RESUMEN
Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.
Asunto(s)
Interleucina-13 , Anticuerpos de Dominio Único , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , CitocinasRESUMEN
Fragment merging is a promising approach to progressing fragments directly to on-scale potency: each designed compound incorporates the structural motifs of overlapping fragments in a way that ensures compounds recapitulate multiple high-quality interactions. Searching commercial catalogues provides one useful way to quickly and cheaply identify such merges and circumvents the challenge of synthetic accessibility, provided they can be readily identified. Here, we demonstrate that the Fragment Network, a graph database that provides a novel way to explore the chemical space surrounding fragment hits, is well-suited to this challenge. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four crystallographic screening campaigns and contrast the results with a traditional fingerprint-based similarity search. The two approaches identify complementary sets of merges that recapitulate the observed fragment-protein interactions but lie in different regions of chemical space. We further show our methodology is an effective route to achieving on-scale potency by retrospective analyses for two different targets; in analyses of public COVID Moonshot and Mycobacterium tuberculosis EthR inhibitors, potential inhibitors with micromolar IC50 values were identified. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search.
Asunto(s)
COVID-19 , Mycobacterium tuberculosis , Humanos , Estudios Retrospectivos , Bases de Datos Factuales , CristalografíaRESUMEN
Despite recent interest in deep generative models for scaffold elaboration, their applicability to fragment-to-lead campaigns has so far been limited. This is primarily due to their inability to account for local protein structure or a user's design hypothesis. We propose a novel method for fragment elaboration, STRIFE, that overcomes these issues. STRIFE takes as input fragment hotspot maps (FHMs) extracted from a protein target and processes them to provide meaningful and interpretable structural information to its generative model, which in turn is able to rapidly generate elaborations with complementary pharmacophores to the protein. In a large-scale evaluation, STRIFE outperforms existing, structure-unaware, fragment elaboration methods in proposing highly ligand-efficient elaborations. In addition to automatically extracting pharmacophoric information from a protein target's FHM, STRIFE optionally allows the user to specify their own design hypotheses.
Asunto(s)
Proteínas , Ligandos , Proteínas/químicaRESUMEN
The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study. We generated analogues of RO-90-7501 and found that one compound, MRT00210423, had increased anti-HCMV activity compared to RO-90-7501. Using a combination of compound analogues, microscopy and biochemical assays we found RO-90-7501 and MRT00210423 interacted with DNA. In single molecule microscopy experiments we found RO-90-7501, but not MRT00210423, was able to compact DNA, suggesting that compaction of DNA was non-obligatory for anti-HCMV effects. Using bioinformatics analysis, we found that there were many putative bisbenzimide binding sites in the HCMV DNA genome. However, using western blotting, quantitative PCR and electron microscopy, we found that at a concentration able to inhibit HCMV replication our compounds had little or no effect on production of certain HCMV proteins or DNA synthesis, but did have a notable inhibitory effect on HCMV capsid production. We reasoned that these effects may have involved binding of our compounds to the HCMV genome and/or host cell chromatin. Therefore, our data expand our understanding of compounds with anti-HCMV activity and suggest targeting of DNA with bisbenzimide compounds may be a useful anti-HCMV strategy.
Asunto(s)
Antivirales/farmacología , Bisbenzimidazol/farmacología , Citomegalovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/química , Sitios de Unión , Bisbenzimidazol/química , Cápside/metabolismo , Línea Celular , Citomegalovirus/fisiología , ADN/biosíntesis , ADN/química , Replicación del ADN/efectos de los fármacos , Humanos , Estructura Molecular , Carga Viral/efectos de los fármacosRESUMEN
A series of Formyl peptide receptor 2 small molecule agonists with a pyrrolidinone scaffold, derived from a combination of pharmacophore modelling and docking studies, were designed and synthesized. The GLASS (GPCR-Ligand Association) database was screened using a pharmacophore model. The most promising novel ligand structures were chosen and then tested in cellular assays (calcium mobilization and ß-arrestin assays). Amongst the selected ligands, two pyrrolidinone compounds (7 and 8) turned out to be the most active. Moreover compound 7 was able to reduce the number of adherent neutrophils in a human neutrophil static adhesion assay which indicates its anti-inflammatory and proresolving properties. Further exploration and optimization of new ligands showed that heterocyclic rings, e.g. pyrazole directly connected to the pyrrolidinone scaffold, provide good stability and a boost in the agonistic activity. The compounds of most interest (7 and 30) were tested in an ERK phosphorylation assay, demonstrating selectivity towards FPR2 over FPR1. Compound 7 was examined in an in vivo mouse pharmacokinetic study. Compound 7 may be a valuable in vivo tool and help improve understanding of the role of the FPR2 receptor in the resolution of inflammation process.