ИСТИНА

A major priority in modern biomedical research is the target-oriented discovery of new drugs. Among the biophysical methods applied to this challenge, nuclear magnetic resonance (NMR) spectroscopy holds a leading position. NMR provides detailed insights into molecular structure, dynamics, and intermolecular interactions – factors essential for elucidating drug mechanisms of action and for identifying novel bioactive compounds. NMR methods are widely used both for the structural characterization of natural products with biological activity and for analyzing the highly specific binding of small molecules to biomolecular targets. These capabilities have driven the development of fragment-based and NMR-guided screening strategies [1]. This presentation will review current approaches to NMR screening and strategies for the rational design of bioactive compounds using fragment-based methods. Both target-observed techniques (SAR-by-NMR) and ligand-observed experiments (STD, WaterLOGSY, FAXS, INPHARMA) will be discussed. The concepts will be illustrated by examples of clinically approved drugs identified through NMR-based approaches, as well as by results from our own studies on potential inhibitors of bacterial enzymes. Specifically, we investigated two enzymes essential for bacterial survival: inorganic pyrophosphatase (Mt-PPase) from Mycobacterium tuberculosis and NAD⁺-dependent formate dehydrogenase (Sa-FDH) from Staphylococcus aureus. Notably, FDH lacks a direct human counterpart, while PPases differ substantially between bacteria and humans, making both enzymes attractive targets for selective inhibition. To date, no clinically used antibiotics act on these enzymes. Mt-PPase is a 110 kDa hexamer, while Sa-FDH is an 84 kDa homodimer. We prepared uniformly 13C/15N/2D-labeled proteins and assigned their backbone resonances. Using NMR screening, we identified compounds binding to functional sites that modulate catalytic activity. Two complementary strategies were applied: monitoring ligand signals (STD and WaterLOGSY) and detecting ligand-induced perturbations in protein TROSY spectra. Optimization of initial hits yielded inhibitors with micromolar IC₅₀ values. Importantly, these compounds display no structural similarity to known PPase or FDH inhibitors, providing promising starting points for the development of novel antibiotics. The study was supported by the Russian Science Foundation grant No. 24-14-00081. 1. Polshakov V.I., Batuev E.A., Mantsyzov A.B. (2019) Russ. Chem. Rev., 88 (1), 59 – 98.