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

• Main Authors: Mohamed Belal Hamed, Ewa Burchacka, Liselotte Angus, Arnaud Marchand, Jozefien De Geyter, Maria S. Loos, Jozef Anné, Hugo Klaassen, Patrick Chaltin, Spyridoula Karamanou, Anastassios Economou
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: Microorganisms
• Subjects: <i>E. coli</i>; alkaline phosphatase; small molecule inhibitors; antibacterials; protein secretion
• Online Access: https://www.mdpi.com/2076-2607/9/3/592

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 <i>E. coli</i> strains with IC₅₀ < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an <i>E. coli</i> 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.