A small molecule that inhibits the evolution of antibiotic resistance

Abstract

Antibiotic resistance rapidly develops against almost all available therapeutics. Therefore, searching for new antibiotics to overcome the problem of antibiotic resistance alone is insufficient. Given that antibiotic resistance can be driven by mutagenesis, an avenue for preventing it is the inhibition of mutagenic processes. We previously showed that the DNA translocase Mfd is mutagenic and accelerates antibiotic resistance development. Here, we present our discovery of a small molecule that inhibits Mfd-dependent mutagenesis, ARM-1 (anti-resistance molecule 1). We found ARM-1 using a high-throughput, small molecule, in vivo screen. Using biochemical assays, we characterized the mechanism by which ARM-1 inhibits Mfd. Critically, we found that ARM-1 reduces mutagenesis and significantly delays antibiotic resistance development across highly divergent bacterial pathogens. These results demonstrate that the mutagenic proteins accelerating evolution can be directly inhibited. Furthermore, our findings suggest that Mfd inhibition, alongside antibiotics, is a potentially effective approach for prevention of antibiotic resistance development during treatment of infections.

Graphical Abstract

Figure 1.

In vivo high-throughput screen identifies ARM-1 as an inhibitor of Mfd. (A) Schematic of the in vivo screen design. When Mfd is present, RNAP complexes stalled at the LacI-bound operator sequence are quickly removed, resulting in little transcription of the Nluc gene and low luminescence output. When Mfd is either absent or inhibited, RNAP complexes stalled at the operator are not removed and are able to proceed during temporary dissociation of LacI, resulting in higher levels of luminescent output. (B) Structure of ARM-1. (C) Normalized luminescence/OD produced by Δmfd E. coli cells containing pRCB-NLuc and either S. enterica mfd (wild type, WT) or an empty vector (Δmfd); 100 μM ARM-1 was included when indicated. Horizontal lines represent the average. Statistical significance was determined via Welch’s ANOVA, *P < 0.05; n = 6 biological replicates (WT − ARM-1 versus WT + ARM-1, P = 0.0027; WT − ARM-1 versus Δmfd − ARM-1, P = 0.0122; Δmfd − ARM-1 versus Δmfd + ARM-1, P = 0.001).

Figure 2.

ARM-1 modulates Mfd’s enzymatic activity. (A) K_D of ARM-1 was determined using microscale thermophoresis. Values represent the average of three replicates except for the highest concentration of ARM-1 (n = 2). Error bars represent the standard error of the mean (SEM). (B) NADH-coupled ATPase assay. Absorbance at 340 nm by NADH is used to monitor the reaction. Data shown represent the average of two replicates except for 0 μM ARM-1 (n = 6). Error bars represent the SEM. (C) Transcription roadblock assay. RNAP was stalled by CTP starvation after which Mfd (pre-incubated with 25 μM ARM-1 when indicated) was added and allowed to remove stalled RNAP for 6 min. Representative gel as well as the average initiation complex (IC) and elongation complex (EC) intensity of five experiments is shown. Error bars represent the SEM.

Figure 3.

ARM-1 binding sites on E. coli Mfd from computational docking. (A) Overall view of the crystal structure of E. coli Mfd (PDB ID 2eyq, apo form), with potential ARM-1 (shown in space filling mode with black carbon atoms) binding sites in the N-terminal UvrB homology domain (righ part of the protein, in blue), and central (top part, in green) and C-terminal regions (left part, in red). (B) Overlay of the Mfd apo (PDB ID 2eyq; pink) and holo structures (PDB ID 6x50; green), affording a close-up view of the first two binding sites in the Mfd apo structure: ‘N-terminal’ and ‘central’. The ARM-1 molecule visible in the center is lodged between the RH and WB-adjacent helix that are colored in magenta and dark pink, respectively, in the apo-Mfd structure. This binding mode of ARM-1 potentially precludes unraveling of RH and a shift by the WB-adjacent helix (colored in yellow and orange, respectively) as seen in Mfd holo structure bound to RNAP and DNA. Selected regions and residues are labeled and ATP/Mg²⁺ and RNAP are visible near the lower edge of the image, middle and right, respectively.

Figure 4.

ARM-1 reduces mutagenesis. (A) Mutation rates of S. enterica strains measured using ciprofloxacin; n = 64 (WT − ARM-1), 64 (WT + ARM-1), 52 (Δmfd − ARM-1) and 52 (Δmfd − ARM-1). Cells were grown in the presence of 100 μM ARM-1 when indicated. Error bars represent 95% confidence intervals. (B) Mutation frequency after infection of human cells measured using 5-fluorouracil; n = 30 (WT − ARM-1), 24 (WT + ARM-1), 18 (Δmfd − ARM-1) and 18 (Δmfd − ARM-1). Cells were grown in the presence of 5 μM ARM-1 when indicated. Error bars represent the SEM. Statistical significance was determined via ordinary one-way ANOVA, *P < 0.05.

Figure 5.

ARM-1 inhibits the evolution of antibiotic resistance. Evolution of indicated species against rifampicin (A) and trimethoprim (B) in the presence of 100 μM ARM-1. Heatmaps show median MIC at the indicated time points. Data for strain/antibiotic combination presented are the result of at least eight biological replicates.