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Drug discovery through biophysical techniques: Methods and applications
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DOI：10.1016/j.pharmthera.2025.108947.png)
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Abstract
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The rapid advancements in drug discovery have underscored the critical demand for sophisticated analytical methodologies capable of delivering comprehensive insights into molecular interactions, thereby accelerating the development of innovative therapeutic agents. As modern drug discovery increasingly depends on precise characterization of molecular dynamics and protein behavior, biophysical techniques have emerged as indispensable tools in pharmaceutical development. These techniques provide robust platforms for investigating protein–ligand interactions, elucidating structural basis, and deciphering cellular mechanisms—all essential components of rational drug design. In this review, we systematically summarize pivotal biophysical technologies shaping contemporary drug discovery, including Förster resonance energy transfer (FRET), fluorescence polarization (FP), thermal shift assays (TSA/CETSA), surface plasmon resonance (SPR), bio-layer interferometry (BLI), microscale thermophoresis (MST), isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), X-ray crystallography, and advanced mass spectrometry (MS)-based approaches such as hydrogen–deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS). For each technique, we trace its historical development, outline fundamental principles, and highlight key innovations that have cemented its role in drug research. We also objectively evaluate the strengths and limitations of each method and discuss emerging trends, including improvements in sensitivity, automation, and high-throughput implementation. These developments promise to maintain their pivotal role in advancing biomedical research and therapeutic innovation, particularly in addressing the growing complexity of drug targets and the urgent need for precision medicine solutions.
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