Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA

Abstract

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

Approaches to the Structure-Based Design of Antivirulence Drugs: Therapeutics for the Post-Antibiotic Era

“…In one study using GOLD, a peptidomimetic fragment library was screened for inhibitors of DsbA [85]. This dithiol oxidoreductase protein is a key component of the oxidative folding system in Gram-negative bacteria, and plays a particularly important role in virulence factor assembly in the periplasm prior to secretion (Figure 1).…”

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents

“…Additional efforts to discover DsbA inhibitors have involved developing peptides [77] and peptidomimetics [78] that prevent the formation of the DsbA-DsbB redox complex. Guided by the crystal structure of this complex [25] (Figure 2d), Duprez et al designed a synthetic peptide (PFATCDS) that mimicked the DsbB periplasmic loop involved in DsbA docking (Figure 5a).…”

“…They reported the crystal structure of a non-covalent complex between PWATCDS and the PmDsbAC30S mutant, which was stabilized by hydrogen bonding to the DsbA cis -proline loop and hydrophobic interactions with the DsbA hydrophobic groove. The possibility of exploiting these interactions for the development of non-covalent DsbA inhibitors, was further pursued using PWATCDS as a template for virtual screening of a peptidomimetic library [78]. The top hit along with nine derivatives were synthesized and found to inhibit EcDsbA at millimolar concentrations (Figure 5b).…”

Strategies for the analysis of thioloxidorductases

“…Opracowano też testy in vitro weryfikujące oddziaływania DsbB lub pochodnych od niego peptydów z DsbA. Można przy ich użyciu monitorować proces utleniania DsbA przez DsbB lub zdolność DsbA do utleniania naturalnych lub syntetycznych substratów w obecności DsbB [2,[14][15].…”