Non-β Lactam Inhibitors of the Serine β-Lactamase blaCTX-M15 in Drug-Resistant Salmonella typhi

Abstract

Antibiotic resistance by bacterial pathogens against widely used β-lactam drugs is a major concern to public health worldwide, resulting in high healthcare cost. The present study aimed to extend previous research by investigating the potential activity of reported compounds against the S. typhi β-lactamase protein. 74 compounds from computational screening reported in our previous study against β-lactamase CMY-10 were subjected to docking studies against blaCTX-M15. Site-Identification by Ligand Competitive Saturation (SILCS)-Monte Carlo (SILCS-MC) was applied to the top two ligands selected from molecular docking studies to predict and refine their conformations for binding conformations against blaCTX-M15. The SILCS-MC method predicted affinities of -8.6 and -10.7 kcal/mol for Top1 and Top2, respectively, indicating low micromolar binding to the blaCTX-M15 active site. MD simulations initiated from SILCS-MC docked orientations were carried out to better characterize the dynamics and stability of the complexes. Important interactions anchoring the ligand within the active site include pi-pi stacked, amide-pi, and pi-alkyl interactions. Simulations of the Top2-blaCTX-M15 complex exhibited stability associated with a wide range of hydrogen-bond and aromatic interactions between the protein and the ligand. Experimental β-lactamase (BL) activity assays showed that Top1 has 0.1 u/mg BL activity, and Top2 has a BL activity of 0.038 u/mg with a minimum inhibitory concentration of 1 mg/mL. The inhibitors proposed in this study are non-β-lactam-based β-lactamase inhibitors that exhibit the potential to be used in combination with β-lactam antibiotics against multidrug-resistant clinical isolates. Thus, Top1 and Top2 represent lead compounds that increase the efficacy of β-lactam antibiotics with a low dose concentration.

Figure 1.

Workflow of computational and experimental work.

Figure 2.

Identification of the drug target region on plasmid including descriptions of the molecular basis for resistance genes with comprehensive antibiotic resistance targets. RGI (resistance genes identifier) circular plot depicting the resistance genes with exact matches to the CARD reference sequences; herein, “perfect” denotes a high degree of sequence similarity between a gene contained in the input data and a reference resistance gene kept in the database. “Loose” hits show discovery of novel resistance genes and the Strict algorithm is used for high-confidence detection of resistance genes.

Figure 3.

Depiction of top docked complexes highlighting the interacting residues of the active site. Ligand is represented in yellow and blue and protein is in cyan ribbon representation (a) Top1 compound (Chembridge ID: 5747390) (b) Top2 compound (Chembridge ID 32025297).

Figure 4.

(a) X-ray crystal orientation of inhibitor C6S from PDB ID 5t66 and SILCS-MC docked orientations of (b) Top 2 and (c) Top 1 in the binding pocket of blaCTX M15. The protein is shown as a white solvent accessible surface along with the SILCS FragMaps for negative (orange), apolar (green), hydrogen bond donor (blue) and hydrogen bond acceptor (red) functional groups. Negative and apolar FragMaps are shown at a GFE cutoff of −0.9 kcal/mol and hydrogen bond donor and acceptor maps at a cutoff of −0.6 kcal/mol. 2D images of the ligands are shown as insets in the respective panels.

Figure 5.

GFE contributions of the rings with their substituents and the linker regions of (a) Top1 and (b) Top2. Atomic GFE contributions are summed over all the classified atoms in the rings and their substituents or over all the classified atoms in the linker regions between the rings. The sum of the GFE contributions yields the LGFE scores for each ligand.

Figure 6.

Root-mean-squared difference (RMSD) of the protein (blue) and ligand (black) non-hydrogen atoms from the (a) Top1 and (b) Top2-complex MD simulations. RMSD values were calculated following alignment of the protein non-hydrogen atoms with the 5T66 crystallographic coordinates.

Figure 7.

Top1 ligand orientations during the MD simulation. (a) Overall binding orientation of the ligand in the pocket at 5 ns (yellow), 50 ns (green), 100 ns (magenta), 150 ns (pink) and 200 ns (orange), (b) zoomed view of ligand conformation with selected functional groups labeled from selected time frames from the MD simulation. Details of the Top1-protein interactions at (c) 50 ns, and (d) 200 ns.

Figure 8.

Top2 ligand orientations during the MD simulation. (a) Overall binding orientation of the ligand in the pocket at 150 ns (yellow) (b) 200 ns (cyan) (c) Details of the Top2-protein interactions at 200 ns. (d) zoomed view of ligand conformation at 200 ns.

Figure 9.

Graphical representation of the hydrolysis of 100 mM nitrocefin used to measure the activity of blaCTX-M15 followed by 5-minute pre-incubation with Top1, Top2 and clavulanic acid inhibitor doses using non-linear regression curve.

Figure 10.

Scanning electron microscopy of Salmonella typhi clinical strains in triplicate against untreated, treated (control) and combinational therapy for compound Top1 and Top2. Red circle highlighting the pores that shows the changes during combination therapy in cell wall against compound Top1 and Top2. During this investigation close morphological changes have been observed upon different scale 2μm and 10μm respictvely.