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Diseño de Fármacos , Pirrolidinonas/farmacología , Receptores de Formil Péptido/agonistas , Receptores de Lipoxina/agonistas , Bibliotecas de Moléculas Pequeñas/farmacología , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The discovery of natural specialized pro-resolving mediators and their corresponding receptors, such as formyl peptide receptor 2 (FPR2), indicated that resolution of inflammation (RoI) is an active process which could be harnessed for innovative approaches to tame pathologies with underlying chronic inflammation. In this work, homology modelling, molecular docking and pharmacophore studies were deployed to assist the rationalization of the structure-activity relationships of known FPR2 agonists. The developed pharmacophore hypothesis was then used in parallel with the homology model for the design of novel ligand structures and in virtual screening. In the first round of optimization compound 8, with a cyclopentane core, was chosen as the most promising agonist (ß-arrestin recruitment EC50 = 20 nM and calcium mobilization EC50 = 740 nM). In a human neutrophil static adhesion assay, compound 8 decreased the number of adherent neutrophils in a concentration dependent manner. Further investigation led to the more rigid cycloleucines (compound 22 and 24) with improved ADME profiles and maintaining FPR2 activity. Overall, we identified novel cyclopentane urea FPR2 agonists with promising ADMET profiles and the ability to suppress the inflammatory process by inhibiting the neutrophil adhesion cascade, which indicates their anti-inflammatory and pro-resolving properties.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Ciclopentanos/farmacología , Inflamación/tratamiento farmacológico , Receptores de Formil Péptido/agonistas , Receptores de Lipoxina/agonistas , Urea/farmacología , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Enfermedades Cardiovasculares/metabolismo , Adhesión Celular/efectos de los fármacos , Ciclopentanos/síntesis química , Ciclopentanos/química , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Inflamación/metabolismo , Modelos Moleculares , Estructura Molecular , Neutrófilos/efectos de los fármacos , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Urea/análogos & derivados , Urea/químicaRESUMEN
Current chemotherapeutics for leishmaniasis have multiple deficiencies, and there is a need for new safe, efficacious, and affordable medicines. This study describes a successful drug repurposing approach that identifies the over-the-counter antihistamine, clemastine fumarate, as a potential antileishmanial drug candidate. The screening for inhibitors of the sphingolipid synthase (inositol phosphorylceramide synthase, IPCS) afforded, following secondary screening against Leishmania major (Lmj) promastigotes, 16 active compounds. Further refinement through the dose response against LmjIPCS and intramacrophage L. major amastigotes identified clemastine fumarate with good activity and selectivity with respect to the host macrophage. On target engagement was supported by diminished sensitivity in a sphingolipid-deficient L. major mutant (ΔLmjLCB2) and altered phospholipid and sphingolipid profiles upon treatment with clemastine fumarate. The drug also induced an enhanced host cell response to infection indicative of polypharmacology. The activity was sustained across a panel of Old and New World Leishmania species, displaying an in vivo activity equivalent to the currently used drug, glucantime, in a mouse model of L. amazonensis infection. Overall, these data validate IPCS as an antileishmanial drug target and indicate that clemastine fumarate is a candidate for repurposing for the treatment of leishmaniasis.
Asunto(s)
Antiprotozoarios , Leishmaniasis , Preparaciones Farmacéuticas , Animales , Antiprotozoarios/farmacología , Clemastina/uso terapéutico , Inositol , Leishmaniasis/tratamiento farmacológico , RatonesRESUMEN
Here, we describe a novel workflow combining informatic and experimental approaches to enable evidence-based prioritising of targets from large sets in parallel. High-throughput protein production and biophysical fragment screening is used to identify those targets that are tractable and ligandable. As proof of concept we have applied this to a set of antibacterial targets comprising 146 essential genes. Of these targets, 51 were selected and 38 delivered results that allowed us to rank them by ligandability. The data obtained against these derisked targets have enabled rapid progression into structurally enabled drug discovery projects, demonstrating the practical value of the fragment-based target screening workflow.
