Fig. 1: Fluorofolin inhibits P. aeruginosa growth through inhibition of DHFR and rapidly accumulates within cells.

a, Schematic of folate metabolism in PA14. Red crossed circles indicate enzymes lacking activity in PA14. b, The structure of fluorofolin. c, C.f.u.s ml⁻¹ of P. aeruginosa PA14 after 4 h treatment with 5% dimethylsulfoxide (DMSO, solvent control), 6.2 μg ml⁻¹ fluorofolin (2× MIC), 250 μg ml⁻¹ TMP (2× MIC) or 4 μg ml⁻¹ polymixin B (2× MIC). Data points represent 3 biological replicates with 3 technical replicates. Mean ± s.d. d, DHFR (FolA) activity measured on purified E. coli FolA by measuring the change in sample absorbance at 340 nm due to DHFR-dependent NADPH consumption. Activity was related to an untreated standard condition using 60 μM NADPH and 100 μM DHF. IC₅₀ values were derived from the Hill equation fits on reactions performed with increasing antibiotic concentrations. e, An analogous assay to Fig. 2b using purified human DHFR. f, Metabolite abundance of dUMP, AICAR and glycinamide ribonucleotide (GAR) of P. Aeruginosa PA14 treated with 5% DMSO (solvent control), 6.3 μg ml⁻¹ fluorofolin (2× MIC) or 250 μg ml⁻¹ TMP (2× MIC) for 15 min. Metabolite abundance was quantified in comparison to the solvent-only control. Data represent mean ± s.d. for 3 biological replicates. *P ≤ 0.05; NS, not significant; calculated using two-sided unpaired t-test using Prism 9. g, Intrabacterial drug metabolism (IBDM) of PA14 treated with 5 μM fluorofolin or TMP. Samples were taken at time t = 0, 30, 60 and 90 min. Data represent mean ± s.d. of 3 technical replicates. h, Cumulative accumulation of drugs over 90 min by AUC of IBDM curves. Data represent mean ± s.d. of 3 technical replicates. ****P < 0.0005, P = 4.5512 × 10⁻⁵; two-sided t-test using Prism 9.

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