Farmacia e CTF: The Role of Advanced Analytical Techniques in Drug Discovery

The present seminar provides an overview of how pharmaceutical analysis supports drug discovery by addressing current medical challenges, with a focus on identifying and developing effective multi-target ligands for complex multifactorial diseases. It highlights the pivotal role of advanced analytical methodologies throughout the drug discovery pipeline. Firstly, the contribution of omics sciences to target identification and validation is presented, focusing on their use in investigating molecular mechanisms underlying disease pathogenesis. In particular, the crucial role of quantitative proteomics is discussed, which enables large-scale identification of differentially expressed proteins and reveals potential drug targets. 1 This is made possible by state-of-the-art technologies, including high-resolution mass spectrometry, optimized sample preparation protocols, and advanced bioinformatics tools for spectral matching, quantification, statistical analysis, and functional interpretation. Since small proteomic changes can cause amplified metabolic shifts, the seminar also introduces a recent multi-omics strategy integrating proteomics and metabolomics to enable more robust and functionally validated identification of disease targets. 2 Next, analytical techniques suitable for high-throughput screening (HTS) that facilitate hit identification are covered, focusing on solution-based spectroscopic assays, particularly fluorescence and luminescence-based methods, which provide rapid and efficient first-line evaluation of inhibitory activity toward enzymatic targets and enable large-scale screening thanks to their sensitivity, reproducibility, and compatibility with automated platforms. These assays allow determination of inhibitory potency (e.g., IC₅₀) and preliminary mechanistic classification (competitive, non-competitive, uncompetitive, and mixed modes). 3,4 In addition, the seminar explores the role of biophysical methods essential for detailed studies of ligand–protein interactions by revealing binding affinity, kinetics, and potential target conformational changes. 5 The combined use of microscale thermophoresis (MST) and surface plasmon resonance (SPR) is presented for comprehensive binding parameter analysis. MST offers efficient affinity screening by allowing rapid determination of dissociation constants (K d ) with low sample consumption and minimal assay development through fluorescently labeled targets in solution without immobilization. SPR complements MST by providing detailed kinetic parameters (k on , k off ) through real-time monitoring of ligand binding to immobilized targets, which is essential for calculating residence times (RT), though it requires more sample and complex preparation. Beyond binding parameters, circular dichroism (CD) spectroscopy is introduced to explore ligand induced changes in protein conformation, which can directly affect protein function and modulate disease pathways. Ultimately, the seminar reveals how integrating diverse data through a multi-methodological approach provides in-depth insight into structure–activity relationships (SARs), thereby supporting the rational design of more effective molecules.

References:
1. Meissner, F., Geddes-McAlister, J., Mann, M. & Bantscheff, M. The emerging role of mass spectrometry-based proteomics in drug discovery. Nat Rev Drug Discov 21, 637–654 (2022).
2. Du, P., Fan, R., Zhang, N., Wu, C. & Zhang, Y. Advances in Integrated Multi-omics Analysis for Drug-Target Identification. Biomolecules 14, 692 (2024).
3. F, M., A, D. S. & V, A. Beta-secretase as a target for Alzheimer’s disease drug discovery: an overview of in vitro methods for characterization of inhibitors. Analytical and bioanalytical chemistry 400, (2011).
4. Baki, A., Bielik, A., Molnár, L., Szendrei, G. & Keserü, G. M. A high throughput luminescent assay for glycogen synthase kinase-3beta inhibitors. Assay Drug Dev Technol 5, 75–83 (2007).
5. Applied Biophysics for Drug Discovery. (John Wiley & Sons, Ltd, 2017). doi:10.1002/9781119099512.

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