RESUMEN
Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Imidazoles/química , Malaria/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Humanos , Malaria/enzimología , Malaria/parasitología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Plasmodium falciparum/enzimología , Conformación Proteica , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Antibody-drug conjugates (ADCs) are a growing class of therapeutics that harness the specificity of antibodies and the cell-killing potency of small-molecule drugs. Beyond cytotoxics, there are few examples of the application of an ADC approach to difficult drug discovery targets. Here, we present the initial development of a non-internalising ADC, with a view to selectively inhibiting an extracellular protein. Employing the wellinvestigated matrix metalloproteinase-9 (MMP-9) as our model, we adapted a broad-spectrum, nonselective MMP inhibitor for conjugation and linked this to a MMP-9-targeting antibody. The resulting ADC fully inhibits MMP-9, and ELISA results suggest antibody targeting can direct a nonselective inhibitor.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Ácidos Hidroxámicos/química , Inmunoconjugados/inmunología , Metaloproteinasa 9 de la Matriz/inmunología , Inhibidores de Proteasas/química , Pirazinas/química , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/metabolismo , Línea Celular , Pruebas de Enzimas , Fluorometría , Humanos , Ácidos Hidroxámicos/metabolismo , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Inhibidores de Proteasas/metabolismo , Unión Proteica , Pirazinas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMEN
Building, curating, and maintaining a compound collection is an expensive operation, beyond the scope of most academic organizations. Here we describe the selection criteria used to compile the LifeArc diversity set from commercial suppliers and the process we undertook to generate our representative LifeArc index set. The aim was to avoid a "junk in, junk out" screen collection to increase chemical tractability going forward, while maximizing diversity. Using historical LifeArc screening data, we demonstrate that the index set was predictive of ligandability and that progressable hits could be identified by mining associated clusters within our larger diversity set. Indeed, a higher percentage of index-derived hit clusters were found to have been progressed into hit-to-lead programs, reflecting better drug-likeness. In practice, the library has been shared widely with academic groups and used routinely within LifeArc to assess the ligandability of novel targets. Its small size is well suited to meet the needs of medium-throughput screening in labs with either limited automation, limited precious or expensive reagents, or complex cellular assays. The strategy of screening a small set in combination with rapid hit analog follow-up has demonstrated the utility of finding active clusters for potential development against challenging targets.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Minería de Datos , Bases de Datos de Compuestos Químicos , Estudios RetrospectivosRESUMEN
Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.
Asunto(s)
Antimaláricos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Imidazoles/farmacología , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Imidazoles/síntesis química , Imidazoles/química , Ligandos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
A series of trisubstituted thiazoles have been identified as potent inhibitors of Plasmodium falciparum (Pf) cGMP-dependent protein kinase (PfPKG) through template hopping from known Eimeria PKG (EtPKG) inhibitors. The thiazole series has yielded compounds with improved potency, kinase selectivity and good in vitro ADME properties. These compounds could be useful tools in the development of new anti-malarial drugs in the fight against drug resistant malaria.
Asunto(s)
Antimaláricos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tiazoles/farmacología , Alquilación , Antimaláricos/química , Humanos , Oxidación-Reducción , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.
Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Aurora Quinasa A/química , Sitios de Unión/efectos de los fármacos , Proteínas de Ciclo Celular/química , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Ligandos , Proteínas Asociadas a Microtúbulos/química , Simulación del Acoplamiento Molecular , Proteínas Nucleares/química , Unión Proteica/efectos de los fármacos , Tiazolidinas/química , Tiazolidinas/farmacologíaRESUMEN
To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Imidazoles/uso terapéutico , Malaria/enzimología , Malaria/transmisión , Piridinas/uso terapéutico , Animales , Línea Celular , Cristalografía por Rayos X , Culicidae , Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Imidazoles/farmacología , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria/tratamiento farmacológico , Ratones Endogámicos BALB C , Modelos Moleculares , Plasmodium chabaudi/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Resultado del TratamientoRESUMEN
HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/inmunología , Digoxina/farmacología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Integración Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Activación de Linfocitos/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
Reactivation of the wild-type p53 pathway is one key goal aimed at developing targeted therapeutics in the cancer research field. Although most p53 protein kinases form 'p53-activating' signals, there are few kinases whose action can contribute to the inhibition of p53, as Casein kinase 1 (CK1) and Checkpoint kinase 1 (CHK1). Here we report on a pyrazolo-pyridine analogue showing activity against both CK1 and CHK1 kinases that lead to p53 pathway stabilisation, thus having pharmacological similarities to the p53-activator Nutlin-3. These data demonstrate the emerging potential utility of multivalent kinase inhibitors.
Asunto(s)
Quinasa de la Caseína I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Pirazoles/química , Piridinas/química , Proteína p53 Supresora de Tumor/agonistas , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Células HCT116 , Humanos , Cinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/toxicidad , Piridinas/síntesis química , Piridinas/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The identification of high-quality hits during the early phases of drug discovery is essential if projects are to have a realistic chance of progressing into clinical development and delivering marketed drugs. As the pharmaceutical industry goes through unprecedented change, there are increasing opportunities to collaborate via pre-competitive networks to marshal multifunctional resources and knowledge to drive impactful, innovative science. The 3D Fragment Consortium is developing fragment-screening libraries with enhanced 3D characteristics and evaluating their effect on the quality of fragment-based hit identification (FBHI) projects.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas/métodos , Bibliotecas de Moléculas Pequeñas/química , Conducta Cooperativa , Industria Farmacéutica/organización & administración , Industria Farmacéutica/tendencias , Humanos , Conformación MolecularRESUMEN
A variety of G-protein-coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated ß-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward ß-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased ß-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter ß-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of ß-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. ß-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through ß-arrestin in response to their native ligand remain to be determined.
Asunto(s)
Arrestinas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Descubrimiento de Drogas/métodos , Células HEK293 , Humanos , Ligandos , Unión Proteica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae , Bibliotecas de Moléculas Pequeñas/análisis , beta-ArrestinasRESUMEN
Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.