Identification of Ribonuclease Inhibitors for the Control of Pathogenic Bacteria

Abstract

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.

Keywords: PNPase; RNase II; RNase R; antimicrobials; virtual high-throughput screening (vHTS).

Figure 1

Structure of the E. coli RNase II catalytic domain and compound docking. (a) Overall structure of RNase II with bound RNA (blue). Orange, yellow, gray, and green coloring identifies the CSD1, CSD2, RNB, and S1 domains, respectively. (b) Predicted docking of SEW04027 in the RNase II catalytic site, where the key residues Ile200, D201, D210, and Y313 are depicted. The green sphere represents the Mg²⁺ ion. (c) Predicted docking of HTS05225 in the RNase II catalytic site, with the D201, T496, and R500 residues depicted. The green sphere denotes the Mg²⁺ ion. The images presented were generated using PyMOL [25].

Figure 2

Structure of the E. coli PNPase core and compound docking. (a) Top view of the E. coli PNPase core trimer, showing the central channel. In each monomer, the RNase PH1 domain is colored in light blue, the α-helical region in yellow, and the RNase PH2 domain in red. RNA-binding domains KH and S1 are not shown. (b) Predicted docking of CD06144 in the PNPase catalytic site, where the key residue D486 is depicted. (c) Predicted docking of SPB04215 in the PNPase catalytic site, where the residue D492 is represented. The images presented were generated using PyMOL [25].

Figure 3

Exoribonucleolytic activity of RNase II in the presence of chemical compounds. A total of 10 nM of RNase II was incubated with 10 nM poly(A) and 10 mM of each compound at 37 °C for 10 min. Samples were taken during the reaction at the time points indicated in the figure. Ctrl, control without enzyme.

Figure 4

Representative viability analysis. MG1655 and MG1655 Δpnp strains were incubated with 10 mM of the chemical compounds (LB media or DMSO were added in the control reactions). Samples were taken at the time points of 120, 420 min, and overnight.

Figure 5

Exoribonucleolytic activity of PNPase in the presence of chemical compounds. A total of 10 nM of PNPase was incubated with 10 nM poly(A) and 10 mM each compound at 37 °C for 10 min. Samples were taken during the reaction at the time points indicated. Ctrl, control without enzyme.

Figure 6

Representative viability analysis. CMA201 and MG1693 strains were incubated with 10 mM of the chemical compounds (LB media or DMSO were added in the control reactions). Samples were taken at the time points of 120, 420 min, and overnight.