Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes.

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

Site-specific covalent labeling of proteins inside live cells using small molecule probes.

The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.

A proteomic strategy for the identification of caspase-associating proteins.

We report the efficient in vivo labelling of caspases expressed inside apoptotic HeLa cells using fluoromethyl ketone (fmk)-containing probes; preliminary results indicated that these probes may be used to identify caspase-associating proteins.

Array-based technologies and their applications in proteomics.

Latest microarray-based technologies, including small molecule-, peptide-, protein- and cell-based arrays, and their applications in the field of proteomics are reviewed.

Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases.

Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P(1) position. It also allows efficient synthesis of vinyl sulfone-containing, activity-based probes of cysteine proteases used in a proteomic experiment.

High expression of a sucrose non-fermenting (SNF1)-related protein kinase from Colletotrichum gloeosporoides f. sp. malvae is associated with penetration of Malva pusilla.

A sucrose non-fermenting (SNF1)-related protein kinase homologue, cgsnf, from Colletotrichum gloeosporoides f. sp. malvae, a hemibiotrophic fungal pathogen of round-leaved mallow (Malva pusilla) was examined. During infection, cgsnf showed a large peak in expression relative to a constitutively expressed fungal actin gene when appressoria had formed during the penetration phase and then showed much lower expression levels during subsequent necrotrophic growth in the host. In pure culture with glucose or glycerol as sole carbon sources, expression levels were similar to that during necrotrophic growth. Expression was consistently higher in glycerol than in glucose cultures, which may reflect a lower cellular energy status in the fungus. These results are consistent with cgsnf having a role in transmitting nutritional signals, which may be involved with host penetration.

Recent advances in gel-based proteome profiling techniques.

This review focuses on recent developments in gel-based proteomics techniques. By combining traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoretic techniques with recent advances in protein labeling using different classes of molecules (i.e., fluorescent dyes, chemical probes, radioisotopes), new technologies have been developed that allow for high-throughput studies of proteins at the whole-proteome scale.

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.

Cell-permeable small molecule probes for site-specific labeling of proteins.

We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.

Site-specific immobilization of biotinylated proteins for protein microarray analysis.

The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide. However, the binding between Ni-NTA and His tag proteins is not strong, causing the immobilized proteins to dissociate from the slide even under simple wash conditions. The authors have developed a novel strategy of using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. Array-scan results not only show successful immobilization of proteins onto slides by antibody detection method but also full retention of biological activities of the immobilized proteins. The strong and specific interaction between biotin and avidin also permits the use of stringent incubation and washing conditions on the protein microchip, thus making it a highly robust method for array studies.

Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays.

The massive throughput offered by array-based technologies can only be realized with the development of equally powerful strategies that offer reproducible consistency. The competence of arrays and efficacy of screening come under scrutiny, with most existing immobilization schemes that do not site-specifically ligate peptides on the arrays. Thus, it is crucial in array-based experiments to orientate peptides in an ordered and uniform fashion. Two new approaches were developed for the directed immobilization of peptides on a microarray, by exploiting measures involving native chemical ligation reactions as well as biotin-streptavidin interactions. This makes it possible to stably immobilize peptides in a consistent manner and in a predetermined orientation on the microarray. The first scheme employs glass slides that are functionalized with avidin for attachment of terminally biotinylated peptides. The second uses slides containing thioester moieties to ligate N-terminal cysteine containing peptides. The authors successfully immobilized peptides on chip using these strategies, and, in extending their method to the study of kinase activity on microarrays, they also developed a novel detection scheme that abrogates the dependence on traditional radioactivity-based kinase screening assays. This method employs fluorescently labeled antiphosphoserine and antiphosphotyrosine antibodies in assessing and monitoring kinase activity on arrays. The above methodologies provide for a fast and sensitive approach with which to conveniently assess kinase activity using peptide microarrays.

A sensitive two-photon probe to selectively detect monoamine oxidase B activity in Parkinson's disease models.

The unusually high MAO-B activity consistently observed in Parkinson's disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level.

Recent advances in microarray technologies for proteomics.

Proteins are fundamental components of all living systems and critical drivers of biological functions. The large-scale study of proteins, their structures and functions, is defined as proteomics. This systems-wide analysis leads to a more comprehensive view of the intricate signaling transduction pathways that proteins engage in and improves the overall understanding of the complex processes supporting the living systems. Over the last two decades, the development of high-throughput analytical tools, such as microarray technologies, capable of rapidly analyzing thousands of protein-functioning and protein-interacting events, has fueled the growth of this important field. Herein, we review the most recent advancements in microarray technologies, with a special focus on peptide microarray, small molecule microarray, and protein microarray. These technologies have become prominent players in proteomics and have made significant changes to the landscape of life science and biomedical research. We will elaborate on their performance, advantages, challenges, and future directions.

One- and two-photon live cell imaging using a mutant SNAP-Tag protein and its FRET substrate pairs.

A small molecule-assisted protein labeling strategy based on a mutant SNAP-Tag (mSNAP) and its FRET substrate pairs has been developed. Both one- and two-photon fluorescence microscopic experiments were successfully demonstrated in living cells.

Chemical modification and organelle-specific localization of orlistat-like natural-product-based probes.

Orlistat, also known as tetrahydrolipstatin (THL), is an FDA-approved anti-obesity drug with potential anti-cancer activity. Previously, we developed a chemical proteomic approach, based on the Orlistat-like probe (1a) for large-scale identification of unknown cellular targets of Orlistat in human hepatocytes. In this article, we report the chemical synthesis and biological evaluation of an expanded set of Orlistat-like compounds, with the intention to further dissect and manipulate potential cellular targets of Orlistat. In doing so, we carried out proteome-wide activity-based profiling and large-scale pull-down/LCMS analysis of these compounds in live HepG2 cells, and successfully identified many putative cellular targets for Orlistat and its structural analogues. By qualitatively assessing the spectra counts of potential protein hits against each of the seventeen Orlistat analogues, we obtained both common and unique targets of these probes. Our results revealed that subtle structural modifications of Orlistat led to noticeable changes in both the cellular potency and target profiles of the drug. In order to further improve the cellular activity of Orlistat, we successfully applied the well-established AGT/SNAP-tag technology to our cell-permeable, benzylguanine (BG)-containing Orlistat variant (4). We showed that the drug could be delivered and effectively retained in different sub-cellular organelles of living cells. This strategy may provide a general and highly effective chemical tool for the potential sub-cellular targeting of small molecule drugs.

Nanodroplet profiling of enzymatic activities in a microarray.

We describe a generic method for the large-scale functional characterization of enzymes in a microarray. Poly-l-lysine and amine reactive slides were coated with fluorogenic substrates sensitive to proteases and phosphatases. Patterning enzymes on the slides by robotic printing produced spatially addressable, segregated droplets that were simultaneously exposed to the on-chip sensors. Multiple enzymes were profiled using this system that provided fluorescence readouts across temporal and stoichiometric dimensions concurrently on a single microarray substrate. This integrated microarray platform is applicable not only for the functional annotation of proteins, but also for the rapid agonist and antagonist discovery and in performing on-chip kinetics.