Enhanced therapeutic window for antimicrobial Pept-ins by investigating their structure-activity relationship

Abstract

The overconsumption and inappropriate use of antibiotics is escalating antibiotic resistance development, which is now one of the 10 top threats to global health. Introducing antibiotics with a novel mode of action into clinical use is urgently needed to address this issue. Deliberately inducing aggregation of target proteins and disrupting protein homeostasis in bacteria via amyloidogenic peptides, also called Pept-ins (from peptide interferors), can be lethal to bacteria and shows considerable promise as a novel antibiotic strategy. However, the translation of Pept-ins into the clinic requires further investigation into their mechanism of action and improvement of their therapeutic window. Therefore, we performed systematic structure modifications of 2 previously discovered Pept-ins, resulting in 179 derivatives, and investigated the corresponding impact on antimicrobial potency, cellular accumulation, and ability to induce protein aggregation in bacteria, in vitro aggregation property, and toxicity on mammalian cells. Our results show that both Pept-in accumulation and aggregation of target proteins in bacteria are requisite for Pept-in mediated antimicrobial activity. Improvement of these two parameters can be achieved via increasing the number of arginine residues, increasing Pept-in aggregation propensity, optimizing the aggregate core structure, adopting β-turn linkers, or forming a disulphide bond. Correspondingly, improvement of these two parameters can enhance Pept-in antimicrobial efficacy against wildtype E. coli BL21 used in the laboratory as well as clinically isolated multidrug-resistant strain E. coli ATCC, A. baumannii, and K. pneumoniae.

Fig 1. Arginine is essential for Pept-in mediated antimicrobial activity.

A-B: MIC of P2 (A) and P33 (B) variants generated by modifying the amount of arginine against E. coli BL21. These two figures are associated with data from S1 and S2 Tables, respectively. Each dot represents the MIC of one Pept-in design. C: Time dependence of pFTAA (0.5 μM) fluorescence intensity of P2 derivatives (50 μM) in the presence of polyP (0.5 mM). (Error bars represent SEM, n = At least 3). D-I: Flow cytometry analysis of E. coli BL21 treated with FAM-labelled Pept-ins for different time points from three independent experiments. Samples from D-E were acquired using BD Fortessa X-20, whereas samples from G-I were acquired using Gallios Flow Cytometry. The percentage of FAM positive cells (D), FAM MFI of FAM positive cell population (E), and the percentage of FAM and HS169 positive cells (F) when treated with P2 or its derivatives with a reduced or an increased amount of arginine residues at the concentration of FAM-P2 RR addition MIC. The percentage of FAM positive cells (G), FAM MFI of FAM positive cell population (H), and the percentage of FAM and HS169 positive cells (I) when treated with P2 or its derivatives with replaced gatekeepers at the concentration of FAM-P2 MIC (25 μg/mL). Error bars represent SEM for (D-I), n = 9 in D, E and F, n = at least 3 in G, H and I. J-K: FTIR spectrum of P2 variants with altered gatekeepers in PBS (6% DMSO), without (J) or with (K) the presence of polyP. The absorbance is normalised between all samples and the spectrum is scaled to the amide I region between 1600–1700 cm⁻¹. Peaks within the left (1689–1696 cm⁻¹) and right (1610–1642 cm⁻¹) grey bar are assigned to β-sheet, while peaks within the grey bar in the middle (1651–1659 cm⁻¹) is assigned to α-helix. The FTIR spectrums are representative of three independent experiments. For A and B, one-sample Wilcoxon signed-rank test was used to compare the MIC median of each Pept-in group to P2 MIC (12.5 μg/mL) or P33 MIC (6.25 μg/mL). For D-I, a two-tailed Student t-test was performed for calculating statistical significance between the mean of P2 variant and P2. Asterisks indicating the level of the p value centred over the error bar mean: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 2. Increased aggregation propensity enhances Pept-in antimicrobial potency by promoting Pept-in accumulation and IB formation.

