3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Potentiation of Antibiotic Activity of Aztreonam against Metallo-ß-Lactamase-Producing Multidrug-Resistant Pseudomonas aeruginosa by 3-O-Substituted Difluoroquercetin Derivatives.

The combination of aztreonam (ATM) and ceftazidime-avibactam (CAZ-AVI; CZA) has shown therapeutic potential against serine-ß-lactamase (SBL)- and metallo-ß-lactamase (MBL)-producing Enterobacterales. However, the ability of CZA to restore the antibiotic activity of ATM is severely limited in MBL-producing multidrug-resistant (MDR) Pseudomonas aeruginosa strains because of the myriad of intrinsic and acquired resistance mechanisms associated with this pathogen. We reasoned that the simultaneous inhibition of multiple targets associated with multidrug resistance mechanisms may potentiate the antibiotic activity of ATM against MBL-producing P. aeruginosa. During a search for the multitarget inhibitors through a molecular docking study, we discovered that di-F-Q, the previously reported efflux pump inhibitor of MDR P. aeruginosa, binds to the active sites of the efflux pump (MexB), as well as various ß-lactamases, and these sites are open to the 3-O-position of di-F-Q. The 3-O-substituted di-F-Q derivatives were thus synthesized and showed hereto unknown multitarget MDR inhibitory activity against various ATM-hydrolyzing ß-lactamases (AmpC, KPC, and New Delhi metallo-ß-lactamase (NDM)) and the efflux pump of P. aeruginosa, presumably by forming additional hydrophobic contacts with the targets. The multitarget MDR inhibitor 27 effectively potentiated the antimicrobial activity of ATM and reduced the MIC of ATM more than four-fold in 19 out of 21 MBL-producing P. aeruginosa clinical strains, including the NDM-producing strains which were highly resistant to various combinations of ATM with ß-lactamase inhibitors and/or efflux pump inhibitors. Our findings suggest that the simultaneous inhibition of multiple MDR targets might provide new avenues for the discovery of safe and efficient MDR reversal agents which can be used in combination with ATM against MBL-producing MDR P. aeruginosa.

Identification of (-)-Epigallocateshin gallate derivatives promoting innate immune activation via 2',3'-cyclic GMP-AMP-stimulator of interferon genes pathway.

(-)-Epigallocatehin-3-gallate (EGCG) is a catechin derived from green tea, which has been widely studied for its anti-oxidant and anti-tumor properties. Although EGCG plays important roles in various biological processes, the its effect on the immune system is not fully understood. In this study, we investigated the potential of EGCG as an activator of the stimulator of interferon genes (STING) pathway in the immune system. The cyclic GMP-AMP synthase (cGAS)-2-3-cyclic GMP-AMP (cGAMP)-STING pathway is crucial in the innate immune response to microbial infections, autoimmunity, and anticancer immunity. We confirmed that EGCG enhanced the immune response of cGAMP and identified E2 from 13 synthetic derivatives of EGCG. E2 specifically activated the interferon (IFN) signaling pathway specifically through STING- and cGAMP-dependent mechanisms. These results demonstrate the potential of EGCG and its derivatives as new STING activators that can stimulate the type I interferon response by boosting cGAMP-mediated STING activity.

Green Tea Catechol (-)-Epigallocatechin Gallate (EGCG) Conjugated with Phenylalanine Shows Enhanced Autophagy Stimulating Activity in Human Aortic Endothelial Cells.

(-)-Epigallocatechin gallate (EGCG) is one of the autophagy stimulators that have been reported to protect vascular endothelial cells from oxidative stress-induced damage. In this study, we attempted potentiation of the autophagy-stimulating activity of EGCG in human aortic epithelial cells (HAECs) by using the EGCG-phenylalanine conjugate, E10. Autophagy-stimulating activity of E10 was evaluated by LC3-II measurement in the absence and presence of the lysosomal blocker chloroquine, CTYO-ID staining, and reporter assay using tandem fluorescence-tagged LC3. These experiments revealed significantly enhanced autophagic flux stimulation in HAECs by E10 compared with EGCG. Further elaboration of E10 showed that activation of AMPK through phosphorylation as the major mechanism of its autophagy stimulation. Like other autophagy stimulators, E10 protected HAECs from lipotoxicity as well as accompanying endothelial senescence. Finally, stimulation of autophagy by E10 was shown to protect HAECs from oxidative stress-induced apoptosis. These findings collectively suggest potential clinical implications of E10 for various cardiovascular complications through stimulation of autophagy.

In Vitro Synergism and Anti-biofilm Activity of Quercetin-Pivaloxymethyl Conjugate against Staphylococcus aureus and Enterococcus Species.

Upon single treatment against Staphylococus aureus, quercetin-pivaloxymethyl conjugate (Q-POM) had antibacterial activities with minimum inhibitory concentrations (MICs) of 16-32 mg/L. Q-POM showed MIC of 32 mg/L against vancomycin-resistant Enterococcus faceium (VRE), which is remarkably lower than other antibiotics investigated (≥256 mg/L). Under sub-MIC concentrations, Q-POM potentiated the activity of ampicillin, cefepime, and vancomycin against S. aureus and Enterococcus (including highly resistant strains such as hetero-resistant vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA), and VRE), by decreasing the MICs of these antibiotics by 4-128 folds. Q-POM was found to be partially synergistic with ampicillin and cefepime against S. aureus and Enterococcus, while it was strongly synergistic with vancomycin. Q-POM at 5 mg/L inhibited the formation of biofilms of S. aureus by 24-83% and VRE by 70%. Additionally, Q-POM inhibited the hemolytic activity of S. aureus in a dose-dependent manner. Cytotoxic activity was evaluated in human liver epithelial cells (HepG2), and the 50% cytotoxicity concentration (CC50) value of Q-POM was higher than 50 mg/L. These results indicate the potential use of Q-POM in treatment of methicillin-resistant Staphylococcus aureus (MRSA) and VRE infections.