RESUMEN
OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.
Asunto(s)
Transportadores de Anión Orgánico/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Transporte Biológico , HumanosRESUMEN
The ectodomain of anthrax toxin receptor 2 (ANTXR2) is composed of a von Willebrand factor A (VWA) domain that binds to anthrax toxin protective antigen (PA) and a newly defined immunoglobulin-like (Ig) domain, in which the disulfide bonds are required for PA pore formation and for the folding of ANTXR2. While the VWA domain has been well characterized, the structure and function of the whole ectodomain (VWA-Ig) are poorly defined, which is mainly due to the limited production of the soluble recombinant protein of the ectodomain. In the present study, the ANTXR2 ectodomain was fused to the C-terminus of bacterial Trigger Factor (TF), a chaperone that mediates the ribosome-associated, co-translational folding of newly synthesized polypeptides in Escherichia coli. Under the control of a cold shock promoter, the fusion protein was overly expressed as a dominant soluble protein at a low temperature in the oxidative cytoplasm of Origami B cells, where formation of the disulfide bonds is favored. Through a series of chromatography, the ANTXR2 ectodomain was purified into homogeneity. The purified ectodomain is functional in binding to PA and mediating PA pore formation on the liposomal membranes, and the yield is applicable for future biochemical and structural characterization.
Asunto(s)
Escherichia coli/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Disulfuros , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Liposomas , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Receptores de Péptidos/química , Receptores de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
Human cancers require fatty acid synthase (FASN)-dependent de novo long-chain fatty acid synthesis for proliferation. FASN is therefore an attractive drug target, but fast technologies for reliable label-free cellular compound profiling are lacking. Recently, MALDI-mass spectrometry (MALDI-MS) has emerged as an effective technology for discovery of recombinant protein target inhibitors. Here we present an automated, mechanistic MALDI-MS cell assay, which monitors accumulation of the FASN substrate, malonyl-coenzyme A (CoA), in whole cells with limited sample preparation. Profiling of inhibitors, including unpublished compounds, identified compound 1 as the most potent FASN inhibitor (1 nM in A549 cells) discovered to date. Moreover, cellular MALDI-MS assays enable parallel profiling of additional pathway metabolites. Surprisingly, several compounds triggered cytidine 5'-diphosphocholine (CDP-choline) but not malonyl-CoA accumulation indicating that they inhibit diacylglycerol generation but not FASN activity. Taken together, our study suggests that MALDI-MS cell assays may become important tools in drug profiling that provide additional mechanistic insights concerning compound action on metabolic pathways.
Asunto(s)
Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células A549 , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Concentración 50 Inhibidora , Células K562 , Lipogénesis , Malonil Coenzima A/metabolismo , Prueba de Estudio ConceptualRESUMEN
Recent advances in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have enabled whole cell-MALDI mass spectrometry biotyping of drug-treated cultured cells for rapid monitoring of known abundant pharmacodynamic protein markers such as polyacetylated histones. In contrast, generic and automated analytical workflows for discovery of such pharmacodynamic markers, in particular lipid markers, and their use in cellular tests of drug-like compounds are still lacking. Here, we introduce such a workflow and demonstrate its utility for cellular drug-response monitoring of BCR-ABL tyrosine kinase inhibitors in K562 leukemia cells: First, low-molecular mass features indicating drug responses are computationally extracted from groups of MALDI-TOF mass spectra. Then, the lipids/metabolites corresponding to these features are identified by MALDI-Fourier transformation mass spectrometry. To demonstrate utility of the method, we identify the potassium adduct of phosphatidylcholine PC(36:1) as well as heme B, a marker for erythroid differentiation, as markers for a label-free MALDI MS-based test of cellular responses to BCR-ABL inhibitors. Taken together, these results suggest that MALDI-TOF mass spectrometry of lipids and other low molecular mass metabolites could support cell-based drug profiling.
Asunto(s)
Factores Biológicos/análisis , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Células K562 , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Some epigenetic modifier proteins have become validated clinical targets. With a few small molecule inhibitors already approved by national health administrations and many more in the pharmaceutical industry pipelines, there is a need for technologies that can promote full comprehension of the molecular action of these drugs. Proteomics, with its relatively unbiased nature, can contribute to a thorough understanding of the complexity of the megadalton complexes, which write, read and erase the histone code, and it can help study the on-target and off-target effect of the drugs designed to modulate their action. This review on the one hand gathers the published affinity probes able to decipher small molecule targets and off-targets in a close-to-native environment. These are small molecule analogues of epigenetic drugs conceived as protein target enrichment tools after they have engaged them in cells or lysates. Such probes, which have been designed for deacetylases, bromodomains, demethylases, and methyltransferases not only enrich their direct protein targets but also their stable interactors, which can be identified by mass spectrometry. Hence, they constitute a tool to study the epigenetic complexes together with other techniques also reviewed here: immunoaffinity purification with antibodies against native protein complex constituents or epitope tags, affinity matrices designed to bind recombinantly tagged protein, and enrichment of the complexes using histone tail peptides as baits. We expect that this toolbox will be adopted by more and more researchers willing to harness the spectacular advances in mass spectrometry to the epigenetic field.