Inhibition of Class A β-Lactamase (TEM-1) by Narrow Fractions of Humic Substances

Mikhnevich Tatyana A., Vyatkina (Turkova) Alexandra V., Grigorenko Vitaly G., Rubtsova Maya Yu, Rukhovich Gleb D., Letarova Maria A., Kravtsova Darya S., Vladimirov Sergey A., Orlov Alexey A., Nikolaev Evgeny N., Zherebker Alexander, Perminova Irina V.

ACS Omega, 2021, , doi: 10.1021/acsomega.1c02841


Antimicrobial resistance is a global threat. A use of biologically active natural products alone or in combination with the clinically proven antimicrobial agents might be a useful strategy to fight resistance. The scientific hypotheses of this study were twofold: (1) the natural humic substances rich in dicarboxyl, phenolic, heteroaryl, and other fragments might possess inhibitory activity against β-lactamases, and (2) this inhibitory activity might be linked by molecular composition of humic ensemble. To test these hypotheses, we used humic substances (HS) from different sources (coal, peat, soil) and of different fractional composition (humic acids, hymatomelanic acids, narrow fractions from solid phase extraction) for inhibiting serine β-lactamase TEM-1. Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize molecular composition of all humic materials used in this study. The kinetic assay with chromogenic substrate CENTA was used for assessment of inhibition activity. The inhibition data have shown that among all humic materials tested the distinct activity was observed within apolar fractions of hymatomelanic acid isolated from lignite. A decrease in the hydrolysis rate in the presence of most active fraction was 42% (with sulbactam – 87%). Of particular importance is that these very fractions caused synergistic effect (two-fold) for the combinations with sulbactam. Linking the observed inhibition effects to molecular composition revealed preferential contribution of low-oxidized aromatic and acyclic components such as flavonoid-, lignin, and terpenoid-like molecules. The binding of single low molecular weight components to the cryptic allosteric site along with supramolecular interactions of humic aggregates with protein surface could be considered as major contributors to the observed inhibition. We believe that fine fractionation of hydrophobic humic materials along with molecular modeling studies on interaction between humic molecules and beta-lactamases might contribute to development of novel beta-lactamase inhibitors of humic nature.Keywords: humic substances, beta-lactamase inhibitors, FT ICR MS, non-lactam inhibitors, synergistic effect, narrow fractions, solid phase extraction