células de Riñón Canino Madin Darby , RatasRESUMEN
A series of 3-aryl(pyrrolidin-1-yl)butanoic acids were synthesized using a diastereoselective route, via a rhodium catalyzed asymmetric 1,4-addition of arylboronic acids in the presence of ( R)-BINAP to a crotonate ester to provide the ( S) absolute configuration for the major product. A variety of aryl substituents including morpholine, pyrazole, triazole, imidazole, and cyclic ether were screened in cell adhesion assays for affinity against αvß1, αvß3, αvß5, αvß6, and αvß8 integrins. Numerous analogs with high affinity and selectivity for the αvß6 integrin were identified. The analog ( S)-3-(3-(3,5-dimethyl-1 H-pyrazol-1-yl)phenyl)-4-(( R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid hydrochloride salt was found to have very high affinity for αvß6 integrin in a radioligand binding assay (p Ki = 11), a long dissociation half-life (7 h), very high solubility in saline at pH 7 (>71 mg/mL), and pharmacokinetic properties commensurate with inhaled dosing by nebulization. It was selected for further clinical investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis.
Asunto(s)
Descubrimiento de Drogas , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Integrinas/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Pirazoles/química , Animales , Antígenos de Neoplasias , Adhesión Celular , Perros , Humanos , Pulmón/metabolismo , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Ratas , Ratas Wistar , Relación Estructura-Actividad , Distribución TisularRESUMEN
The synthesis of potent amide-containing phthalazinone H1 histamine receptor antagonists is described. Three analogues 3e, 3g, and 9g were equipotent with azelastine and were longer-acting in vitro. Amide 3g had low oral bioavailability, low brain-penetration, high metabolic clearance, and long duration of action in vivo, and it was suitable for once-daily dosing intranasally, with a predicted dose for humans of approximately 0.5 mg per day.
RESUMEN
The arginyl-glycinyl-aspartic acid (RGD) integrin subfamily contains five members that partner with the αv subunit: αvß1, αvß3, αvß5, αvß6, and αvß8. Within the αv integrins, the epithelially restricted αvß6 has been identified as playing a key role in the activation of transforming growth factor ß that is hypothesized to be pivotal in the development of idiopathic pulmonary fibrosis (IPF). As part of a drug discovery program to identify a selective αvß6 RGD mimetic for IPF, cell adhesion and radioligand binding assays were investigated to screen compounds to determine affinity and αv integrin selectivity. In this study, a pan-αv radioligand was characterized against all the αv integrins and used to determine accurate selectivity profiles for literature and novel RGD ligands, as well as enable an early readout on αvß6 dissociation kinetics. It has been shown that while cell adhesion offers a high throughput and reliable format for ranking compounds, there are downsides to this format when comparing selectivity across αv integrins. By accurately defining the relationship between these assay formats, a medicinal chemistry effort has identified novel, high-affinity, and selective αvß6 RGD mimetics with slow dissociation kinetics, with the potential to be developed into clinical candidates for IPF.
Asunto(s)
Integrina alfaV/metabolismo , Ensayo de Unión Radioligante/métodos , Animales , Células CHO , Adhesión Celular/efectos de los fármacos , Cricetinae , Cricetulus , Humanos , Células K562 , Cinética , Ligandos , Oligopéptidos/química , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Tritio/metabolismoRESUMEN
A novel molecular scaffold has been synthesized, and its incorporation into new analogues of biologically active molecules across multiple target classes will be discussed. In these studies, we have shown use of the tricyclic scaffold to synthesize potent inhibitors of the serine peptidase DPP-4, antagonists of the CCR5 receptor, and highly potent and selective PI3K δ isoform inhibitors. We also describe the predicted physicochemical properties of the resulting inhibitors and conclude that the tractable molecular scaffold could have potential application in future drug discovery programs.
Asunto(s)
Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/farmacología , Inhibidores de la Dipeptidil-Peptidasa IV/química , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Receptores CCR5/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
A novel series of potent quinoline-based human H1 and H3 bivalent histamine receptor antagonists, suitable for intranasal administration for the potential treatment of allergic rhinitis associated nasal congestion, were identified. Compound 18b had slightly lower H1 potency (pA2 8.8 vs 9.7 for the clinical goldstandard azelastine), and H3 potency (pKi 9.1vs 6.8 for azelastine), better selectivity over α1A, α1B and hERG, similar duration of action, making 18b a good back-up compound to our previous candidate, but with a more desirable profile.
Asunto(s)
Descubrimiento de Drogas , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores Histamínicos H3/farmacología , Quinolinas/farmacología , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H3/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de los Receptores Histamínicos H1/síntesis química , Antagonistas de los Receptores Histamínicos H1/química , Antagonistas de los Receptores Histamínicos H3/síntesis química , Antagonistas de los Receptores Histamínicos H3/química , Humanos , Ligandos , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
The integrin αvß6 is a potential target for treatment of idiopathic pulmonary fibrosis (IPF). Equilibrium dialysis (ED) was investigated for its ability to report ligand binding in an αvß6 inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against αvß6, and the fraction bound (f b) and percentage saturation determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Quantitation of the inhibitor in the two chambers of the ED cartridge revealed an uneven distribution in the presence of αvß6, corresponding to near saturation binding to the protein (93 ± 3%), while the control (without integrin) showed an equal partitioning of the inhibitor on either side of the dialysis membrane. A competitive ED assay with a 12 component mixture of antagonists was conducted, and the results compared with an established cell adhesion assay for quantifying αvß6 inhibition of individual antagonists. Compounds clustered into three groupings: those with pIC 50 values between ca. 5.0 and 5.5, which possessed ED f b values indistinguishable from the controls, those with pIC 50s of 6.5 ± 0.2, which exhibited detectable integrin binding (f b 13-25%) in the ED assay, and a single compound of pIC 50 7.2 possessing an f b value of 38%. A good correlation between ED-derived f b and pIC 50 was observed despite the two assays utilizing quite different outputs. These results demonstrate that ED with LC-MS detection shows promise as a rapid αvß6 integrin antagonist screening assay for mixtures of putative ligands.
RESUMEN
Antagonism of αvß6 is emerging as a potential treatment of idiopathic pulmonary fibrosis based on strong target validation. Starting from an αvß3 antagonist lead and through simple variation in the nature and position of the aryl substituent, the discovery of compounds with improved αvß6 activity is described. The compounds also have physicochemical properties commensurate with oral bioavailability and are high quality starting points for a drug discovery program. Compounds 33S and 43E1 are pan αv antagonists having ca. 100 nM potency against αvß3, αvß5, αvß6, and αvß8 in cell adhesion assays. Detailed structure activity relationships with these integrins are described which also reveal substituents providing partial selectivity (defined as at least a 0.7 log difference in pIC50 values between the integrins in question) for αvß3 and αvß5.