Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted β-Secretase Using Single-Molecule Imaging

Abstract

Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against β-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds.

Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary Constraints

“…In addition, the simulations revealed that the structural rearrangements required for ligand entry and exit are significantly different for GR and MR. As a consequence, the ligand binding kinetics should differ for the two receptors. Using both SPR and SMM (Gunnarsson et al, 2015), we measured the residence time of both dexamethasone and dibC by monitoring the time-resolved change in receptor binding to a surface-immobilized co-regulator peptide upon addition of >10-fold concentration excess of a reference compound ( Figure S6). The data from all experiments are summarized in Table 2.…”

Hydrolase-Like Activity Provided by Zinc(II) and Oleoyl-Histidine at Liposome Membrane Surface

“…The kinetic parameter obtained from conventional approaches (e.g., Lineweaver-Burk plot) might be different from the correct one [24]. A single-molecule assay will also shed a light into the enzymatic reaction at the membrane interface [25]. Such approaches will contribute to the investigation of the true role of functional ligands incorporated into liposome membranes.…”

Biophysics in drug discovery: impact, challenges and opportunities

“…Although the well-known single-molecule patch clamp technique has been used in drug discovery since the 1970s, the application of other singlemolecule experiments in drug discovery is scarce owing to the complexity of experiments and the limitations of the methods 101 . Further developments -including higher-throughput detection on microfluidic chips 102 , progress in fluorophore chemistry, and technical improvement of instrumentation, software for data collection and analysis -are still needed to enable wider application of single-molecule technologies in an industrial environment.…”