A: MIC (left Y-axis) against E. coli BL21 and Tango score (right Y-axis) of P2 derivatives generated by alanine scanning of the APR region. This figure is associated with data from S3 Table. The red bars represent the APR TANGO score, and the red line is the baseline of the P2 APR Tango score. The blue squares represent MIC values, and the blue line is the baseline of P2 MIC. B: MIC of P2 variants generated by aggregation propensity modification against E. coli BL21. This figure is associated with data from S5 Table. Each dot represents the MIC of one Pept-in design. C-G: P2 variants with decreased aggregation propensity: P2_0 Tango, P2_Br2, P2_Br_3; with increased aggregation propensity: P2_VFV, P2_IM. C: Time dependence of pFTAA (0.5 μM) fluorescence intensity of P2 derivatives (50 μM) in the presence of polyP (0.5 mM) (n = at least 6). D-G: Flow cytometry analysis of E. coli BL21 treated with FAM-labelled Pept-ins for different time points at FAM-P2-MIC from three independent experiments. Samples were acquired using Gallios Flow Cytometry. MFI of FAM⁺ cells (D), and the percentage⁺ of FAM and HS169⁺ cells (E) when treated with P2 and its derivatives with decreased antimicrobial potency. MFI of FAM⁺ cells (F), and the percentage⁺ of FAM and HS169⁺ cells (G) when treated with P2 and its derivatives with increased antimicrobial potency. Error bars represent SEM. H-I: FTIR spectrum of P2 variants with altered aggregation propensity in PBS (6% DMSO), without (H) or with (I) the presence of polyP. The absorbance is normalised between all samples and the spectrum is scaled to the amide I region between 1600–1700 cm⁻¹. Peaks within the left (1689–1696 cm⁻¹) and right (1610–1642 cm⁻¹) grey bar are assigned to β-sheet, while peaks within the grey bar in the middle (1651–1659 cm⁻¹) is assigned to α-helix. The FTIR spectrums are representative of three independent experiments. For B, one-sample Wilcoxon signed-rank test was used to compare the MIC median of each Pept-in group to P2 MIC (12.5 μg/mL). For D-G, a two-tailed Student t-test was performed for calculating statistical significance between the mean of P2 variant and P2 (n = at least 6). Asterisks indicating the level of the p value centred over the error bar mean: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 3. Optimizing the aggregate core structure enhances Pept-in antimicrobial potency.

A: The most likely structure predicted by Cordax for the amyloid core formed between P2 (left) and the APR of target proteins. The impact of introducing a mutant in the APR (e.g., V7Y) (right) can then be evaluated using the FoldX force field. B: Free energy of the APR structure calculated by FoldX after introducing a single mutation in the APR region. Blue bars represent mutations that are incompatible with the cross-beta core structure, while purple bars represent the ones that are more compatible with the cross-beta core structure. C: MIC of P2 variants generated by modifying the aggregate core structure against E. coli BL21. This figure is associated with data from S6 Table. Each dot represents the MIC of one Pept-in design. D-H: P2 variants which are incompatible with the cross-beta core structure: L6G (P2); V7S (P2); P2 variants which are more compatible with the cross-beta core structure more compatible: V7Y (P2), P2_A5F. D-G: Flow cytometry analysis of E. coli BL21 treated with FAM-labelled Pept-ins for different time points at FAM-P2-MIC from three independent experiments. Samples were acquired using Gallios Flow Cytometry. MFI of FAM⁺ cells (D), and the percentage⁺ of FAM and HS169⁺ cells (E) when treated with P2 and its derivatives with decreased antimicrobial potency. MFI of FAM⁺ cells (F), and the percentage⁺ of FAM and HS169⁺ cells (G) when treated with P2 and its derivatives with increased antimicrobial potency. Error bars represent SEM. H: Time dependence of pFTAA (0.5 μM) fluorescence intensity of P2 derivatives (50 μM) in the presence of PolyP (0.5 mM). (n = at least 6). I-J: FTIR spectrum of P2 variants with altered compatibility to the aggregate core structure in PBS (6% DMSO), without (I) or with (J) the presence of PolyP. The absorbance is normalised between all samples and the spectrum is scaled to the amide I region between 1600–1700 cm⁻¹. Peaks within the left (1689–1696 cm⁻¹) and right (1610–1642 cm⁻¹) grey bar are assigned to β-sheet, while peaks within the grey bar in the middle (1651–1659 cm⁻¹) is assigned to α-helix. The FTIR spectrums are representative of three independent experiments. For C, one-sample Wilcoxon signed-rank test was used to compare the MIC median of each Pept-in group to P2 MIC (12.5 μg/mL). For D-G, a two-tailed Student t-test was performed for calculating statistical significance between the mean of P2 variant and P2 (n = at least 6). Asterisks indicating the level of the p value centred over the error bar mean: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 4. Beta-hairpin promoting motifs can improve antimicrobial potency.

