RESUMEN
Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery.
Asunto(s)
Descubrimiento de Drogas/métodos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Disponibilidad Biológica , Transporte Biológico , Células HEK293 , Células HL-60 , Humanos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinéticaRESUMEN
The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.
Asunto(s)
Administración Intravenosa , Animales , Administración Oral , Ratones , Relación Estructura-Actividad , Humanos , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Estructura MolecularRESUMEN
Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium-derived measurements of IC50, EC50, or KD. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype; however, these parameters are yet to be fully adopted in early drug discovery. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. Here, we describe an intracellular NanoBRET competition assay that can be used to quantify cellular kinetic rates of compound binding to nanoluciferase-fused bromodomain and extra-terminal (BET) proteins. Comparative rates are generated using a cell-free NanoBRET assay and by utilizing orthogonal recombinant protein-based methodologies. A screen of known pan-BET inhibitors is used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.
Asunto(s)
Luciferasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia , Células Cultivadas , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Luciferasas/química , Proteínas del Tejido Nervioso/química , Receptores de Superficie Celular/químicaRESUMEN
The bromodomain and extra terminal (BET) family of proteins are an integral part of human epigenome regulation, the dysregulation of which is implicated in multiple oncology and inflammatory diseases. Disrupting the BET family bromodomain acetyl-lysine (KAc) histone protein-protein interaction with small-molecule KAc mimetics has proven to be a disease-relevant mechanism of action, and multiple molecules are currently undergoing oncology clinical trials. This work describes an efficiency analysis of published GSK pan-BET bromodomain inhibitors, which drove a strategic choice to focus on the identification of a ligand-efficient KAc mimetic with the hypothesis that lipophilic efficiency could be drastically improved during optimization. This focus drove the discovery of the highly ligand-efficient and structurally distinct benzoazepinone KAc mimetic. Following crystallography to identify suitable growth vectors, the benzoazepinone core was optimized through an explore-exploit structure-activity relationship (SAR) approach while carefully monitoring lipophilic efficiency to deliver I-BET432 (41) as an oral candidate quality molecule.
Asunto(s)
Lisina , Factores de Transcripción , Humanos , Lisina/metabolismo , Ligandos , Dominios Proteicos , Histonas/metabolismoRESUMEN
Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.
Asunto(s)
Aminoquinolinas/química , Diseño de Fármacos , Proteínas/metabolismo , Administración Oral , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacocinética , Aminoquinolinas/uso terapéutico , Animales , Benzoatos/química , Benzoatos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Semivida , Humanos , Masculino , Ratones , Conformación Molecular , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Proteínas/antagonistas & inhibidores , Ratas , Relación Estructura-ActividadRESUMEN
Bromodomain containing proteins and the acetyl-lysine binding bromodomains contained therein are increasingly attractive targets for the development of novel epigenetic therapeutics. To help validate this target class and unravel the complex associated biology, there has been a concerted effort to develop selective small molecule bromodomain inhibitors. Herein we describe the structure-based efforts and multiple challenges encountered in optimizing a naphthyridone template into selective TAF1(2) bromodomain inhibitors which, while unsuitable as chemical probes themselves, show promise for the future development of small molecules to interrogate TAF1(2) biology. Key to this work was the introduction and modulation of the basicity of a pendant amine which had a substantial impact on not only bromodomain selectivity but also cellular target engagement.
RESUMEN
Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.
Asunto(s)
Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Animales , Diseño de Fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Semivida , Humanos , Masculino , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Células THP-1RESUMEN
5-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2(1H)-quinolinones and 3,4-dihydro-2(1H)-quinolinones have been identified with different combinations of 5-HT(1) autoreceptor antagonist and hSerT potencies and excellent rat PK profiles. The availability of tool compounds with a range of profiles at targets known to play a key role in the control of synaptic 5-HT levels will allow exploration of different pharmacological profiles in a range of animal behavioral and disease models.
Asunto(s)
Quinolonas/química , Receptores de Serotonina 5-HT1/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinética , Animales , Autorreceptores/antagonistas & inhibidores , Autorreceptores/efectos de los fármacos , Quinolonas/farmacocinética , Ratas , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Sinapsis/químicaRESUMEN
Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.
Asunto(s)
Bioensayo , Epigénesis Genética/genética , Relación Estructura-Actividad , Ensamble y Desensamble de Cromatina/genética , Metilación de ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Código de Histonas/genética , Humanos , Luciferasas/químicaRESUMEN
Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.
RESUMEN
8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT(1A/B/D) receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.
Asunto(s)
Benzoxazinas/química , Piperazinas/química , Inhibidores Selectivos de la Recaptación de Serotonina/química , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas de la Serotonina/química , Animales , Benzoxazinas/metabolismo , Benzoxazinas/farmacología , Células CHO , Cricetinae , Cricetulus , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Piperazinas/metabolismo , Piperazinas/farmacología , Ratas , Receptores de Serotonina 5-HT1/metabolismo , Antagonistas de la Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
5-HT1 receptor antagonists have been discovered with good selectivity over the 5-HT transporter. This is the first report of highly potent, selective ligands for the 5-HT1A/B/D receptors with low intrinsic activity, which represent a useful set of molecules for further understanding the roles of the 5-HT1 receptor subtypes and providing new approaches for the treatment of depression.
Asunto(s)
Piperazinas/síntesis química , Quinolinas/síntesis química , Antagonistas del Receptor de Serotonina 5-HT1 , Animales , Barrera Hematoencefálica/metabolismo , Corteza Cerebral/metabolismo , Humanos , Técnicas In Vitro , Piperazinas/farmacocinética , Piperazinas/farmacología , Quinolinas/farmacocinética , Quinolinas/farmacología , Ensayo de Unión Radioligante , Ratas , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT(1A/1B/1D) receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.
