Thermal unfolding methods in drug discovery

Abstract

Thermal unfolding methods, applied in both isolated protein and cell-based settings, are increasingly used to identify and characterize hits during early drug discovery. Technical developments over recent years have facilitated their application in high-throughput approaches, and they now are used more frequently for primary screening. Widespread access to instrumentation and automation, the ability to miniaturize, as well as the capability and capacity to generate the appropriate scale and quality of protein and cell reagents have all played a part in these advances. As the nature of drug targets and approaches to their modulation have evolved, these methods have broadened our ability to provide useful chemical start points. Target proteins without catalytic function, or those that may be difficult to express and purify, are amenable to these methods. Here, we provide a review of the applications of thermal unfolding methods applied in hit finding during early drug discovery.

FIG. 1.

Technologies that can be utilized to monitor the thermal unfolding of isolated protein samples. Several technologies can be utilized to monitor the thermal unfolding of isolated protein samples. Differential scanning calorimetry (DSC) detects the change in enthalpy of the transition between folded and unfolded states and is the most direct method; however, it is only capable of low throughput analysis. nanoDSF can analyze up to 48 samples in parallel and measures the intrinsic fluorescence of tryptophan residues within the protein structure as their exposure to the environment changes during protein unfolding. Differential scanning fluorimetry (DSF), while less direct, is capable of higher sample throughput and can be used to analyze samples prepared in a multi-well plate format. This dye-based method can be used to simultaneously analyze up to 384 or 1536 samples using multi-well plate-compatible thermocycler/PCR instruments. This form of DSF measures the extrinsic fluorescence generated by the inclusion of environmentally sensitive dyes such as Sypro^® Orange, which bind to exposed hydrophobic regions of the protein structure during unfolding.

FIG. 2.

(a) Typical dye-based DSF thermal denaturation curve for protein sample. At the starting temperature, the protein is present in its folded, native state. Fluorescence emission increases with temperature as the protein unfolds and SYPRO^® Orange co-operatively binds to exposed hydrophobic residues. The melting temperature (Tm) (dotted line) can be defined as the midpoint of the transition, or the temperature at which 50% of the protein is unfolded. Fluorescence decreases after the peak is largely attributed to protein aggregation causing shielding and exclusion of dye from hydrophobic residues. (b) Thermal denaturation profile of protein showing two distinct transitions (T_m1 and T_m2) indicating the presence of multiple structural domains. While T_m1 remains the same as in the native protein, T_m2 shifts to a higher temperature in the presence of a ligand, offering information about potential binding site location. (c) Thermal denaturation profiles of a protein in the absence and presence of a ligand. Ligand presence causes the Tm to shift positively (positive ΔT_m) indicative of thermal stabilization due to binding. Raw fluorescence emission is plotted as a function of temperature. (d) First derivative of thermal denaturation curve plotted as a function of temperature. The T_m (dotted line) can also be defined as the inflection point, or peak, of the first derivative trace of the thermal shift curve, the temperature at which the steepest increase in fluorescence signal occurs.

FIG. 3.

A range of technologies can be used to detect unfolding of the protein target(s) of interest for CETSA. Detection of the endogenous protein is enabled by mass spectrometry or antibody-based methods, whilst detection of genetically tagged target is most often based on NanoLuciferase technologies. Left to right images represent the target detection by (1) Western blot, (2) AlphaScreen anti-target antibody pairs, (3) immunofluorescence microscopy, (4) mass spectrometry, (5) acoustically dispensed reverse phase protein array, (6) TIRF of anti-target antibody, bound to immobilized target, released following single cell lysis within a microfluidic device, (7) monitoring of NanoLuciferase formed following the binding of LgBiT to the HiBiT-tagged target, and (8) monitoring of NanoLuciferase engineered to be thermostable using a Gly-Ser linker, and thus suitable for real time CETSA. (b) When applying CETSA to drug discovery, the concentration of the compound, the temperature of the melt event, or the treatment of the cells may all be varied to elucidate further information about the compound's mechanism of action.