Bead-based screening in chemical biology and drug discovery.

High-throughput screening is an important component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds requires assays amenable to miniaturisation and automization. Combinatorial chemistry holds a unique promise to deliver structurally diverse libraries for early drug discovery. Among the various library forms, the one-bead-one-compound (OBOC) library, where each bead carries many copies of a single compound, holds the greatest potential for the rapid identification of novel hits against emerging drug targets. However, this potential has not yet been fully realized due to a number of technical obstacles. In this feature article, we review the progress that has been made in bead-based library screening and its application to the discovery of bioactive compounds. We identify the key challenges of this approach and highlight key steps needed for making a greater impact in the field.

Oxidative Modification of Tryptophan-Containing Peptides.

We herein present a broadly useful method for the chemoselective modification of a wide range of tryptophan-containing peptides. Exposing a tryptophan-containing peptide to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) resulted in a selective cyclodehydration between the peptide backbone and the indole side chain of tryptophan to form a fully conjugated indolyl-oxazole moiety. The modified peptides show a characteristic and significant emission maximum at 425 nm, thus making the method a useful strategy for fluorescence labeling.

A selectivity filter at the intracellular end of the acid-sensing ion channel pore.

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the "GAS belt" in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel.

Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition.

A photolabile linker for the solid-phase synthesis of peptide hydrazides and heterocycles.

A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino acids, including those with side-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis.

Bacteria-triggered release of antimicrobial agents.

Medical devices employed in healthcare practice are often susceptible to microbial contamination. Pathogenic bacteria may attach themselves to device surfaces of catheters or implants by formation of chemically complex biofilms, which may be the direct cause of device failure. Extracellular bacterial lipases are particularly abundant at sites of infection. Herein it is shown how active or proactive compounds attached to polymeric surfaces using lipase-sensitive linkages, such as fatty acid esters or anhydrides, may be released in response to infection. Proof-of-concept of the responsive material is demonstrated by the bacteria-triggered release of antibiotics to control bacterial populations and signaling molecules to modulate quorum sensing. The self-regulating system provides the basis for the development of device-relevant polymeric materials, which only release antibiotics in dependency of the titer of bacteria surrounding the medical device.

Solid-phase synthesis of structurally diverse heterocycles by an amide-ketone condensation/N-acyliminium pictet-spengler sequence.

An efficient approach for the solid-phase synthesis of structurally diverse heterocyclic compounds is presented. Under acidic reaction conditions, peptidic levulinamides undergo intramolecular ketone-amide condensation reactions to form cyclic N-acyliminium intermediates. In the presence of a tethered nucleophile, a second cyclization reaction results in the formation of a fused bicyclic ring system. The scope of the methodology was demonstrated by several combinations of substituted ketones and nucleophiles, the latter conveniently originating from amino acids with functionalized side chains, such as tryptophan, substituted phenylalanines, and cysteine. The cyclization sequence provides diastereomerically pure products in high yields. In one extension of the methodology, the resulting relative stereochemistry of the products enables the formation of bridged ring systems by a unique cyclative release mechanism.