Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen

Abstract

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

Revealing the Effects of Three Different Antimicrobial Agents on E. coli Biofilms by Using Soft-Probe Scanning Electrochemical Microscopy

“…The UV/Vis absorption spectra of AgNPs are shown in Figure S5a-c. The maximum absorption results of the studied nanocomposites were found at 473 nm, 442 nm, and 414 nm for citrate-capped AgNPs, PVP-capped AgNPs in water, and PVP-capped AgNPs in EG, respectively [55,66]. It has been reported that the absorption spectrum of spherical silver nanoparticles showed a maximum between 400 nm and 500 nm with a blue or red shift when the particle size diminished or increased, respectively [67,68].…”

“…Under aerobic conditions, oxygen is the final electron acceptor. This results in the transport of up to eight protons [55]. The ETC comprises membrane-bound cytochromes (a-, b-, d-, o-type), dehydrogenases, quinones, and flavins.…”

“…The ETC comprises membrane-bound cytochromes (a-, b-, d-, o-type), dehydrogenases, quinones, and flavins. The sites of electron transfer of the anodic electron transport are cytochrome bo, cytochrome bd, Ubiquinone-pool, FAD reduction, and NADH oxidation, according to Equations (1)-(3) (Figure S3, E 0 given at 25 • C and pH 7 [55]):…”

“…The reported potentials (ox/red) of cytochromes a-, b-, d-, and o-type are 0.290 V, 0.080 V, 0.024 V, and 0.200 V, respectively [55]. In the aerobic respiration of E. coli, oxygen (E 0 = 0.82 V) acts as an electron acceptor [55].…”

Revealing the effects of three different antimicrobial agents on E. coli biofilms by using Soft-Probe Scanning Electrochemical Microscopy

“…Biofilms on a coverslip were incubated in a solution with 4 mM sodium azide, a potent inhibitor of bacterial growth [49]. Sodium azide inhibits bacterial growth by inhibiting the activity of SecA, an…”