RESUMEN
Approximately 20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2), providing an actionable target for many different therapies. In the metastatic setting, prognosis has improved greatly with the use of anti-HER2 drugs such as trastuzumab, pertuzumab, and trastuzumab-emtansine. In the third line setting and beyond, several emerging treatments have shown benefits, including novel small molecule targeted agents and antibody-drug conjugates. Systemic treatment of brain metastases in HER2-positive patients and the role of endocrine-based treatment for patients with hormone receptor (HR) positive disease remain areas of research interest. This article will review the current approach to systemic management of metastatic HER2-positive breast cancer in Canada, and present novel treatments that may be available in the near future.
Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Ado-Trastuzumab Emtansina , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Humanos , Inmunoconjugados/uso terapéutico , Trastuzumab/uso terapéuticoRESUMEN
One of the most significant hurdles to developing new chemical probes of biological systems and new drugs to treat disease is that of understanding the mechanism of action of small molecules discovered with cell-based small-molecule screening. Here we have assembled an ordered, high-expression clone set of all of the essential genes from Escherichia coli and used it to systematically screen for suppressors of growth inhibitory compounds. Using this chemical genomic approach, we demonstrate that the targets of well-known antibiotics can be identified as high copy suppressors of chemical lethality. This approach led to the discovery of MAC13243, a molecule that belongs to a new chemical class and that has a unique mechanism and promising activity against multidrug-resistant Pseudomonas aeruginosa. We show that MAC13243 inhibits the function of the LolA protein and represents a new chemical probe of lipoprotein targeting in bacteria with promise as an antibacterial lead with Gram-negative selectivity.
Asunto(s)
Escherichia coli/genética , Genoma Bacteriano/genética , Lipoproteínas/genética , Antibacterianos/farmacología , División Celular/efectos de los fármacos , Cromatografía Liquida/métodos , Cicloserina/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacología , Genes Bacterianos/efectos de los fármacos , Genómica/métodos , Lipoproteínas/antagonistas & inhibidores , Espectrometría de Masas/métodos , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/efectos de los fármacos , Proteínas de Unión Periplasmáticas/efectos de los fármacos , Proteínas de Unión Periplasmáticas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Sulfametoxazol/farmacología , Supresión Genética , Trimetoprim/farmacologíaRESUMEN
A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of â¼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.
Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Evaluación Preclínica de Medicamentos/métodos , Nanotecnología/métodos , Análisis por Matrices de Proteínas/métodos , Animales , Electrophorus , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Fluorometría , Cinética , Dióxido de Silicio/química , SolucionesRESUMEN
There is an increasing interest in new strategies to rapidly detect analytes of clinical and environmental interest without the need for sophisticated instrumentation. As an example, the detection of acetylcholinesterase (AChE) inhibitors such as neurotoxins and organophosphates has implications for neuroscience, drug assessment, pharmaceutical development, and environmental monitoring. Functionalization of surfaces with multiple reagents, including enzymes and chromogenic reagents, is a critical component for the effective development of "dipstick" or lateral flow biosensors. Herein, we describe a novel paper-based solid-phase biosensor that utilizes piezoelectric inkjet printing of biocompatible, enzyme-doped, sol-gel-based inks to create colorimetric sensor strips. For this purpose, polyvinylamine (PVAm, which captures anionic agents) was first printed and then AChE was overprinted by sandwiching the enzyme within two layers of biocompatible sol-gel-derived silica on paper. AChE inhibitors, including paraoxon and aflatoxin B1, were detected successfully using this sensor by measuring the residual activity of AChE on paper, using Ellman's colorimetric assay, with capture of the 5-thio-2-nitrobenzoate (TNB(-)) product on the PVAm layer. The assay provided good detection limits (paraoxon, approximately 100 nM; aflatoxin B1, approximately 30 nM) and rapid response times (<5 min). Detection could be achieved either by eye or using a digital camera and image analysis software, avoiding the need for expensive and sophisticated instrumentation. We demonstrate that the bioactive paper strip can be used either as a dipstick or a lateral flow-based biosensor. The use of sol-gel-based entrapment produced a sensor that retained enzyme activity and gave reproducible results after storage at 4 degrees C for at least 60 days, making the system suitable for storage and use in the field.
Asunto(s)
Técnicas Biosensibles/métodos , Neurotoxinas/análisis , Inhibidores de la Colinesterasa/química , Organofosfatos/química , Transición de Fase , Polivinilos/química , Tiras Reactivas/química , Tiras Reactivas/metabolismoRESUMEN
With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.