RESUMO
BACKGROUND: The central role of proteins in diseases has made them increasingly attractive as therapeutic targets and indicators of cellular processes. Protein microarrays are emerging as an important means of characterising protein activity. Their accurate downstream analysis to produce biologically significant conclusions is largely dependent on proper pre-processing of extracted signal intensities. However, existing computational tools are not specifically tailored to the nature of these data and lack unanimity. RESULTS: Here, we present the single-channel Protein Microarray Analysis Pipeline, a tailored computational tool for analysis of single-channel protein microarrays enabling biomarker identification, implemented in R, and as an interactive web application. We compared four existing background correction and normalization methods as well as three array filtering techniques, applied to four real datasets with two microarray designs, extracted using two software programs. The normexp, cyclic loess, and array weighting methods were most effective for background correction, normalization, and filtering respectively. CONCLUSIONS: Thus, here we provided a versatile and effective pre-processing and differential analysis workflow for single-channel protein microarray data in form of an R script and web application ( https://metaomics.uct.ac.za/shinyapps/Pro-MAP/ .) for those not well versed in the R programming language.
Assuntos
Análise Serial de Proteínas , Software , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linguagens de Programação , Fluxo de Trabalho , Perfilação da Expressão Gênica/métodosRESUMO
In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.
Assuntos
Proteínas de Ligação a DNA/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Escherichia coli/metabolismo , Peptídeo Sintases/química , Peptídeos/química , Fatores de Transcrição/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Cristalografia por Raios X , Receptores ErbB/metabolismo , Proteínas de Escherichia coli/química , Etilmaleimida/química , Biblioteca Gênica , Células HEK293 , HIV , Células HeLa , Humanos , Concentração Inibidora 50 , Iodoacetamida/química , Peptídeo Hidrolases/química , Plasmídeos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Especificidade por SubstratoRESUMO
Cyclic peptides are an emerging class of molecular therapeutics that are increasingly viewed as ideal backbones for modulation of protein-protein interactions. A split-intein based method, termed SICLOPPS, enables the rapid generation of genetically encoded cyclic peptide libraries of around a hundred million members. Here we review recent approaches using SICLOPPS for the discovery of bioactive compounds.
Assuntos
Peptídeos Cíclicos/química , Descoberta de Drogas , Biblioteca de Peptídeos , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 µM by binding to UEV with a Kd of 11.9 ± 2.8 µM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of â¼2 µM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenvolvimento de Medicamentos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Escherichia coli/genética , Células HEK293 , HIV/química , HIV/efeitos dos fármacos , Células HeLa , Humanos , Peptídeos Cíclicos/toxicidade , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Liberação de Vírus/efeitos dos fármacosRESUMO
The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-protein interactions. Here we discuss a range of methods used for the creation of cyclic peptide libraries and detail examples of their successful implementation.