RESUMEN
High Content Analysis (HCA) has become a cornerstone of cellular analysis within the drug discovery industry. To expand the capabilities of HCA, we have applied the same analysis methods, validated in numerous mammalian cell models, to microbiology methodology. Image acquisition and analysis of various microbial samples, ranging from pure cultures to culture mixtures containing up to three different bacterial species, were quantified and identified using various machine learning processes. These HCA techniques allow for faster cell enumeration than standard agar-plating methods, identification of "viable but not plate culturable" microbe phenotype, classification of antibiotic treatment effects, and identification of individual microbial strains in mixed cultures. These methods greatly expand the utility of HCA methods and automate tedious and low-throughput standard microbiological methods.
Asunto(s)
Bacterias/metabolismo , Aprendizaje Automático , Antibacterianos/farmacología , Bacillus megaterium/efectos de los fármacos , Bacillus megaterium/ultraestructura , Bacterias/química , Bacterias/efectos de los fármacos , Proteínas Bacterianas/análisis , Bradyrhizobium/efectos de los fármacos , Bradyrhizobium/crecimiento & desarrollo , Bradyrhizobium/metabolismo , Bradyrhizobium/ultraestructura , Recuento de Colonia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/ultraestructura , Pseudomonas fluorescens/efectos de los fármacos , Pseudomonas fluorescens/ultraestructuraRESUMEN
Historically, monitoring nematode movement and mortality in response to various potential nematicide treatments usually involved tedious manual microscopic analysis. High-content analysis instrumentation enables rapid and high-throughput collection of experimental data points on large numbers of individual worms simultaneously. The high-throughput platform outlined here should accelerate discovery of unique classes and types of promising lead molecules and sample types to control these plant pests. Also, the ability to automate the data analysis pipeline rather than relying on manual scoring reduces a potential source of data variance. Here we describe a high-throughput process based on high-content imaging. We demonstrate the use of time-lapse image acquisition to measure movement, and viability staining to confirm nematode mortality (versus paralysis) in targeted plant-pathogenic nematodes. We present screening results from a microbial-exudate library generated from approximately 2,300 microbial fermentations that demonstrate the robustness of this high-throughput process. The described methods should be applicable to other relevant nematode parasites with human, crop, or animal hosts.
Asunto(s)
Antinematodos , Productos Biológicos , Nematodos/crecimiento & desarrollo , Control Biológico de Vectores , Plantas/parasitología , Animales , Antinematodos/química , Antinematodos/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , HumanosRESUMEN
A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Indoles/síntesis química , Quinazolinas/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/química , Humanos , Indoles/farmacología , Modelos Moleculares , Unión Proteica , Quinazolinas/farmacología , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Descubrimiento de Drogas , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , ortoaminobenzoatos/administración & dosificación , ortoaminobenzoatos/farmacología , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Conformación Molecular , Profármacos/farmacocinética , Especificidad por Sustrato , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacocinéticaRESUMEN
In the course of our Heat Shock 90 program, certain carbazole compounds were identified which had an off-target antiproliferative activity. To understand the off-target activity, we studied one analog with strong activity. We discovered that it had an effect on tubulin polymerization kinetics and was competitive with colchicine. Additional analogs were made, and a number of potent compounds were identified.
Asunto(s)
Antimitóticos/química , Carbazoles/química , Indoles/química , Antimitóticos/síntesis química , Antimitóticos/farmacología , Carbazoles/síntesis química , Carbazoles/farmacología , Línea Celular Tumoral , Colchicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Indoles/síntesis química , Indoles/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
Inhibition of Hsp90 has emerged as a therapeutic strategy to target NSCLC subtypes, which are refractory to epidermal growth factor receptor (EGFR) inhibitor-based treatment. We report on a novel small molecule inhibitor of Hsp90, SNX-2112, and an orally bioavailable prodrug analog, SNX-5422. In cellular models of wild-type or mutant EGFR (L858R and T790M mutations), SNX-2112 alone and in combination with erlotinib inhibited EGF activation of pAKT(473) and pSTAT3(705). pERK1/2 and pS6 were also potently inhibited by similar treatments. SNX-2112 reduced EGF cross-talk and activation of the c-Met receptor by causing c-Met degradation. In NCI-H1975 xenograft models, SNX-5422 showed activity as a single agent and in combination with erlotinib resulted in prolonged animal survival at reduced compound concentrations relative to either compound alone. These results support the advanced evaluation of SNX-5422 as a treatment for non-small cell lung cancer (NSCLC), especially in cases where the cancer is driven by c-Met amplification or mutated EGFR forms that are resistant to EGFR inhibitors.
Asunto(s)
Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA). METHODS: SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-kappaB nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor alpha, or interleukin-1beta stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment. RESULTS: SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-kappaB nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal. CONCLUSION: The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.
Asunto(s)
Antiinflamatorios/farmacología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Benzamidas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Administración Oral , Animales , Antiinflamatorios/farmacocinética , Artritis Reumatoide/inmunología , Benzamidas/farmacocinética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Femenino , Fibroblastos/citología , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Células Jurkat , Macrófagos/citología , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Células 3T3 NIH , Neovascularización Fisiológica/fisiología , Óxido Nítrico/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Transducción de Señal/inmunología , Membrana Sinovial/citología , omega-ConotoxinasRESUMEN
Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.