ИСТИНА

Antibiotic resistance became a global problem, while microorganisms developed several adaptive tools to escape the action of antibiotics. The most efficient tool is enzymatic degradation of β-lactam antibiotics realized via release of β-lactamases. To overcome this problem, inhibitors of β-lactamases are widely used in clinical practice. However, they are quickly losing activity for new β-lactamase mutants. Molecular ensemble of humic substances (HS) has dynamic composition which might hinder molecular recognition of HS as inhibitor by bacteria. The aim of this work was to isolate and characterize the most active components of HS. For achieving the goal of this study, we have synthesized and used CENTA [1] as a chromogenic substrate for studying enzymatic activity of β-lactamase TEM-1 in the presence of sulbactam, HS or mixture of them. To isolate the most active components of HS, the hymatomelanic acids (CHM) were isolated from commercial sample of coal potassium/sodium humate (Humate-Extra) using ethanol extraction. The narrow fractions of CHM were obtained by polarity driven separation with a use of solid-phase extraction and gradient elution with water-methanol eluate with a stepwise increase in the amount of methanol from 30 to 100%. All narrow fractions were characterized with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and fluorescence spectroscopy. No significant inhibitory effect was found for CHM and its narrow fractions in this study, however, a regular synergistic increase in inhibition was observed in the presence of sulbactam (Fig. 1.) Fig. 1. Kinetic curves of CENTA (100 μM) hydrolysis by β-lactamase TEM-1 (7 nM): in the absence of inhibitors (control, highlighted in black) and in the presence of sulbactam (5 μM, red), CHM and its narrow fractions (50 mg∙L-1, purple, yellow, light green, green and blue). Some data are omitted to avoid overlap of the curves. Molecular compositions of the fractions varied substantially in chemical diversity. The most non-polar fractions are characterized by a higher content of aromatic and low-oxidized compounds. Also, with an increase in the nonpolarity of the fractions, a decrease in the ratio of the short-wave and long-wave parts in the fluorescence spectrum is observed, which may indicate an increase in the degree of humification and condensation of HS molecules. Thus, a relationship was established between the aromaticity and nonpolarity of HS components and their synergistic enhancement of sulbactam. Establishing the mechanism of the observed effect is the subject of future research. Acknowledgements This work was supported by grant from the Russian Science Foundation № 21-73-20202. FTICR MS measurements were carried out using the equipment of the Shared Use Center of the Institute of Organic Chemistry of the Russian Academy of Sciences. [1] Bebrone C. et al. Antimicrob. Agents Chemother. 2001, 45 (6), 1868–1871.