Validation of an Optical Microplate Label-Free Platform in the Screening of Chemical Libraries for Direct Binding to a Nuclear Receptor

Abstract

Optical microplate-based biosensors combine the advantages of label-free detection with industry-standard assay laboratory infrastructure and scalability. A plate-based label-free platform allows the same basic platform to be used to quantify molecular interactions of macromolecules and to screen and characterize drug-like small-molecule interactions. The ligand-binding domain of orphan estrogen-related nuclear receptor-γ (ERRγ) is utilized, as a model system of a challenging type of target, to illustrate the rapid development and utility of a range of biochemical assay formats on these biosensors. Formats in which either the domain, or a peptide derived from its cognate corepressor, RIP140, were immobilized were utilized. The direct binding of small drug molecules to the domain was characterized using immobilized domain. Subsequent addition of peptide distinguished whether compounds acted as either antagonists of peptide binding, or as agonists promoting a ternary complex. The format with peptide immobilized gave a more sensitive procedure for establishing the effect of compounds on the domain-peptide interaction. Using a direct-binding format, a diverse chemical library of 1,408 compounds in DMSO was screened for ability to bind to biosensors coated with ERRγ ligand-binding domain. Hits were then characterized using the other biosensor assay formats. The standard requirements for a full primary screening campaign were fulfilled by the acceptable hit-rate, quality-performance parameters, and throughput of the direct-binding assay format. Such a format allows direct screening of targets, such as orphan receptors, without the requirement for prior knowledge of a validated ligand.

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface

“…Peptides were eluted with the following gradients: 0 -4 min, 98% A and 2% B; 4 -48 min, 2% A and 98% B; 48 -50 min, 98% A. MS 2 spectra were collected by use of collision-induced dissociation with the normalized collision energy set to 35 kV. Binding of TM Peptides to Immobilized Rho-To evaluate the binding of TM peptides to Rho, we also used a label-free assay that measures changes in the refractive index upon the addition of a ligand (58). Rho (75 g/ml) dissolved in 20 mM HEPES, pH 8.0, containing 1 mM DDM was immobilized on the surface of an EnSpire-LFB 384-well plate (a label-free biochemical sensor plate with amine coupling preactivated (PerkinElmer Life Sciences)) for 1 h at room temperature before overnight incubation at 4°C.…”

Strategies for membrane interaction proteomics: No mass spectrometry required

“…Along with numerous drugs that act on proteins at other cell membranes such as the nuclear envelope, this staggering figure confirms the importance of membrane proteins as drug targets. Although beyond the scope of this review, there is a plethora of published studies aimed at screening for novel chemical–protein interactions of this highly druggable set of proteins in order to develop new, clinically useful drugs [].…”