RESUMEN
Mass spectrometry-based in vitro diagnostic devices that measure proteins and peptides are underutilized in clinical practice, and none has been cleared or approved by the Food and Drug Administration (FDA) for marketing or for use in clinical trials. One way to increase their utilization is through enhanced interactions between the FDA and the clinical mass spectrometry community to improve the validation and regulatory review of these devices. As a reference point from which to develop these interactions, this article surveys the FDA's regulation of mass spectrometry-based devices, explains how the FDA uses guidance documents and standards in the review process, and describes the FDA's previous outreach to stakeholders. Here we also discuss how further communication and collaboration with the clinical mass spectrometry communities can identify opportunities for the FDA to provide help in the development of mass spectrometry-based devices and enhance their entry into the clinic.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Espectrometría de Masas/normas , United States Food and Drug Administration/legislación & jurisprudencia , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/normas , Técnicas de Laboratorio Clínico/instrumentación , Humanos , Espectrometría de Masas/instrumentación , Estados UnidosRESUMEN
Tumor types can be defined cytologically by their regions of chromosomal amplification, which often results in the high expression of both mRNA and proteins of certain genes contained within the amplicon. An important strategy for defining therapeutically relevant targets in these situations is to ascertain which genes are amplified at the protein level and, concomitantly, are key drivers for tumor growth or maintenance. Furthermore, so-called passenger genes that are amplified with driver genes and a manifest on the cell surface can be attractive targets for an antibody-drug conjugate approach (ADC). We employed a tandem mass spectrometry proteomics approach using tumor cell lines to identify the cell surface proteins whose expression correlates with the 11q13 amplicon. The 11q13 amplicon is one of the most frequently amplified chromosomal regions in human cancer, being present in 45% of head and neck and oral squamous cell carcinoma (OSCC) and 13-21% of breast and liver carcinomas. Using a panel of tumor cell lines with defined 11q13 genomic amplification, we identified the membrane proteins that are differentially expressed in an 11q13 amplified cell line panel using membrane-enriched proteomic profiling. We found that DSG3, CD109, and CD14 were differentially overexpressed in head and neck and breast tumor cells with 11q13 amplification. The level of protein expression of each gene was confirmed by Western blot and FACS analysis. Because proteins with high cell surface expression on selected tumor cells could be potential antibody drug conjugate targets, we tested DSG3 and CD109 in antibody piggyback assays and validated that DSG3 and CD109 expression was sufficient to induce antibody internalization and cell killing in 11q13-amplified cell lines. Our results suggest that proteomic profiling using genetically stratified tumors can identify candidate antibody drug conjugate targets. Data are available via ProteomeXchange with the identifier PXD002486.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 11 , Neoplasias de Cabeza y Cuello/genética , Proteínas de la Membrana/genética , Proteómica , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/química , Espectrometría de Masas en TándemRESUMEN
While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms.
Asunto(s)
Iones , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/sangre , Suero/química , Secuencia de Aminoácidos , Biomarcadores , Humanos , Masculino , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/sangre , Péptidos/química , Antígeno Prostático Específico/química , Neoplasias de la Próstata/sangre , Espectrometría de Masas en TándemAsunto(s)
Antirreumáticos/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Agammaglobulinemia Tirosina Quinasa , Secuencia de Aminoácidos , Animales , Antirreumáticos/química , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Sitios de Unión , Modelos Animales de Enfermedad , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/metabolismo , Roedores , Relación Estructura-ActividadRESUMEN
A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.
Asunto(s)
Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/farmacología , Tripsina/clasificación , Tripsina/metabolismo , Línea Celular , Humanos , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Estructura Molecular , Inhibidores de Tripsina/químicaRESUMEN
The synthesis of a series of highly potent and selective inhibitors of cathepsin K based on the 3,4-disubstituted azetidin-2-one warhead is reported. A high degree of potency and selectivity was achieved by introducing a basic nitrogen into the distal part of the P3 element of the molecule. Data from kinetic and mass spectrometry experiments are consistent with the interpretation that compounds of this series transiently acylate the sulfhydrile of cathepsin K.
Asunto(s)
Azetidinas/farmacología , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Azetidinas/síntesis química , Azetidinas/química , Catepsina K , Catepsinas/química , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Cinética , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting approximately 1% of the adult population worldwide, with an estimated heritability of 60%. To identify genes involved in RA susceptibility, we investigated the association between putative functional single-nucleotide polymorphisms (SNPs) and RA among white individuals by use of a case-control study design; a second sample was tested for replication. Here we report the association of RA susceptibility with the minor allele of a missense SNP in PTPN22 (discovery-study allelic P=6.6 x 10(-4); replication-study allelic P=5.6 x 10(-8)), which encodes a hematopoietic-specific protein tyrosine phosphatase also known as "Lyp." We show that the risk allele, which is present in approximately 17% of white individuals from the general population and in approximately 28% of white individuals with RA, disrupts the P1 proline-rich motif that is important for interaction with Csk, potentially altering these proteins' normal function as negative regulators of T-cell activation. The minor allele of this SNP recently was implicated in type 1 diabetes, suggesting that the variant phosphatase may increase overall reactivity of the immune system and may heighten an individual carrier's risk for autoimmune disease.
Asunto(s)
Artritis Reumatoide/genética , Mutación Missense , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Fosfatasas/genética , Frecuencia de los Genes , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , ARN Mensajero/fisiologíaRESUMEN
Unraveling the functional roles of proteins is a major challenge facing the postgenome researcher. Advances towards this goal have been made through the development of both chemical and biochemical tools for monitoring protein activity. Recently, a myriad of fluorescence-based imaging tools have emerged for in vitro, in vivo and whole animal applications. These tools have provided methods to monitor the spatial and temporal distribution of proteins and bioorganic molecules dynamically. Here, recent advances in chemical and biochemical techniques that allow the detection of enzymatic activity within intact cells and in vivo are reviewed. Such technologies have the potential to be integrated into drug-development programs to facilitate both the functional validation of pharmaceutical targets and the treatment of human disease.
Asunto(s)
Enzimas/metabolismo , Microscopía Fluorescente/métodos , Espectroscopía Infrarroja Corta/métodos , Animales , Endopeptidasas/metabolismo , Enzimas/análisis , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Fosfotransferasas/metabolismoRESUMEN
Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.
Asunto(s)
Sondas Moleculares , Proteínas/química , Colorantes Fluorescentes/química , Hidrólisis , Indicadores y Reactivos/química , Péptido Hidrolasas/químicaRESUMEN
The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteómica/métodos , Animales , Evaluación Preclínica de Medicamentos , HumanosRESUMEN
The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.