Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

Abstract

The review highlights how protein–protein interactions (PPIs) have determining roles in most life processes and how interactions between protein partners are involved in various human diseases. The study of PPIs and binding interactions as well as their understanding, quantification and pharmacological regulation are crucial for therapeutic purposes. Diverse computational and analytical methods, combined with high-throughput screening (HTS), have been extensively used to characterize multiple types of PPIs, but these procedures are generally laborious, long and expensive. Rapid, robust and efficient alternative methods are proposed, including the use of Microscale Thermophoresis (MST), which has emerged as the technology of choice in drug discovery programs in recent years. This review summarizes selected case studies pertaining to the use of MST to detect therapeutically pertinent proteins and highlights the biological importance of binding interactions, implicated in various human diseases. The benefits and limitations of MST to study PPIs and to identify regulators are discussed.

Development of Receptor for Advanced Glycation End Products (RAGE) ligands through target directed dynamic combinatorial chemistry: a novel class of possible antagonists

“…We next used microscale thermophoresis (MST) [38][39] to evaluate the affinities of our DCC hits for our target receptor, as indicated in Table 1. Some interesting findings revealed the fact that A 2 H 1 and A 2 H 4 N-acylhydrazones bearing Azeliragon's fragments, seem to present a similar affinity, compared to our reference (Entry 14, Table 1), with K d for RAGE of 38 μM and 49.9 μM, respectively (Entries 1 and 2, Table 1), proportional with their docking score of À 7.2 kcal/mol and À 6.9 kcal/mol (Figure S18 and S20).…”

Applications of the Microscale Thermophoresis Binding Assay in COVID-19 Research

“…A protein (e.g., RBD) that is bound to a ligand (e.g., a peptide) will exhibit a different rate of motion in response to laser exposure than an unbound protein because its size, charge, and/or hydration shell will change after binding. For each dilution, fluorescence is measured at the same time point, resulting in an affinity curve which is then used to determine the dissociation constant (KD) (Figure 1c) [8][9][10][11][12][13]. There are a variety of alternative biochemical assays that can be used to determine the dissociation constant (KD) of protein-ligand interactions such as thermal shift assay [14], surface plasmon resonance (SPR) [15], circular dichroism [14], isothermal titration calorimetry [16], small-angle X-ray scattering [17], and nuclear magnetic resonance spectrometry [18], among others.…”

“…A protein (e.g., RBD) that is bound to a ligand (e.g., a peptide) will exhibit a different rate of motion in response to laser exposure than an unbound protein because its size, charge, and/or hydration shell will change after binding. For each dilution, fluorescence is measured at the same time point, resulting in an affinity curve which is then used to determine the dissociation constant (K D ) ( Figure 1 c) [ 8 , 9 , 10 , 11 , 12 , 13 ].…”

“…For further information on this versatile MST binding assay, please refer to previous publications showing a variety of additional applications of this cutting-edge technique [ 9 , 10 , 11 , 12 , 21 ].…”

Recent Advances in Protein–Protein Interactions

“…The contribution of Magnez et al [ 9 ] describes microscale thermophoresis (MT) as a technique to study protein–protein as well as protein–ligand interactions. MT allows to quantify binding interactions by determining the binding constant values, offers the possibility of fast screening of large libraries, and may become the technology of choice in the drug discovery programs.…”