A-B: MIC of P2 variants generated by linker modification (A) or cysteine addition (B) against E. coli BL21. These two figures are associated with data from S7 and S8 Tables, respectively. Each dot represents the MIC of one Pept-in design. C: Time dependence of pFTAA (0.5 μM) fluorescence intensity of P2 derivatives (50 μM) in the presence of polyP (0.5 mM). (n = At least 3). D-E: Flow cytometry analysis of E. coli BL21 treated with FAM-labelled Pept-ins for different time points at FAM-P2-MIC from three independent experiments. Samples were acquired using BD Fortessa X-20. MFI of FAM positive cell population (D), and the percentage of FAM and HS169 positive cells (E) when treated with P2 and its derivatives with increased antimicrobial potency at the concentration of FAM-P2 MIC. Error bars represent SEM (n = 6). F-I: FTIR spectrum of P2 variants with altered linkers in PBS (6% DMSO), without (F, H) or with (G, I) the presence of PolyP. The absorbance is normalised between all samples and the spectrum is scaled to the amide I region between 1600–1700 cm⁻¹. Peaks within the left (1689–1696 cm⁻¹) and right (1610–1642 cm⁻¹) grey bar are assigned to β-sheet, while peaks within the grey bar in the middle (1651–1659 cm⁻¹) is assigned to α-helix. The FTIR spectrums are representative of three independent experiments. For A-B, one-sample Wilcoxon signed-rank test was used to compare the MIC median of each Pept-in group to P2 MIC (12.5 μg/mL). For D-E, a two-tailed Student t-test was performed for calculating statistical significance between the mean of P2 variant and P2. Asterisks indicating the level of the p value centred over the error bar mean: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 5. Improved antimicrobial activity against laboratory-derived P2-resistant strains and clinically isolated multi-drug resistant strains.

A-B: Flow cytometry analysis of ancestors and P2-resistant E. coli treated with FAM-labelled Pept-ins for different time points at the concentration of FAM-P2-MIC from three independent experiments. FAM MFI of FAM positive cell population (A), and the percentage of FAM and Draq7 positive cells (B) when treated with P2 and P2-VFV. Error bars represent SEM (n = 3). C: SIM images of ancestors and P2-resistant strains treated with P2 and P2-VFV for 4 h at the concentration of FAM-P2-MIC. Amyloid-specific dye pFTAA was incubated with bacteria for 1.5 h. D-F is associated with S12 Table, the number of tested isolates for E. coli, A. baumannii, and K. pneumoniae is 34, 12, and 12, respectively. D: The percentage of E. coli, A. baumannii, or K. pneumoniae isolates which had a MIC at >32, 32, 16 or 8 μg/mL for P2, P2_4R_L and P2_4R_GV. The colours white to blue in the heatmap indicate an increased percentage of isolates. E: The percentage of E. coli, A. baumannii, or K. pneumoniae isolates which had a MIC at >32, 32, 16, 8, or ≤4 μg/mL for P2, P2_A5F and V7Y (P2). The colours white to blue in the heatmap indicate an increased percentage of isolates. F: The percentage of E. coli, A. baumannii, or K. pneumoniae isolates which had a MIC at >32, 32, 16, 8, or ≤4 μg/mL for P2, P2_H12_PEG and P2_H12_GV. The colours white to blue in the heatmap indicate an increased percentage of isolates. G: SIM of K. pneumoniae treated by P2 and P2_H12_GV at 32 ug/mL for 2 h. Amyloid-specific dye pFTAA was incubated with bacteria for 1.5 h. Scale bar: 10 μm.

Fig 6. Enhancing antimicrobial potency while exhibiting no toxicity to mammalian cells.

A-B: Concentration-dependent toxicity by CellTiter Blue assay on HEK 293T cells for P2 and derivatives with improved antimicrobial potency. A: Derivatives with increased aggregation propensity or compatibility to the aggregate core structure. B: Derivatives of linker modification or cysteine addition. C: Derivatives of altered number of arginine residues. Error bars represent SEM. Two-tailed Student t-test was performed for calculating statistical significance between the mean of P2 variant and P2 at the corresponding concentration (n = at least 6). Asterisks indicating the level of the p-value centred over the error bar mean: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.