In vivo affinity maturation of the CD4 domains of an HIV-1-entry inhibitor.

Human proteins repurposed as biologics for clinical use have been engineered through in vitro techniques that improve the affinity of the biologics for their ligands. However, the techniques do not select against properties, such as protease sensitivity or self-reactivity, that impair the biologics' clinical efficacy. Here we show that the B-cell receptors of primary murine B cells can be engineered to affinity mature in vivo the human CD4 domains of the HIV-1-entry inhibitor CD4 immunoadhesin (CD4-Ig).

Surveying GPCR solubilisation conditions using surface plasmon resonance.

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor.

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response.

A mutant O-GlcNAcase enriches Drosophila developmental regulators.

Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis.

Discovery of New Bromodomain Scaffolds by Biosensor Fragment Screening.

The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains.

Surface plasmon resonance analysis of seven-transmembrane receptors.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for around a third of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. Current receptor assay systems are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding.

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization.

A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues.

O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis.

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor.