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. 2022 Aug 24;8(8):1145-1158.
doi: 10.1021/acscentsci.2c00598. Epub 2022 Aug 10.

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections

Erica N Parker  1 , Brett N Cain  1 , Behnoush Hajian  2 , Rebecca J Ulrich  1 , Emily J Geddes  1 , Sulyman Barkho  2 , Hyang Yeon Lee  1 , John D Williams  3 , Malik Raynor  3 , Diana Caridha  3 , Angela Zaino  2 , Mrinal Shekhar  2 , Kristen A Muñoz  1 , Kara M Rzasa  2 , Emily R Temple  2 , Diana Hunt  2   4 , Xiannu Jin  3 , Chau Vuong  3 , Kristina Pannone  3 , Aya M Kelly  1 , Michael P Mulligan  1 , Katie K Lee  2 , Gee W Lau  5 , Deborah T Hung  2   4 , Paul J Hergenrother  1
Affiliations

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections

Erica N Parker et al. ACS Cent Sci. .

Erratum in

Abstract

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.

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Conflict of interest statement

The authors declare the following competing financial interest(s): The University of Illinois and the Broad Institute have filed patents on some compounds described herein.

Figures

Figure 1
Figure 1
FabI inhibitors. (A) Debio-1452 is highly potent against S. aureus; Debio-1452-NH3 retains this potency and gains modest activity against many Gram-negative pathogens. (B) The structure–activity relationship (SAR) of Debio-1452 showing regions amenable to substitution (highlighted in green) and those critical for antibacterial activity (highlighted in red).
Figure 2
Figure 2
Debio-1452 analogue synthesis and antibacterial activity. The general synthetic route utilized to synthesize amine-containing compounds, and their antimicrobial activities against Gram-positive and Gram-negative bacteria. (†) indicates dose-independent trailing growth observed; see Supporting Information, Extended Data Figure S1. MIC values were determined using the microdilution broth method, as outlined by CLSI. All experiments were performed in biological triplicate. E. coli ΔtolC = JW5503.
Figure 3
Figure 3
Optimized synthesis of fabimycin. The synthetic route used to access gram-scale quantities of fabimycin, utilizing dynamic kinetic resolution (DKR) to install the critical stereogenic center.
Figure 4
Figure 4
Antimicrobial activity of fabimycin against clinical isolates. (A) The susceptibility of clinical isolates (Gram-negative species and S. aureus) to fabimycin, Debio-1452-NH3, and Debio-1452. MICs performed in biological triplicate. (B) Further exploration of the breadth of fabimycin’s antibacterial activity against diverse clinical isolate panels of K. pneumoniae and A. baumannii, as compared to levofloxacin. MICs performed in biological duplicate.
Figure 5
Figure 5
Fabimycin mode of action studies. (A) Spontaneous resistance frequencies of S. aureus, E. coli, and A. baumannii versus fabimycin. Data represent three replicates for each pathogen with error bars representing the SEM. (B) Point mutations in FabI observed in fabimycin-resistant colonies, and the corresponding MIC values of fabimycin versus the mutants. All MICs were performed in biological triplicate.
Figure 6
Figure 6
Co-crystal structures of fabimycin and its enantiomer with FabI. (A) Co-crystal structure of fabimycin with E. coli FabI with NADH cofactor (PDB 7UMW). (B) Co-crystal structure of (R)-7 in E. coli FabI with NADH cofactor (7UM8). (C) Water network surrounding fabimycin in the E. coli FabI active site. (D) Water network surrounding (R)-7 in the E. coli FabI active site.
Figure 7
Figure 7
Computational and biophysical evaluation of fabimycin and its enantiomer. (A) Molecular dynamic simulations of fabimycin and its enantiomer using the co-crystal structures in E. coli FabI, demonstrating the enhanced flexibility (decreased stability) of (R)-7. (B) The determined enthalpy changes upon binding to FabI as assessed by isothermal titration calorimetry (ITC), as well as observed stabilization in a differential scanning fluorimetry assay of FabI (from E. coli and A. baumannii) upon compound binding relative to the holoenzyme. Tm values are means of technical triplicates with error shown as the standard deviation.
Figure 8
Figure 8
Plasma stability and in vivo efficacy of fabimycin. (A) Assessment of fabimycin stability in plasma. Data shown as the mean and standard deviation from two experiments. (B) Acute pneumonia infections initiated in CD-1 mice with A. baumannii AR-0299 (1.6 × 108 CFUs per mouse intranasally). Mice were treated with vehicle (8 mice) or FabI inhibitor (8 mice per group) 4, 23, and 41 h postinfection (50 mg/kg intramuscular) and the bacterial burden evaluated at 48h postinfection. (C) Neutropenic mouse thigh infection initiated in CD-1 mice with A. baumannii AR-0299 (1.22 × 106 CFUs per mouse intramuscular in thigh) were treated with vehicle (8 mice) or FabI inhibitor (8 mice per group) 2, 6, and 11 h postinfection (50 mg/kg intramuscular), and the bacterial burden was evaluated 26h postinfection. (D) Neutropenic mouse thigh infection initiated in CD-1 mice with S. aureus USA300 LAC (2.3 × 106 CFU per mouse intramuscular in thigh) were treated with vehicle (eight mice) or FabI inhibitor (eight mice per group) 2 and 7 h postinfection (5 mg/kg retro-orbital IV), and the bacterial burden was evaluated 24 h postinfection. Debio-1452-tosylate used. FabI inhibitors formulated with 20% SBE-β-CD in H2O. In B, C, and D statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons. NS, not significant. ****P < 0.0001. Error bars represent standard deviation.
Figure 9
Figure 9
In vivo efficacy of fabimycin in a murine UTI model. After inducing diuresis, infection initiated in C3H/HeJ mice (8 per arm, 1.38 * 109 CFU/mouse transurethral) with E. coli AR-0055 and treated with fabimycin (IV) at varying concentrations three times daily with bacterial enumeration at 168h postinfection. Fabimycin formulated in 17% Cremophor EL, 3% SBE-β-CD in H2O which was the formulation used in the vehicle arm (administered intravenously on the same schedule as fabimycin). Colistin was formulated in H2O with 0.9% NaCl and administered subcutaneously. Percentage in red indicates the percentage of animals with bacterial counts below the limit of detection (LOD, indicated by the dotted horizontal line). In A—D statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons. NS, not significant. *P = 0.0243, ***P < 0.001, ****P < 0.0001. Data represented as the mean with s.e.m.
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