Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae

Abstract

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.

Fig. 1.. Structural model of the Lpt machinery.

(A) Protein assembly of the Lpt in Gram-negative bacteria. (B) Schematic representation of the postulated mode of action based on this work: Thanatin inhibits the LptA-LptA and LptC-LptA protein-protein interactions and disassembles the Lpt periplasmic protein bridge. Note that two LptA molecules are depicted although the exact number is unknown.

Fig. 2.. Thanatin and optimized analogs.

(A) Chemical structures of thanatin and of the new antibiotic peptide analogs 6 and 7 as modified during the structure-activity relationship (SAR) exploration. Structural modifications implemented during the medicinal chemistry optimization are highlighted in green. (B) Amino acid sequences (one letter code) of the described peptides. Thanatin and thanatin analogs residues are marked with an apostrophe. DDab, d-2,3-diaminobutyric acid.

Fig. 3.. Energy-minimized average structures from MD simulations of thanatin (yellow) bound to LptAm (green) and compound 7 (brown) bound to LptAm^Q62L (blue).

(A) Important interactions involving Q62 that helps to anchor the N-terminal helix to the β-jellyroll. (B) Schematic presentations of the fold of the LptAm-thanatin and the LptAm^Q62L-7 complex. Roman numbers I to V refer to the five principal sites of interaction. (C) Structural details of interactions at sites I to V for the LptAm-thanatin and the LptAm^Q62L-7 complexes. For a comprehensive comparison of all complexes described in this work, see fig. S23.

Fig. 4.. In vivo efficacy of compound 7.

Compound 7 activity against E. coli (Ec) American Type Culture Collection (ATCC) 25922 in mouse neutropenic thigh infection model, K. pneumoniae (Kp) AR-BANK#0160 and K. pneumoniae ATCC 43816 in mouse neutropenic lung infection models. Colony-forming units (CFU) counts 24 hours after first administration of 7 (blue), vehicle only (gray), and standard of care (red) are compared. The geometric mean value of each group is depicted as a black dash.

Fig. 5.. Disassembly of E. coli dimers.

(A) Schematic overview of the disassembly process. A, LptA; C, LptC^AA. (B) SEC trace from the mLptA-LptAm (top) and LptAm-LptC^AA (bottom) mixtures in presence of compound 7 (bold black lines). SEC traces of the protein dimers (green lines) or from the 7 complexes with LptAm, mLptA, or LptC^AA are shown (thin blue and red lines). (C) [¹⁵N,¹H]-HSQC spectra from the mLptA-LptAm (top) and LptAm-LptC^AA (bottom) mixtures in presence of 7. In presence of 7, the peaks of mixtures of the dimeric proteins superimpose with those of the corresponding monomeric proteins. In both NMR and SEC experiments, the protein concentration was 200 μM, and 1.5 equivalent of peptide were added. For a complete set of all SEC or NMR data, see fig. S21.