RESUMEN
Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene,Gch1, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers.Gch1is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target inducedGch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell-derived sensory neurons. Graphical overview.
RESUMEN
Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents.
Asunto(s)
Neoplasias Pulmonares , Neuralgia , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Biopterinas/análogos & derivados , Receptores ErbB/genética , Receptores ErbB/metabolismo , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed "cortical" here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.
Asunto(s)
Proteínas de Unión al ADN/genética , Células Madre Embrionarias de Ratones/citología , Neocórtex/citología , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Sirtuina 1/antagonistas & inhibidores , Animales , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , Factores de Transcripción/metabolismoRESUMEN
Dysfunction or death of pancreatic ß cells underlies both types of diabetes. This functional decline begins with ß cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate ß cell stress nor prevent, let alone reverse, ß cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature ß cells, is down-regulated in the early stages of T2D in mice and when ß cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature ß cell state, we screen for factors that reverse ß cell de-differentiation. We find that a small molecule inhibitor of TGFß receptor I (Alk5) protects cells from the loss of key ß cell transcription factors and restores a mature ß cell identity even after exposure to prolonged and severe diabetes.
Asunto(s)
Desdiferenciación Celular/efectos de los fármacos , Células Secretoras de Insulina/patología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Biomarcadores/metabolismo , Citocinas/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Resistencia a la Insulina , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Estrés Fisiológico/efectos de los fármacos , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Urocortinas/metabolismoRESUMEN
Loss of ß-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote ß-cell regeneration is a priority. cAMP is an intracellular second messenger that modulates ß-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate ß-cell growth. To identify cAMP-stabilizing medications that promote ß-cell replication, we performed high-content screening of a phosphodiesterase (PDE) inhibitor library. PDE3, -4, and -10 inhibitors, including dipyridamole, were found to promote ß-cell replication in an adenosine receptor-dependent manner. Dipyridamole's action is specific for ß-cells and not α-cells. Next we demonstrated that norepinephrine (NE), a physiologic suppressor of cAMP synthesis in ß-cells, impairs ß-cell replication via activation of α(2)-adrenergic receptors. Accordingly, mirtazapine, an α(2)-adrenergic receptor antagonist and antidepressant, prevents NE-dependent suppression of ß-cell replication. Interestingly, NE's growth-suppressive effect is modulated by endogenously expressed catecholamine-inactivating enzymes (catechol-O-methyltransferase and l-monoamine oxidase) and is dominant over the growth-promoting effects of PDE inhibitors. Treatment with dipyridamole and/or mirtazapine promote ß-cell replication in mice, and treatment with dipyridamole is associated with reduced glucose levels in humans. This work provides new mechanistic insights into cAMP-dependent growth regulation of ß-cells and highlights the potential of commonly prescribed medications to influence ß-cell growth.
Asunto(s)
División Celular/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Páncreas/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Regeneración/efectos de los fármacos , Animales , División Celular/fisiología , Células Secretoras de Insulina/fisiología , Masculino , Norepinefrina/farmacología , Páncreas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease, characterized by motor neuron (MN) death, for which there are no truly effective treatments. Here, we describe a new small molecule survival screen carried out using MNs from both wild-type and mutant SOD1 mouse embryonic stem cells. Among the hits we found, kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK-3 and HGK kinases. Furthermore, kenpaullone also strongly improved the survival of human MNs derived from ALS-patient-induced pluripotent stem cells and was more active than either of two compounds, olesoxime and dexpramipexole, that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Células Madre Embrionarias/citología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/patología , Animales , Benzazepinas/química , Benzazepinas/farmacología , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colestenonas/química , Colestenonas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Indoles/química , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/enzimología , Mutación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Relación Estructura-Actividad , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.
Asunto(s)
Glucocorticoides/farmacología , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Anilidas/farmacología , Animales , Células COS , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Chlorocebus aethiops , Interacciones Farmacológicas , Fluocinolona Acetonida/farmacología , Glucocorticoides/química , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Receptores Patched , Piridinas/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptor SmoothenedRESUMEN
Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo's actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Cilios/efectos de los fármacos , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transporte de Proteínas/efectos de los fármacos , Receptor SmoothenedRESUMEN
Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing ß cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase ß-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary ß cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of ß cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases ß-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.
Asunto(s)
Adenosina Quinasa/farmacología , Regulación de la Expresión Génica , Células Secretoras de Insulina/citología , Animales , Femenino , Fibroblastos/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Glucosa/metabolismo , Hepatocitos/citología , Insulina/metabolismo , Islotes Pancreáticos/citología , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Receptores de Glucagón/metabolismo , Porcinos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2-3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC(50) of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood-brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells, potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73) were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel therapeutic candidates for ALS.
