Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions

Abstract

Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.

Fluorescence-Based Protein Stability Monitoring—A Review

“…An interesting approach to combine good properties of DSF and ICD was recently introduced by using a FRET-Probe [162]. This assay is suitable mainly for PLI studies, and unlike typical ICD, it is performed in a single optimized denaturant concentration following the denaturation process over time.…”

“…Alcohols such as ethanol, methanol, propanol, and butanol have been proposed for the chemical denaturation of proteins due to their high content of hydrocarbon and water miscibility. These properties facilitate the unfolding of native structures at a relatively low dose [162]. Less used denaturants in the context of ICD are pH and SDS.…”

“…SDS is often used as a denaturant in gel electrophoresis, but its chemical nature as a surfactant makes SDS difficult for ICD due to the formation of micelles. The buffer pH is known to have a significant effect on protein stability, which needs to be additionally considered in all assay designs [162]. In specific cases, investigations at various pH levels can give valuable information, as shown, for example, with cytochrome c (cyt c).…”

“…ICD has a limited sensitivity and micromolar range which is similar to DSF-type assays. However, whilst DSF is performed in a single condition, ICD measurements are made by titrating the denaturant using varying concentrations for a single experiment, increasing the protein consumption significantly [162]. It has been suggested that a combination of DSF and ICD would be feasible to reduce the protein amount required to assess the physical stability in various formulations but still provide a sufficient prediction quality [180].…”

“…It has been suggested that a combination of DSF and ICD would be feasible to reduce the protein amount required to assess the physical stability in various formulations but still provide a sufficient prediction quality [180]. Indeed, assays performed with the FRET-Probe using a single denaturant concentration supports this idea [162]. A comparison of the sensitivity of DSF and ICD in detecting the ligand binding of reduced nicotinamide adenine dinucleotide (NADH) with malate dehydrogenase not only validates ICD as a reliable method for screening for ligands, but also indicates that, in some cases, ICD is more sensitive than DSF at detecting binding at lower concentrations of ligands [181].…”