An anti-virulence drug targeting the evolvability protein Mfd protects against infections with antimicrobial resistant ESKAPE pathogens

Abstract

The increasing incidence of antibiotic resistance and the decline in the discovery of novel antibiotics have resulted in a global health crisis, particularly, for the treatment of infections caused by Gram-negative bacteria, for which therapeutic dead-ends are alarming. Here, we identify and characterize a molecule, NM102, that displays antimicrobial activity exclusively in the context of infection. NM102 inhibits the activity of the non-essential Mutation Frequency Decline (Mfd) protein by competing with ATP binding to its active site. Inhibition of Mfd by NM102 sensitizes pathogenic bacteria to the host immune response and blocks infections caused by the clinically-relevant bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa, without inducing host toxicity. Finally, NM102 inhibits the mutation and evolvability function of Mfd, thus reducing the bacterial capacity to develop antimicrobial resistance. These data provide a potential roadmap for the development of drugs to combat antimicrobial resistance.

Fig. 1. Identification of NM102 as an ATP competitor for Mfd activity.

A In vitro high-throughput screening for inhibitors of Mfd-C ATPase activity. The indicated compounds were tested at a final concentration of 100 mg/mL. The data were normalized to those of the DMSO control. Mfd-C ATPase activity was measured at 0.35 µM at an ATP concentration of 1 mM. The results are the average of two independent experiments done in duplicate with standard deviation. B Michaelis Menten plot for inhibition activity of NM102 (0–100 µM) on E. coli Mfd-C (0.35 µM) ATPase activity with ATP (0–0.3 mM). The results are the average of at least three independent experiments with standard deviation. The IC₅₀ was computed using GraphPad Prism7.05. C Lineweaver-Burk plot showing NM102 inhibition of Mfd-C ATPase activity through a competitive mode of action. The results are the average of at least three independent experiments with standard deviation. Source data are provided as a source data file. D Chemical structure of the NM102 compound.

Fig. 2. NM102 affinity to Mfd.

A ITC measurement of binding of NM102 to Mfd. The heat rate in the presence of NM102 was compared with and without NM102 and plotted as a function of time (higher panel), and the normalized fit was measured with an increased mole ratio (lower panel). B ITC thermodynamic parameters associated with the interaction of Mfd with ATP in the absence or presence of NM102. K_d, dissociation constant K_d, K_a, association constant, A, ratio of K_a in the presence vs absence of inhibitor NM102.

Fig. 3. NM102 specifically binds the ATP binding site of Mfd.

A NM102 inhibition of bacterial Mfd and eukaryotic proteins (ERCC3, ERCC6, XPD, yUpf1) ATPase activity. ATPase activity of the proteins, in the absence and presence of 100 µM of NM102, was assessed by using BIOMOL® Green reagent microtiter-plate assay. Data were normalized to that of the DMSO control. The graph shows the mean of at least two independent experiments with standard deviation. B Michaelis Menten plot for inhibition activity of NM102 (0 to 100 µM) on E. coli RecG (0.35 µM) ATPase activity with ATP (0 to 0.3 mM). The results are the average of two independent experiments done in duplicate with standard deviation. C Binding energy measured in silico in kcal/mol between ATP or NM102 and Mfd (left) anf Upf1 (right). The bindings of ATP and NM102 are shown in cyan, and orange, respectively. The graph shows the distribution of binding energies obtained for twenty poses of the substrate/ligand couple by AutoDock. Source data are provided as a source data file.

Fig. 4. NM102 docking in the ATP binding site of Mfd.

A Upper part: strip of the eight domains with the following color code blue D1a-D2-D1b, orange D3, pink D4, yellow D5, green D6, and red D7. Lower part: Mfd rendered surfaces of conformations L₀ inactive (pdb id 2eyq) and L₁ active (pdb id 6 × 26), and their corresponding view at 90°. The color code of the surface corresponds to the strip of domains shown above. Right part, top insert: Close-up of Mfd from E. coli with the docking of ATP (in stick with carbon colored in cyan) and down insert: similar view with NM102 (in stick with carbon colored in deepteal). The residues involved in the sphere of binding of ligands are shown as sticks. The color code of the domains is respected with residues of D5 and D6 in yellow and green, respectively, and residues of motifs I and II in red and blue, respectively. B Close-up of the docking of ATP (cyan sticks) and NM102 (orange stick) in Mfd of E. coli harboring the site-directed mutations F597A, F599A, K634A, and E730Q. For the sake of comparison, position of ATP in the non-mutated Mfd is shown as black lines.

Fig. 5. Antimicrobial effect of NM102 on Gram-negative bacteria during NO stress.

Strains were grown to exponential phase in LB medium. The bacteria solution was prepared in RPMI medium and dispatched in 96-wells plate. Bacteria were exposed for 4 h at 37 °C to 50 µL of increasing concentrations of NM102 alone A K. pneumoniae DOU or with NOC-5 as a NO donor, B K. pneumoniae DOU, C E. coli ATCC 25922, D P. aeruginosa CIP27853). The bacteria survival rate was calculated by normalizing bacteria load against control without NM102. The results reported are mean ± SD of four independent experiments each in triplicates, P values are calculated against the condition without NM102, using One Way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, the exact P values are provided as source data). IC₅₀ was computed using Graph Pad 7.05. Source data are provided as a source data file.

Fig. 6. Antimicrobial effect of NM102 in vivo in insects and mice.

A P. aeruginosa CIP27853 wt and ∆mfd strains were grown to late exponential phase in LB medium at 37 °C and injected into forth instar silkworm larvae without or with NM102 (2.78 µg/larvae). Boosts of NM102 (6.97 µg/larvae) were administrated at 4 h and 8 h post-infection. At 24 h post-infection larvae were crushed and the content of each tube was serially diluted on LB plates for CFU numeration. Each graph represents the values obtained for n insects with the mean value indicated. Each dot represents one larva (Pa wt n = 10, Pa wt +NM102 n = 10, Pa ∆mfd n = 15, Pa 3/18/25mfd + NM102 n = 15). P values are calculated against the condition wild type-strain without NM102, using Mann-Whitney test (p < 0.0001). For mice experiments, K. pneumoniae ATCC 700603 wt and ∆mfd (B), K. pneumoniae ALE (C), P. aeruginosa CIP27853 (D) were grown to late exponential phase and diluted in PBS. Intranasal (i.n.) bacterial administration was performed through slow instillation of 20 µL of bacterial suspension (1 × 10⁷ CFU). 200 µL of NM102 nanoformulation (NF-NM102 at 1.5 mg/kg or 6 mg/kg) or empty nanoformulation (control) were administrated in mice via the intraperitoneal (i.p.) route (B). Alternatively, 20 µl of a mixture containing bacterial suspension (1×10⁷ CFU) and NM102 nanoformulation (1.5 or 6 mg/kg) or empty nanoformulation was administered via the i.n. route (C, D). MPN (10 mg/kg) was inoculated via the i.p. route. Mice were sacrificed by cervical dislocation after 24 h and the log CFU in the lung was calculated per gram of organ. Each graph represents the bar plot obtained for n mice with mean value indicated: (B) CTR n = 9, NM102 1.5 mg/kg n = 4, NM102 6 mg/kg n = 9, MPN 10 mg/kg n = 6, ∆mfd n = 6, ∆mfd + NM102 n = 10; C CTR n = 8, NM102 1.5 mg/kg n = 8, NM102 6 mg/kg n = 8, MPN 10 mg/kg n = 8; (D) CTR n = 7, NM102 6 mg/kg n = 7, MPN 10 mg/kg n = 8, NM102 1.5 mg/kg+ MPN n = 8, NM102 6 mg/kg + MPN n = 7. P values are calculated using a two-tailed Mann-Whitney test (***p < 0.0005; **p < 0.001; *p < 0.005). The exact P values, minima, maxima, center, bound of box, and whisker are all provided as source data. Source data are provided as a source data file.

Fig. 7. NM102 is not toxic to the host.

A HeLa and Vero cells were cultured in DMEM at 37 °C and 5% CO₂. Cells were treated for 1 h with MPN at 10 and 100 µM, NM102 at 10 and 300 µM, NM102 nanoformulation at 1.29 and 5.16 mM or their respective control 10% DMSO or empty nanoformulation. Cytotoxicity was assessed using CellTiter96®AQ_ueous. Values for treated cells were normalized to the untreated control. The results reported are mean ± SD of three independent experiments each in triplicates. B Mice were i.p. treated with NM102 (6 mg/kg) or control. Changes in the mice body weight were assessed for 7 days. Source data are provided as a source data file.

Fig. 8. NM102 does not impact the gut microbiome.

Gut microbiome diversity is unaffected by NM102. Mice were infected with K. pneumoniae and left untreated or treated with NM102 nanoformulation (NF-NM102) or the empty nanoformulation (NF) as control. After 24 h, feces were collected and the microbiome diversity was analyzed by 16S rRNA gene sequencing. Relative abundance of bacterial families (A), Shannon index (B), and weighted and unweighted UniFrac distances (C) were determined. The bar colors represent bacterial families with mean abundance >1%. Each dot (B, C) and each column (A) represents the value obtained for one mouse. Error bars in Fig. 6B represent mean ± SEM. The sample size in Fig. 6 A, B, C is n = 7 mice per group. The statistical test in Fig. 6B is permutational anova (PERMANOVA). The statistical test in Fig. 6C is a two-tailed Wilcoxon test with Benjamini-Hochberg correction for multiple comparisons. Source data are provided as a source data file.

Fig. 9. NM102 inhibits Mfd evolvability activity.

A The mutation rate of E. coli wt, Δmfd, and Ω∆mfd/mfd strains were measured following exposure to NM102 (100 µM) or the DMSO control, using the frequency of spontaneous accumulation of resistant mutants to rifampicin as reference measurement. Data in this figure correspond to 12 independent cultures. Error bars show SEM. Lea Coulson analysis was used on webSalvador to determine significance using an efficiency parameter of ε = 0,5 (^∗p < 0.05; ^∗∗p < 0.01; ^****p < 0.001, the exact P values are provided as source data). Resistance evolution of E. coli to rifampicin (B) and streptomycin (C) was measured following exposure to NM102 (100 µM) or the DMSO control. Line plots show the median antibiotic concentration at each sampled time point for three independent experiments. Source data are provided as a source data file.