In This Issue Cite This: ACS Chem. Biol. 2018, 13, 495−495
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PADS GET A NEW LABEL
Protein arginine deiminases (PADs) catalyze citrullination of proteins, a post-translational modification whereby positively charged arginine side chains are converted to neutral citrulline groups. Increased PAD activity and citrullinated proteins are associated with autoimmune diseases including rheumatoid arthritis (RA). As anticitrulline antibodies appear and an immune response is mounted in RA, more PADs are released into joints, setting up a positive feedback loop. This makes PADs a possible drug target, but understanding their activity in the cell demands new tools. In this issue, Nemmara et al. (DOI: 10.1021/acschembio.7b00957) deliver next generation probes for activity-based profiling of PADs. A conserved active site cysteine becomes the covalent target for PAD-specific benzimidazole probes that also harbor an alkyne group for subsequent functionalization. In this study, the alkyne is modified with either a fluorophore for PAD detection in mammalian cell lysates or a biotin group for affinity purification. Conveniently, biotinylation experiments coupled with mass spectrometry help identify off-target proteins that react with the PAD probes, a key piece of information for drug design studies.
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Fishing out a biologically active small molecule from a chemically diverse library often involves plating test cells in microtiter wells and performing a screen for each compound’s activity individually. In contrast, modern screens involving genetic modifiers such as transgene expression, RNA interference, or CRISPR-Cas9 take advantage of viruses as individually programmable delivery vessels, allowing cultured cells to be grown together. These technologies combined with the advent of economical DNA synthesis and sequencing make ambitious pooled genetic screens possible. Now, Yozwiak et al. (DOI: 10.1021/acschembio.8b00043) borrow from the genetic toolkit to develop a pooled chemical screening framework. In lieu of a virus, they turn to functionalized silica microparticles that can be internalized into cells to deliver their small molecule cargo. Triggering apoptosis is the goal of the screen, so the particles have a quenched fluorophore as a sensor for caspase proteolytic activity by virtue of a cleavable linker. Fluorescent cells are those that internalized biologically active small molecules and are selected by cell sorting. Finally, an oligonucleotide is covalently attached to the particles as a barcode for identifying selected compounds.
INSTALLING THIOESTERS IN PROTEINS
Thioesters are at the heart of many biochemical pathways, from the Krebs cycle to cell wall synthesis, yet the natural catalog of amino acids lacks a member with a thioester group. For protein chemists, thioesters are key reactants for chemical synthesis or semisynthesis of proteins. Nature even has its own version of this synthetic route with intein splicing. Now, Xuan et al. (DOI: 10.1021/acschembio.7b00998) give the thioester its debut as an amino acid side chain via sitespecific incorporation of a non-natural amino acid. Their starting point was the tRNA and charging enzyme for pyrrolysine, a natural amino acid found in some species of archaea and bacteria. After randomizing several active site residues, a screen in E. coli uncovers a mutant synthetase that can charge a suppressor tRNA with a thioester-containing aspartic acid derivative, ThioD. The study goes on to show that a recombinant GFP containing ThioD can be readily modified with β-aminothiol derivatives, opening up the door for a variety of new modifications or branched proteins. © 2018 American Chemical Society
SMALL MOLECULES GET IN THE POOL
Published: March 16, 2018 495
DOI: 10.1021/acschembio.8b00207 ACS Chem. Biol. 2018, 13, 495−495
