ABSTRACT
The multi-component Ugi reaction has been employed to assemble a small library of affinity-based probes (AfBPs) that target potential protein tyrosine phosphatases. The probes showed good labelling of PTP1B and MptpB, and were subsequently used to label endogenous PTP1B in MCF-7 cell lysates.
Subject(s)
Affinity Labels/chemical synthesis , Affinity Labels/metabolism , Drug Design , Protein Tyrosine Phosphatases/metabolism , Affinity Labels/chemistry , Cell Line, Tumor , Humans , KineticsABSTRACT
Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.
Subject(s)
Proteins/chemistry , Proteins/metabolism , Humans , Molecular Structure , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Array Analysis , Protein Kinases/metabolism , Proteome/chemistryABSTRACT
Synthesis of a novel unnatural amino acid (2-FMPT) for the solid-phase synthesis of peptide-based probes suitable for target-specific activity-based profiling of protein tyrosine phosphatases from crude proteomes is reported.
Subject(s)
Peptides/chemistry , Peptides/metabolism , Phosphotyrosine/analogs & derivatives , Protein Tyrosine Phosphatases/metabolism , Proteomics , Animals , Cell Line , Humans , Molecular Structure , Proteome/metabolism , Substrate SpecificityABSTRACT
A approximately 3500-member library of bidentate inhibitors against protein tyrosine phosphatases (PTPs) was rapidly assembled using click chemistry. Subsequent high-throughput screening had led to the discovery of highly potent (K(i) as low as 150 nM) and selective MptpB inhibitors, some of which represent the most potent MptpB inhibitors developed to date.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Protein Tyrosine Phosphatases/antagonists & inhibitors , Small Molecule Libraries , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A key challenge in current drug discovery is the development of high-throughput (HT) amenable chemical reactions that allow rapid synthesis of diverse chemical libraries of enzyme inhibitors. The Cu(I)-catalyzed, 1,3-dipolar cycloaddition between an azide and an alkyne, better known as "click chemistry", is one such method that has received the most attention in recent years. Despite its popularity, there is still a lack of robust and efficient chemical strategies that give access to diverse libraries of azide-containing building blocks (key components in click chemistry). We report herein a highly robust and efficient strategy for high-throughput synthesis of a 325-member azide library. The method is highlighted by its simplicity and product purity. The utility of the library is demonstrated with the subsequent "click" synthesis of the corresponding bidentate inhibitors against PTP1B.
Subject(s)
Azides/chemical synthesis , Small Molecule Libraries/chemical synthesis , Azides/pharmacology , Combinatorial Chemistry Techniques , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Molecular Structure , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Small Molecule Libraries/pharmacologyABSTRACT
We report herein a novel phosphopeptide microarray capable of noncovalently "trapping" catalytically inactive mutants of protein tyrosine phosphatases (PTPs), and its application in high-throughput determination of PTP substrate specificity.
Subject(s)
Biocatalysis , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Phosphopeptides/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Drug Design , Enzyme Inhibitors/pharmacology , Humans , Mutant Proteins/antagonists & inhibitors , Phosphopeptides/chemistry , Protein Array Analysis , Protein Tyrosine Phosphatases/antagonists & inhibitors , Substrate SpecificityABSTRACT
A highly efficient solid-phase strategy for assembly of small molecule inhibitors against protein tyrosine phosphatase 1B (PTP1B) is described. The method is highlighted by its simplicity and product purity. A 70-member combinatorial library of analogues of a known PTP1B inhibitor has been synthesized, which upon direct in situ screening revealed a potent inhibitor ( Ki = 7.0 microM) against PTP1B.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Amides/chemistry , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Time FactorsABSTRACT
This protocol details methodologies for the site-specific biotinylation of proteins using in vitro, in vivo and cell-free systems for the purpose of fabricating functional protein arrays. Biotinylation of recombinant proteins, in vitro as well as in vivo, relies on the chemoselective reaction between cysteine-biotin and a reactive thioester group at the C-terminus of a protein generated via intein-mediated cleavage. The cell-free system utilizes low concentrations of biotin-conjugated puromycin. Unlike other approaches that require tedious and costly downstream steps of protein purification, C-terminal biotinylated proteins can be captured directly onto avidin-functionalized slides from a mixture of other cellular proteins to generate the corresponding protein array. These methods were designed to maintain the integrity and activity of proteins in a microarray format, which potentially allows simultaneous functional assays of thousands of proteins. Assuming that the target proteins have been cloned into the expression vector, transformation of bacterial strain and growth of starter culture would take approximately 2 days. Expression and in vitro protein purification and biotinylation will take approximately 3 days whereas the in vivo method would take approximately 2 days. The cell-free protein biotinylation strategy requires only 6-8 h.
Subject(s)
Biotin/chemistry , Protein Array Analysis/methods , Proteins/chemistry , Cell-Free System , Cysteine/chemistry , Proteins/isolation & purificationABSTRACT
We review intein-mediated approaches for the site-specific modifications of proteins and highlight their applications in (1) the site-specific in vitro and in vivo biotinylation of proteins for protein arrays and (2) the site-specific in vivo labeling of proteins in living cells.
Subject(s)
Inteins/physiology , Protein Modification, Translational/physiology , Proteins/chemistry , Biotin/chemistry , Biotinylation/methods , Cell-Free System , Green Fluorescent Proteins/chemistry , Protein Array Analysis/methods , Protein Engineering/methods , Proteomics/methodsABSTRACT
One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. We previously developed intein-mediated strategies for protein biotinylation and site-specific protein microarray generation. Herein, we report new findings of these strategies, which improve the biotinylation efficiency of proteins by up to 10-folds.
Subject(s)
Inteins , Proteins/chemistry , Biotin/chemistry , Biotin/metabolism , Biotinylation , Cloning, Molecular , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Inteins/physiology , Protein Array Analysis/methods , Proteins/genetics , Proteins/metabolismABSTRACT
This review focuses on recent developments in microarray-based technologies for high-throughput screenings of enzymes. Novel methods of protein immobilization, detection of enzymatic activities, and inhibitions were highlighted.
Subject(s)
Enzymes/chemistry , Oligonucleotide Array Sequence Analysis , Amino Acid Sequence , Enzymes/geneticsABSTRACT
We present a new approach to site-specifically biotinylate protein in a cell-free protein synthesis system with puromycin-containing small molecules. With this new method, biotinylated proteins were generated from the DNA templates in a matter of hours, making it useful for protein microarray generation. We also validated that the method is compatible with other high-throughput cloning/proteomics methods.
Subject(s)
Biotinylation/methods , Proteins/genetics , Proteins/metabolism , Binding Sites/physiology , Proteins/chemical synthesisABSTRACT
Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.