Monitoring the antibiotic darobactin modulating the β-barrel assembly factor BamA

Abstract

Highlights d Mechanical, kinetic, and energetic properties of BamA d Properties change upon binding to an antibiotic d Structural regions change mechanical stability and lifetime d Structural regions change free-energy and mechanical rigidity

Activity and cryo-EM structure guided biosynthetic pathway engineering yields non-natural Darobactin antibiotics with superior activity against Gram-negative pathogens

“…Taken together, the different Darobactin-BamA binding interactions of Darobactin A, D9 and D22 likely reveal the structural basis for enhanced activity of engineered Darobactins, which are more closely paired with BamA to mimic the ß-sheet prolongation and to lock the BAM complex in the inactive state by sealing the lateral gate tightly (Figure 1, 3). [13] These structure and activity based findings are reinforced by the results of analyzing the different binding kinetics for Darobactin A, D9 and D22 revealing K D values of 0.278 µM, 0.3 µM and 0.159 µM, respectively (Figure S 43).…”

“…Preceding Darobactin SAR studies focussed on native Darobactins [16,21] or were based upon observations from them. [19] Revealing the MoB of Darobactin A and D9 to BamA, combined with structural data of recently published Darobactins, [13,15,19,21] here enabled the first concise SAR study, in which we constructed twenty new derivatives, including twelve derivatives with terminal L-tryptophan exhibiting similar or better activity than Darobactin A (Figure S2). In our SAR study we found, that structural changes at position 6 and 7 appear to be the most important, necessary for interactions with β-hairpins of BamA, while the minor influence of position 4 on activity and BamA target interaction gives a valuable starting point for future structural modifications to further improve the pharmaceutical properties of Darobactins.…”

“…D22 shows rapid bacterial killing of E. coli MG1655 at 4x and 8x MIC after an early static phase of up to 2 hours followed by bactericidal activity with more than 3-log killing colony-forming units (CFU/mL) within 2 to 4 h. The observed initial bacteriostatic effect can possibly be explained by data generated by Ritzmann et al, who demonstrated that binding of Darobactin on the lateral gate leads to an increased stability by "freezing" BamA and consequently no immediate bactericidal effect. [13] However, Darobactin A and D22 at 2x, 4x and 8x MIC acted bactericidal over the first 8 h in time-kill experiments using E. coli MG1655, and the extent of bactericidal activity was concentrationdependent. Nevertheless, re-growth of bacteria was observed at 2x and 4x MIC conditions after 24 h, while this was not the case at 8x MIC (MIC values reported for Darobactin A and D22 were 2 µg/mL and 1 µg/mL, respectively).…”

“…Further studies will explore the optimized effects of D22 regarding mechanistic, kinetic and energetic properties, e.g. regarding the linker region and the eight β-hairpins, as detailed by Ritzmann et al [13] for Darobactin A. Future studies should also include a comparison to the structurally variable BAM complex of A. baumannii strains, of which a co-structure with Darobactins has not been solved yet.…”

“…[10] Due to the selective binding to the ß-barrel of the outer 2 membrane protein (OMP) BamA, Darobactin furthermore exhibits a unique mode of action (MoA), preventing OMP incorporation into the bacterial cell wall by closing the lateral gate of BamA and affecting mechanical, kinetic, and energetic properties of the entire complex. [12,13] BamA is an entirely novel antibacterial target, and in vitro generated Darobactin-resistant E. coli mutants appear with massively reduced fitness and virulence, indicating a lack of pre-existing resistance and a reduced risk of fast resistance development, at least in E. coli. [14][15][16] The Darobactin production in the native host bacterium only allows for a low production rate, not sufficient for industrial fermentation and production of compounds.…”