Synthetic Approaches of Epoxysuccinate Chemical Probes.

Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity-based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl-epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain-like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity- or affinity-based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate-based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.

Dual-light control of nanomachines that integrate motor and modulator subunits.

A current challenge in the field of artificial molecular machines is the synthesis and implementation of systems that can produce useful work when fuelled with a constant source of external energy. The first experimental achievements of this kind consisted of machines with continuous unidirectional rotations and translations that make use of 'Brownian ratchets' to bias random motions. An intrinsic limitation of such designs is that an inversion of directionality requires heavy chemical modifications in the structure of the actuating motor part. Here we show that by connecting subunits made of both unidirectional light-driven rotary motors and modulators, which respectively braid and unbraid polymer chains in crosslinked networks, it becomes possible to reverse their integrated motion at all scales. The photostationary state of the system can be tuned by modulation of frequencies using two irradiation wavelengths. Under this out-of-equilibrium condition, the global work output (measured as the contraction or expansion of the material) is controlled by the net flux of clockwise and anticlockwise rotations between the motors and the modulators.

Luminogenic "clickable" lanthanide complexes for protein labeling.

Development of lanthanide-based luminescent "switch-on" systems via azide-alkyne [3+2] cycloaddition is described. We used these for non-specific protein labeling and as tags for specific and selective activity-based protein labeling.

A general solid phase method for the synthesis of sequence independent peptidyl-fluoromethyl ketones.

We present here a new, general, solid phase strategy for the synthesis of sequence independent peptidyl-fluoromethyl ketones using standard Fmoc peptide chemistry. Our method is based on the synthesis of bifunctional linkers which allows the incorporation of amino acid fluoromethyl ketone unit at the C-terminal end of peptide sequences. Application of this approach for the synthesis of activity based probes for SENPs is also described.

Glycine fluoromethylketones as SENP-specific activity based probes.

We report here the synthesis and biochemical properties of a new peptidyl activity-based probe 1 for SUMO proteases, SENPs. The activity-based probe has at its C terminus a glycine-derived fluoromethylketone moiety as a reactive group designed to target the active-site cysteine of SENPs. Based on a study of the interactions between SENPs and SUMOs, we introduced further design elements that allow the activity-based probe to selectively target SENPs at low micromolar to high nanomolar concentrations. Moreover, 1 out-competes SUMO1 from the reversible SUMO1-SENP1 complex, thus suggesting that 1 and SUMO1 share a common binding site on SENP1.

Metal ion mediated self-assembly directed formation of protein arrays.

Self-assembled inorganic-protein arrays with well-defined and controllable size and structure were obtained through the Fe(II) complexation of protein-conjugated terpyridine units (ligand). The atom-level control of the ligand is obtained through residue-specific conjugation between the complexing unit (terpy) containing an activity-based probe and a corresponding active enzyme (papain). The Fe(II)-based self-assembly performed on this unique building block (ligand) leads to chemical species of unprecedented constitution. The first example presented herein opens the way to a shape and size regime usually reserved to polymers.

Protein-inorganic array construction: design and synthesis of the building blocks.

Herein we describe the design and synthesis of the first series of di-functional ligands for the directed construction of inorganic-protein frameworks. The synthesized ligands are composed of a metal-ion binding moiety (terpyridine-based) conjugated to an epoxysuccinyl peptide, known to covalently bind active cysteine proteases through the active-site cysteine. We explore and optimize two different conjugation chemistries between the di-functionalized metal-ion ligand and the epoxysuccinyl-containing peptide moiety: peptide-bond formation (with limited success) and Cu(I)-catalysed click chemistry (with good results). Further, the complexation of the synthesized ligands with Fe(II) and Ni(II) ions is investigated: the di-functional ligands are confirmed to behave similarly to the parent terpyridine. As designed, the peptidic moiety does not interfere with the complexation reaction, in spite of the presence of two triazole rings that result from the click reaction. ES-MS together with NMR and UV/Vis studies establish the structure, the stoichiometry of the complexation reactions, as well as the conditions under which chemically sensitive peptide-containing polypyridine ligands can undergo the self-assembly process. These results establish the versatility of our approach and open the way to the synthesis of di-functional ligands containing more elaborated polypyridine ligands as well as affinity labels for different enzyme families. As such, this paper is the first step towards the construction of robust supramolecular species that cover a size-regime and organization level previously unexplored.

Identification of twinfilin-2 as a factor involved in neurite outgrowth by RNAi-based screen.

Using RNA interference (RNAi) to suppress gene expression, we attempted to identify tyrosine kinases involved in the extension of neurites from SH-SY5Y cells. A comprehensive analysis of gene "knock-down" profiles with small interfering RNAs (siRNAs) revealed candidate proteins that might control neurite extension. Phenotype-based screening of differentiating SH-SY5Y cells following retinoic acid (RA) stimulation indicated that twinfilin-2 is a protein that is involved in neurite outgrowth, as confirmed by morphological analysis of twinfilin-2-overexpressing cells.

Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined.

On-chip transfection of PC12 cells based on the rational understanding of the role of ECM molecules: efficient, non-viral transfection of PC12 cells using collagen IV.

Transfection microarrays (TMA) are important emerging tools for the study of genetic events in living cells in a high-throughput fashion and with significant material economy. However, the difficulty to transfect various relevant cell types on-chip hinders the use of TMAs. Herein we present the realization of a transfection microarray applicable to PC12 cells that heavily relies on the use of ECM molecules. Collagen IV and at a lesser extent laminin or collagen I, but not fibronectin or poly-l-lysine were found to significantly increase the solution-phase as well as on-chip transfection efficiency of PC12 cells. The highest transfection efficiency obtained was consistently above 60%. The observed correlations between the transfection efficiencies and the differential adhesion-induced events triggered by the studied ECMs provides the basis for the rationalization of the role of ECMs on the transfection process.

Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown.

The transfection efficiency of primary cells is the bottleneck for their use with miniaturized formats for gene validation assays. We have found that when formulations containing various reporter plasmids were microarrayed on glass slides (chips), hMSCs cultivated on the chip incorporated and expressed the microarrayed plasmid DNAs with high efficiency and virtually total spatial resolution. Fibronectin, as the key formulation component, was found to significantly increase the on-chip transfection efficiency in hMSCs as well as many other cells. Further, we have conclusively proven that when siRNA was co-arrayed with the target plasmid DNA, a concentration-dependent gene knockdown was observed. Thus, massively miniaturized RNAi gene knockdown experiments can now be performed in primary cells, previously unusable with transfection microarrays (TMA).

Immobilized culture of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified surface.

Microarrays of living cells are an emerging tool in systems such as reverse transfection. These studies are limited to adherent cells partly because of the difficulty of cell immobilization. Using a newly developed reagent, the biocompatible anchor for membrane (BAM), we show herein the rapid and strong attachment of living nonadherent cells and adherent cells on BAM-modified surfaces. Normal cellular growth was observed for over 7 days on BAM-modified surfaces. We expect this methodology to greatly expand the scope of current cell microarray technology.