Interrogating Fragments Using a Protein Thermal Shift Assay

Abstract

Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand–protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein–drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein–protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD campaign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree.

Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift

“…Considering that the Δ T m from compound binding may result in relatively small changes (1–2 °C), ,, the high assay reproducibility of NaLTSA is critical for discriminating true hits within a relatively narrow screening window ( Z ′ = 0.80 as determined with positive and negative controls, Figure a). The relative stability of Nluc-MAPK14 was 32 ± 2.1% (DMSO control; 54 °C), allowing hits to be specified as >40% stability (absent anomalous effect on the unfused Nluc control) (Figure a,b).…”

Applications of Solution NMR in Drug Discovery

“…In practice, many biophysical techniques have been used to conduct the first round of screening, which include surface plasmon resonance (SPR), thermal shift, weak affinity chromatography - mass spectroscopy (WAC-MS), X-ray crystallography, microscale thermophoresis (MST), and NMR, etc. [ 65 , 66 , 67 , 68 , 69 ]. The advantages and drawbacks of these techniques are briefly summarized in Table 1 .…”

Meltdown: A Tool to Help in the Interpretation of Thermal Melt Curves Acquired by Differential Scanning Fluorimetry

“…Thermal shift analyses may be performed in many ways; one of the more popular techniques uses a real-time polymerase chain reaction (RT-PCR) machine, and this version (known as Thermofluor or more generally as differential scanning fluorimetry [DSF]) is becoming widely used in structural biology and biophysics, driven in part by its simplicity, low cost, and the wide availability of the hardware used in the assay. Initially, these miniaturized thermal analyses were used to investigate ligand binding to a protein target, 1,2 but DSF has been adopted as a general method to assess relative protein stability. 3–5…”

“…Initially, these miniaturized thermal analyses were used to investigate ligand binding to a protein target, 1,2 but DSF has been adopted as a general method to assess relative protein stability. [3][4][5] In the DSF experiment, an RT-PCR machine is used to measure the fluorescence of an array containing different protein/fluorescent dye mixtures as they are heated.…”