The physical-chemical characterization of interactions between proteins and ligands is crucial for understanding biological systems and for the rational design of drug molecules. Since ligands, proteins, and their complexes are dynamic systems, it is essential to study them in an environment that preserves their natural dynamics. Studying these interactions in an aqueous environment better resembles physiological conditions than studying crystal structures, while preserving the dynamics of the system.
In our study, we investigated the binding of ligands to two protein targets: sterol 14α-demethylase (CYP51), an essential enzyme in the sterol biosynthesis pathway and the main target of antifungal drugs, and muramyl ligase D (MurD), a multidomain bacterial enzyme involved in peptidoglycan synthesis and considered a promising target for the development of new antibacterial agents. The main experimental method we used was nuclear magnetic resonance (NMR).
By determining binding affinities and using ligand-based NMR methods, we studied the binding of pyridylethanol(phenylethyl)amines to CYP51 from Candida albicans (CaCYP51) and human CYP51 (hCYP51), and identified key functional groups that allow selective binding to CaCYP51 while preventing inhibition of hCYP51 to avoid the accumulation of toxic sterols in the body. We identified fragments that contribute to improved binding affinity. We determined the binding mode of selected derivatives at the active site of CaCYP51 and found that it differs from the binding of clinically used azole inhibitors. The most promising selective derivatives exhibited inhibitory and antifungal activity. GIS-528 proved to be the most promising and can serve as a lead compound for further optimization.
In the Escherichia coli MurD protein, we assigned the 1HN and 15N nuclei of the backbone and the 1H and 13C nuclei of the methyl groups of the AILVproR amino acid residues of the free and bound enzyme in the NMR spectra. We then studied the dynamics of the MurD and its influence on ligand binding on different time scales. We presented a new analytical approach to the analysis of spectral density relationships using principal component analysis, which provided us with mechanistic insight into the dynamics at the level of individual residues. In apo and bound MurD, we detected compensatory effects (ps–ms time scale) and conformational exchange dynamics (µs–ms time scale), the latter of which was also measured independently. The presence of conformational dynamics on the µs-ms time scale in the apo state and the state with bound AMP-PCP indicates that conformational selection is the binding mechanism of MurD. In MurD with bound inhibitor, we have defined in detail the dynamic variability of structural elements that limits binding efficiency. The results of molecular dynamics simulations confirmed the experimental findings and revealed additional diversity in the structural clusters of apo states and MurD complexes.
In this study, we succeeded in designing a new structural type of selective CaCYP51 inhibitors and investigating the structural-dynamic aspect of MurD inhibitor binding. New guidelines for the rational design of CYP51 and MurD inhibitors may contribute to the development of innovative antimicrobial agents and fill a gap in the neglected area of finding solutions to microbial resistance.
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