RpoN-Based stapled peptides with improved DNA binding suppress Pseudomonas aeruginosa virulence

Abstract

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 μM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.

Fig. 1. The model for stapled peptide binding to DNA informs library design. A. A model of the RpoN stapled peptide binding to DNA major groove glnAP2 promoter, based on PDB 2O8K, highlights the design modifications used in this library. B. Helical wheel of the stapled peptide sitting in the major groove, based on PDB 2O8K.

Fig. 2. An E. coli cell-based assay was used to evaluate all library members. A. Cell-based screening assay workflow for peptide library, indicating weak transcriptional inhibitors or potent transcriptional inhibitors. B. Stapled peptide library sequence with lead peptides highlighted in grey (additional details on the sequences and the structures are given in Table S1†). C. Normalized fluorescence graph subdivided in staple walk (), termini modifications (), DNA-interacting residues (), non-DNA-interacting residues () and amphipathic network (), peptide groups. All peptides were tested at 10 μM. Data are represented as n = 3 (except for PBS and 2 where n = 12), means ± STD. Fluorescence is normalized to untreated samples. The dotted line is set at the mean value of 2. Statistical significance is determined by an independent two-tailed t-test compared to the PBS control. *p = ≤0.05; **p = ≤0.01; ***p = ≤0.001, ****p = ≤0.0001.

Fig. 3. Stapled peptide selectivity, binding affinity, and hemolysis. (A) Lead stapled peptides selectivity inhibit RpoN-mediated transcription. Bar graph showing the ratio of the normalized GFP expression from the lacUV5 and glnAp2 promoter screens. (B) Binding affinity (K_d, nM) determined through fluorescence polarization assay (FPA) and hemolysis (%) with 100 μM stapled peptide against human red blood cells (HBC). Highlighted in black are the changes made from 2.

Fig. 4. In vivo wax worm model of P. aeruginosa infection. Log-rank Mantel–Cox test survival (%) of wax worms infected with P. aeruginosa (PA) and treated with stapled peptide or PBS vehicle control at 30 °C: A. stapled peptide 10, B. stapled peptide 14, C. stapled peptide 31, D. stapled peptide 33.