Actions of Pharmaceutical Lactones and Lactams on the Enzymes with Lactonase Activity: In Silico Prediction and In Vitro Study

Pharmaceutical pollutants that contain lactone or lactam rings in their structure are frequently detected in wastewater. Enzymes with lactonase activity, synthesized by the microorganisms in aerobic activated sludge, are expected to be able to participate in the hydrolysis of these pharmaceutical pollutants, but the actual activity of many lactonases towards such compounds remains poorly characterized. In this work, a systematic molecular docking study of 12 enzymes and 39 pharmaceutical pollutants (β-lactam antibiotics, macrolides, coumarins, statins) was undertaken. The binding affinity, active-site occupancy, total enzyme surface occupancy and geometric characteristics of the molecules (the distance from the catalytic nucleophile to the carbonyl carbon of the lactone ring, and the attack angle formed between the nucleophilic atom of the enzyme and the C–N/C–O bond of the substrate) were evaluated in silico for a set of hydrolases displaying lactonase activity (AidC, AaL, hexahistidine-tagged organophosphate hydrolase (His6-OPH), etc.) and metallo-β-lactamases (MβLs) (NDM-1, MIM-1, SAM-1). Lactamases exhibited favorable intermolecular interaction geometry for the majority of β-lactams. No enzyme was found that satisfied the geometric criteria corresponding to the biodegradation of macrolides. With coumarins, only Paraoxonase PON2 provided an optimal geometry of biocatalysis; however, this enzyme is absent from activated sludge but may be of interest for in vivo applications. For several statins widely used in practice, combinations with enzymes having virtually ideal geometry for biocatalysis were identified: AidC/rosuvastatin lactone, AaL/lovastatin, His6-OPH/mevastatin, and PON2/simvastatin. For the first time, several pharmaceutical lactones were identified in silico and confirmed in vitro as inhibitors of MβL and His6-OPH. Competitive inhibition of NDM-1 by lactone-containing signaling molecules of Gram-negative bacteria present in activated sludge was revealed for the first time both in silico and in vitro. The obtained results‘ may be taken into account when implementing monitoring of biological stages at water treatment stations and improve the efficiency of water purification. The revealed characteristics of MβL inhibition by macrolides may be taken into account as one of possible explanations for the effective application of β-lactams in combinations with macrolides known from multiple paratactical investigations.