Asunto(s)
Ciclohexanonas/química , Ciclohexanonas/farmacología , Ciclopropanos/química , Éteres Fenílicos/química , Superóxido Dismutasa/antagonistas & inhibidores , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Barrera Hematoencefálica/metabolismo , Ciclohexanonas/uso terapéutico , Ciclohexanonas/toxicidad , Ciclopropanos/uso terapéutico , Ciclopropanos/toxicidad , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Mutación , Neuronas/efectos de los fármacos , Células PC12 , Éteres Fenílicos/uso terapéutico , Éteres Fenílicos/toxicidad , Ratas , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.
Asunto(s)
Descubrimiento de Drogas/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Atrofia Muscular Espinal/tratamiento farmacológico , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Adulto , Animales , Benzazepinas/farmacología , Células Cultivadas , Preescolar , Células Madre Embrionarias , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/farmacología , Ratones , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Mutación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Transcripción STAT1 , Bibliotecas de Moléculas Pequeñas , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, most often from respiratory failure. The only FDA-approved drug for the treatment of ALS, riluzole, only extends the median survival in patients by 2-3 months. There is an urgent need for novel therapeutic strategies for this devastating disease. Using a high-throughput screening assay targeting an ALS cultured cell model (PC12-G93A-YFP cell line), we previously identified three chemotypes that were neuroprotective. We present a further detailed analysis of one promising scaffold from that group, pyrimidine-2,4,6-triones (PYTs), characterizing a number of PYT analogues using SAR and ADME. The PYT compounds show good potency, superior ADME data, low toxicity, brain penetration, and excellent oral bioavailability. Compounds from this series show 100% efficacy in the protection assay with a good correlation in activity between the protection and protein aggregation assays. The modifications of the PYT scaffold presented here suggest that this chemical structure may be a novel drug candidate scaffold for use in clinical trials in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Proteínas Mutantes/química , Mutación , Multimerización de Proteína/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacología , Superóxido Dismutasa/química , Animales , Humanos , Modelos Moleculares , Proteínas Mutantes/genética , Células PC12 , Estructura Cuaternaria de Proteína , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Ratas , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC(50) of 170nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio=0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Pirazolonas/farmacología , Superóxido Dismutasa/antagonistas & inhibidores , Animales , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Espectrometría de Masa por Ionización de Electrospray , Superóxido Dismutasa/genéticaRESUMEN
The underlying cause of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, remains unknown. However, there is strong evidence that one pathophysiological mechanism, toxic protein misfolding and/or aggregation, may trigger motor neuron dysfunction and loss. Since the clinical and pathological features of sporadic and familial ALS are indistinguishable, all forms of the disease may be better understood and ultimately treated by studying pathogenesis and therapy in models expressing mutant forms of SOD1. We developed a cellular model in which cell death depended on the expression of G93A-SOD1, a mutant form of superoxide dismutase found in familial ALS patients that produces toxic protein aggregates. This cellular model was optimized for high throughput screening to identify protective compounds from a >50,000 member chemical library. Three novel chemical scaffolds were selected for further study following screen implementation, counter-screening and secondary testing, including studies with purchased analogs. All three scaffolds blocked SOD1 aggregation in high content screening assays and data on the optimization and further characterization of these compounds will be reported separately. These data suggest that optimization of these chemicals scaffolds may produce therapeutic candidates for ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Diseño de Fármacos , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Benzoquinonas/farmacología , Muerte Celular/efectos de los fármacos , Citoprotección , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Lactamas Macrocíclicas/farmacología , Leupeptinas/farmacología , Macrólidos/farmacología , Proteínas Mutantes/metabolismo , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bibliotecas de Moléculas Pequeñas , Superóxido Dismutasa/genéticaRESUMEN
This editorial looks at how a fully integrated structure that performs all aspects in the drug discovery process, under one company, is slowly disappearing. The steps in the drug discovery paradigm have been slowly increasing toward virtuality or outsourcing at various phases of product development in a company's candidate pipeline. Each step in the process, such as target identification and validation and medicinal chemistry, can be managed by scientific teams within a 'virtual' company. Pharmaceutical companies to biotechnology start-ups have been quick in adopting this new research and development business strategy in order to gain flexibility, access the best technologies and technical expertise, and decrease product developmental costs. In today's financial climate, the term virtual drug discovery has an organizational meaning. It represents the next evolutionary step in outsourcing drug development.
RESUMEN
Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines"), inhibited both purified ENR (50% inhibitory concentration [IC(50)] = 3 to 25 micro M) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 micro g/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [(14)C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift). Synthesis of a variety of compounds in this chemical series revealed a correlation between IC(50) and MIC, and the results provided initial structure-activity relationships. Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development.