Bacterial pyrophosphatase and formate dehydrogenase as promising antibiotic targets: NMR studies and the search for inhibitors

Polshakov, V.I.; Mariasina, S.S.; Rudenko, A.Y.; Mantsyzov, A.B.; Romanov, R.S.; Vasiliev, V.G.; Pometun, A.A.; Tishkov, V.I.

Авторы: Polshakov V.I., Mariasina S.S., Rudenko A.Y., Mantsyzov A.B., Romanov R.S., Vasiliev V.G., Pometun A.A., Tishkov V.I.
Сборник: The 30th International Conference on Magnetic Resonance in Biological Systems. Abstract e-Book
Тезисы
Год издания: 2024
Место издания: Seoul, Korea (South)
Первая страница: 486
Последняя страница: 486
Номер статьи: 9-0658
Аннотация: The challenge of discovering new antibiotics and finding effective treatments for bacterial infections is becoming increasingly urgent due to the rising resistance to existing drugs. We have studied two enzymes that play a crucial role in the survival of bacteria: inorganic pyrophosphatase (Mt-PPase) from Mycobacterium tuberculosis and NAD+-dependent formate dehydrogenase (sa-FDH) from Staphylococcus aureus. While FDH does not have an exact counterpart in human cells, the structure of PPases varies significantly between bacteria and humans. This means that these enzymes could be potential targets for selective inhibition. Currently, there are no antibiotics in medical practice that inhibit these enzymes.Mt-PPase is a hexamer with a molecular weight of 110 kDa and FDH is an 84 kDa homodimer. We have obtained 13C, 15N, 2D-labeled enzymes, designed effective protocols for the back exchange of amide protons, and obtained NMR signal assignments for protein backbone nuclei. In case of FDH, assignments were determined for both the apo form of the protein and its complex with NAD+. Chemical shift information was used for verification of the secondary structure of the proteins in solution. Analysis of amide 15N relaxation parameters made it possible to establish the nature of protein backbone dynamics.We then used NMR screening techniques to identify compounds that could bind to the enzymes at sites that influence their catalytic activity. We used two complementary approaches, based on the observation of ligand signals (STD and WaterLOGSY experiments) and ligand-induced perturbations in protein chemical shifts in TROSY spectra. After optimizing the hit compounds, we were able to obtain inhibitors with IC50 values in the micromolar range. These compounds do not have any structural similarities to known FDH or PPase inhibitors, making them potential starting points for the design of new antibiotics.Russian Science Foundation (grant 24-14-00081) supported the study.
Добавил в систему: Польшаков Владимир Иванович

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