High-throughput screening of BAM inhibitors in native membrane environment

Abstract

The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin displayed up to tenfold higher potency than in synthetic membranes, highlighting the impact of native-like conditions in antibiotics development. The assay was successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of novel antibiotics targeting BAM.

The translocation assembly module (TAM) catalyzes the assembly of bacterial outer membrane proteinsin vitro

“…5a-b). Furthermore, while PMSF blocked TAM-mediated assembly of OmpA, consistent with previous results 79,80 the protease inhibitor did not affect BAM activity (Supplementary Fig. 5c).…”

ATP‐independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next‐generation therapeutics

“…It also shows synergistic activity with rifampicin. VUF15259 (Selvam et al, 2023) (e), compound 2 (Rath et al, 2023) (f), and compound 14 (Rath et al, 2023) (g) were discovered through high throughput screening assays. They decrease OMP levels and increase OM permeability, presumably by targeting BamA.…”

“…High throughput screening (HTS) assays have exploited the activity of BAM complex as an OMP insertase, and OM perturbation and triggering of stress response by BAM complex inhibitors, to identify new compounds (Rath et al, 2023; Steenhuis et al, 2019). For example, VUF15259 (Figure 6e) was identified as an inhibitor of autotransporter secretion by monitoring the σ E stress response (Selvam et al, 2023).…”

“…They escalate the σ E and Rcs stress response pathways, reduce OMP levels and autotransporter secretion in vivo, and impair BAM complex activity in vitro. Though toxic to human cells, both molecules can enhance the effects of vancomycin by affecting OM integrity (Rath et al, 2023).…”

Daropeptide natural products

“…monoaryl) crosslinked cyclophanes [9,79], typically introduced by the rSAM enzymes. In addition, crosslinks involving endogenous heteroatom, e.g., Csp 2 -N or Csp 3 -N crosslink [11,18,19,80], have also been found, demonstrating the immense biosynthetic potential of the RiPP cyclophane synthases. As a recent addition to the already impressive collection of cyclophanes, daropeptides, characterized by their unique ether (i.e.…”