RESUMEN
Neuropathic pain is a chronic and debilitating condition characterised by episodes of hyperalgesia and allodynia. It occurs following nerve damage from disease, inflammation or injury and currently impacts up to 17% of the UK population. Existing therapies lack efficacy and have deleterious side effects that can be severely limiting. Galanin receptor 2 (GalR2) is a G-protein coupled receptor (GPCR) implicated in the control and processing of painful stimuli. Within the nervous system it is expressed in key tissues involved in these actions such as dorsal root ganglia (DRG) and the dorsal horn of the spinal cord. Stimulation of GalR2 is widely reported to have a role in the attenuation of inflammatory and neuropathic pain. Several studies have indicated GalR2 as a possible drug target, highlighting the potential of specific GalR2 agonists to both provide efficacy and to address the side-effect profiles of current pain therapies in clinical use. A strong biological target for drug discovery will be well validated with regards to its role in the relevant disease pathology. Ideally there will be good translational models, sensitive probes, selective and appropriate molecular tools, translational biomarkers, a clearly defined patient population and strong opportunities for commercialisation. Before GalR2 can be considered as a drug target suitable for investment, key questions need to be asked regarding its expression profile, receptor signalling and ligand interactions. This article aims to critically review the available literature and determine the current strength of hypothesis of GalR2 as a target for the treatment of neuropathic pain.
Asunto(s)
Neuralgia , Receptor de Galanina Tipo 2 , Humanos , Receptor de Galanina Tipo 2/agonistas , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Hiperalgesia/metabolismo , Médula Espinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ganglios Espinales/metabolismoRESUMEN
The Anti- Wolbachia (A·WOL) consortium at the Liverpool School of Tropical Medicine (LSTM) has partnered with the Global High-Throughput Screening (HTS) Centre at AstraZeneca to create the first anthelmintic HTS for neglected tropical diseases (NTDs). The A·WOL consortium aims to identify novel macrofilaricidal drugs targeting the essential bacterial symbiont ( Wolbachia) of the filarial nematodes causing onchocerciasis and lymphatic filariasis. Working in collaboration, we have validated a robust high-throughput assay capable of identifying compounds that selectively kill Wolbachia over the host insect cell. We describe the development and validation process of this complex, phenotypic high-throughput assay and provide an overview of the primary outputs from screening the AstraZeneca library of 1.3 million compounds.
Asunto(s)
Antibacterianos/farmacología , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno/efectos de los fármacos , Wolbachia/efectos de los fármacos , Antibacterianos/química , Técnicas de Cultivo de Célula/métodos , Descubrimiento de Drogas , Filariasis Linfática/tratamiento farmacológico , Humanos , Citometría de Imagen , Oncocercosis/tratamiento farmacológico , Wolbachia/patogenicidad , Wolbachia/ultraestructuraRESUMEN
Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (AâWOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.
Asunto(s)
Descubrimiento de Drogas , Filaricidas/análisis , Ensayos Analíticos de Alto Rendimiento , Aedes , Animales , Línea Celular , WolbachiaRESUMEN
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Piridinas/uso terapéutico , Quinina/análogos & derivados , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Éteres/química , Éteres/farmacocinética , Éteres/farmacología , Éteres/uso terapéutico , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Quinina/química , Quinina/farmacocinética , Quinina/farmacología , Quinina/uso terapéutico , Tuberculosis/metabolismoRESUMEN
Quantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Perfilación de la Expresión Génica/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Neoplasias Experimentales/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Diseño de Equipo , Análisis de Falla de Equipo , Genes Reporteros/genética , Humanos , Miniaturización , Neoplasias Experimentales/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z' of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Virus Sincitiales Respiratorios/efectos de los fármacos , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Genes Reporteros , Humanos , Pruebas de Sensibilidad Microbiana , Virus Sincitiales Respiratorios/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.