The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. The Sec preprotein export pathway is an attractive potential alternative target. It is essential for bacterial viability and includes components that are absent from eukaryotes. Here we used a new high throughputin vivoscreen 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, fourteen HTS secretion inhibitors (HSI), belonging to 8 structural classes, were identified (IC50−and Gram+bacterial species (including those from the WHO top pathogens list). Seven of them, HSI#6, 9; HSI#1, 5, 10 and HSI#12, 14 representing three structural families were microbicidals. HSI#6 was the most potent (IC50of 0.4-8.7 μM), against 13 species of both Gram−and Gram+bacteria. HSI#1, 5, 9 and 10 inhibited viability of Gram+bacteria with IC50~6.9-77.8 μM. HSI#9, 12 and 14 inhibited viability ofE. colistrains with IC50E. colistrain missing TolC to improve permeability with IC504-14 μM, indicating their inability to penetrate the outer membrane.In vitroassays revealed that antimicrobial activity was not related to inhibition of the SecA component of the translocase and hence HSI molecules may target new unknown components that affect secretion. The results provide proof of principle for our approach, and new starting compounds for optimization.
