Ugi reaction-assisted rapid assembly of affinity-based probes against potential protein tyrosine phosphatases.

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.

Current chemical biology tools for studying protein phosphorylation and dephosphorylation.

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.

Peptide-based activity-based probes (ABPs) for target-specific profiling of protein tyrosine phosphatases (PTPs).

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.

High-throughput discovery of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitors using click chemistry.

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.

High-throughput synthesis of azide libraries suitable for direct "click" chemistry and in situ screening.

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).

High-throughput screening of catalytically inactive mutants of protein tyrosine phosphatases (PTPs) in a phosphopeptide microarray.

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.

Solid-phase assembly and in situ screening of protein tyrosine phosphatase inhibitors.

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.

Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays.

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.

Intein-mediated, in vitro and in vivo protein modifications with small molecules.

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.

Improving the intein-mediated, site-specific protein biotinylation strategies both in vitro and in vivo.

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.

Application of microarrays in high-throughput enzymatic profiling.

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.

Expanding the scope of site-specific protein biotinylation strategies using small molecules.

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.

Strategies for immobilization of biomolecules in a microarray.

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.