Design of a new class of broad-spectrum therapeutics targeted to drug-resistant bacteria

Figures & data

Figure 1. (a) Bacteria-Targeting Concept: Spacer-linked Anthraquinone-TPP (AQ-TPP) Conjugates. (b) Anthraquinone natural product: emodin.

Scheme 1. Reagents and conditions: (a) 4-amino-1-butanol, DMSO, 95°C; (b) 1,3-di-4-piperidylpropane, DMSO, 95°C; (c) TFA, diethyl ether; (d) (4-carboxybutyl)triphenylphosphonium bromide, DCC, DMAP, CH₂Cl₂; (e) 4-(carboxybutyl)triphenylphosphonium bromide, PyBOP, HOBt, DIPEA, DMF.

Table 1. Minimum inhibitory and bactericidal concentrations of compounds (4) and (5) screened against M. smeg mc²155 and MRSA (NCTC 13616)*.

	M. smegmatis mc^2155		MRSA (NCTC 13616)
Compound	MIC	MBC	MIC	MBC
4	4 µg/mL (5.6 μM)	4 µg/mL (5.6 μM)	1 µg/mL (1.4 μM)	2 µg/mL (2.8 μM)
5	4 µg/mL (4.8 μM)	8 µg/mL (9.6 μM)	1 µg/mL (1.2 μM)	1 µg/mL (1.2 μM)

*Data was obtained using EUCAST serial 2-fold dilution protocols for MIC and MBC determinations (EUCAST 2022); the number of observations for compounds 4 and 5 in each strain = 3. No variation in MIC or MBC values was observed between experiments performed in triplicate.

Figure 2. Confocal microscopy live cell imaging of HCT-116 colon cells, treated with 2 μM of compound (4) for 2 h. Panel (i) (A) Compound (4) stained with LysoTracker^TM Green DND26 (60 nM), (B) LysoTracker^TM Green DND26 (60 nM), and (C) merged image showing no colocalization. Panel (ii) (A) Compound (4) stained with MitoTracker^TM Green FM (50 nM), (B) MitoTracker^TM Green FM (50 nM), and (C) merged image showing clear colocalization. Areas where compound (4) and mitochondria colocalize appear as yellow/orange (C). Captured at 63 × magnification, scale bar 10 μm.

Figure 3. Cytotoxic effect of different MOIs of M. smeg mc²155 infection on differentiated THP-1 cells over 24 h, determined by the MTT assay. Results are mean ± SD of three independent determinations. NTC = no treatment control, 0.1% Triton-X-100 was the positive control. Statistical Analysis was performed using Graph Pad Prism 9.3.1. One-way ANOVA using Dunnett’s multiple comparison test, where **p < 0.01.

Figure 4. Colony counts on Mueller Hinton agar plates following treatment of intracellular infections. THP-1 cells were infected with M. smeg mc²155 for 4 h at an MOI of 1:5, then washed and treated for 24 h. Colony counts were measured following the growth of M. smeg mc²155 on solid agar after incubation for 72 h. Results are mean ± SD of three independent determinations. Statistical Analysis was performed using Graph Pad Prism 9.3.1. One-way ANOVA using Dunnett’s multiple comparison test, where *p < 0.05.

Figure 5. Colony counts on Mueller Hinton agar plates following treatment of intracellular infections. THP-1 cells were infected with M. smeg mc²155 for 4 h at an MOI of 1:5, then washed and treated for 24 h. Colony counts were measured following the growth of M. smeg mc²155 on solid agar after incubation for 72 h. In vitro intracellular LC₅₀ determination for compound (4) was calculated to be 3.8 µg/mL.

Figure 6. Cytotoxic effect of Compounds 4 (A) & 5 (B) on differentiated THP-1 cells over 4, 24 and 48 h. Results are mean ± SD of three independent determinations (each repeat done in triplicate), NTC = no treatment control. Statistical Analysis was performed using Graph Pad Prism 9.3.1. Two-way ANOVA using Tukey’s multiple comparison test, where *p < 0.05 and **p < 0.01

EUCAST. 2022. EUCAST reading guide for broth microdilution. V4. https://www.eucast.org/ast_of_bacteria/previous_versions_of_documents.

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Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials. Raw data has been shared to https://figshare.com/ at https://doi.org/10.6084/m9.figshare.25335139

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