Asunto(s)
Benzoxazoles/síntesis química , Piperazinas/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antagonistas de la Serotonina/síntesis química , Antagonistas de la Serotonina/farmacología , Animales , Antidepresivos/síntesis química , Antidepresivos/farmacología , Benzoxazoles/farmacología , Diseño de Fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Modelos Químicos , Piperazina , Piperazinas/química , Piperazinas/farmacología , Ratas , Antagonistas del Receptor de Serotonina 5-HT1RESUMEN
The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains-BRD2, BRD3, BRD4, and BRDT-each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.
Asunto(s)
Descubrimiento de Drogas , Proteínas Nucleares/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Homología de SecuenciaRESUMEN
An increase in brain 5-HT levels is thought to be the key mechanism of action which results in an antidepressant response. It has been proven that selective serotonin re-uptake inhibitors are effective antidepressants but the delay to therapeutic onset of these agents is thought to be due to the time required for 5-HT1A, and possibly 5-HT1B, autoreceptor desensitisation. Therefore an agent incorporating 5-HT re-uptake inhibition coupled with 5-HT1A and/or 5-HT1B autoreceptor antagonism may provide a fast acting clinical agent. The current studies describe the in vitro profile of SB-649915 (6-[(1-{2-[(2-methylquinolin-5-yl)oxy]ethyl}piperidin-4-yl)methyl]-2H-1,4-benzoxazin-3(4H)-one), a novel compound which has high affinity for human recombinant 5-HT1A, 5-HT1B and 5-HT1D receptors (pKi values of 8.6, 8.0, 8.8, respectively) and the human recombinant 5-HT transporter (pKi value of 9.3). SB-649915 also displays high affinity for rat, guinea pig, mouse and marmoset native tissue 5-HT1A, 5-HT1B and 5-HT1D receptors and rat native tissue 5-HT transporters (pKi values>or=7.5). In functional [35S]GTPgammaS binding studies, SB-649915 (up to 1 microM) does not display intrinsic activity in HEK293 cells expressing human recombinant 5-HT1A receptors but acts as a partial agonist at human recombinant 5-HT1B and 5-HT1D receptors with intrinsic activity values of 0.3 and 0.7, respectively, as compared to the full agonist 5-HT. From Schild analysis, SB-649915 caused a concentration-dependent, rightward shift of 5-HT-induced stimulation of basal [35S]GTPgammaS binding in cells expressing human recombinant 5-HT1A or 5-HT1B receptors to yield pA2 values of 9.0 and 7.9, respectively. In electrophysiological studies in rat dorsal raphe nucleus, SB-649915 did not affect the cell firing rate up to 1 microM but attenuated (+)8-hydroxy-2-(di-n-propylamino) tetralin-induced inhibition of cell firing with an apparent pKb value of 9.5. SB-649915 (1 microM) significantly attenuated exogenous 5-HT-induced inhibition of electrically-stimulated [3H]5-HT release from guinea pig cortex. In studies designed to enhance endogenous 5-HT levels, and therefore increase tone at 5-HT1B autoreceptors, SB-649915 significantly potentiated [3H]5-HT release at 100 and 1000 nM. In LLCPK cells expressing human recombinant 5-HT transporters and in rat cortical synaptosomes, SB-649915 inhibited [3H]5-HT re-uptake with pIC50 values of 7.9 and 9.7, respectively. In summary, SB-649915 is a novel, potent 5-HT1A/1B autoreceptor antagonist and 5-HT re-uptake inhibitor in native tissue systems and represents a novel mechanism that could offer fast acting antidepressant action.
Asunto(s)
Piperidinas/farmacología , Quinolinas/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas de la Serotonina/farmacología , Animales , Autorreceptores/agonistas , Autorreceptores/antagonistas & inhibidores , Benzoxazinas , Unión Competitiva/efectos de los fármacos , Callithrix , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Humanos , Técnicas In Vitro , Ratones , Ensayo de Unión Radioligante , Núcleos del Rafe/citología , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/fisiología , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1A/genética , Receptor de Serotonina 5-HT1B/genética , Receptor de Serotonina 5-HT1D/genética , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Serotonina/metabolismo , Agonistas del Receptor de Serotonina 5-HT1 , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.
Asunto(s)
Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Descubrimiento de Drogas/métodos , Células HeLa , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Permeabilidad , Espectrometría de Masas en Tándem/métodosRESUMEN
The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.
RESUMEN
A series of 5-(piperidinylethyloxy)quinoline 5-hydroxytryptamine(1D) (5-HT(1D)) receptor antagonists have been discovered from elaboration of the series of dual 5-hydroxytryptamine(1)-selective serotonin reuptake inhibitors (5HT(1)-SSRIs) reported previously. This is the first report of highly potent, selective antagonists for the 5-HT(1D) receptor, which represents an extremely useful set of pharmacological tools for further understanding the roles of the 5-HT(1) receptor subtypes.
Asunto(s)
Piperazinas/síntesis química , Quinolinas/síntesis química , Antagonistas del Receptor de Serotonina 5-HT1 , Animales , Unión Competitiva , Células CHO , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cricetinae , Cricetulus , Técnicas In Vitro , Piperazinas/química , Piperazinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Ensayo de Unión Radioligante , Ratas , Agonistas del Receptor de Serotonina 5-HT1 , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Relación Estructura-ActividadRESUMEN
The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.