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. 2024 Feb 20;19(3):743–752. doi: 10.1021/acschembio.3c00773

Antibacterial Marinopyrroles and Pseudilins Act as Protonophores

Gabriel Castro-Falcón †, Jan Straetener ‡, Jan Bornikoel ‡, Daniela Reimer †, Trevor N Purdy †, Anne Berscheid ‡, Florence M Schempp †, Dennis Y Liu §, Roger G Linington §, Heike Brötz-Oesterhelt ‡,∥,⊥,*, Chambers C Hughes †,‡,∥,⊥,*
PMCID: PMC10949930  PMID: 38377384

Abstract

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Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. Marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way they exert this antibacterial activity has not been established. In this study, we explore their structure–activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds.

Marinopyrrole A (MarA, 1),1 pentabromopseudilin (PBP, 2),2,3 and pentachloropseudilin (PCP, 3)4,5 are densely halogenated hybrid pyrrole-phenol natural products isolated from various bacteria (Figure 1). Since the compounds have pronounced cytotoxicity against cancer cell lines, various studies have focused on the antieukaryotic properties of 1–3, wherein actin,6 Mcl-1,7 and others8 have been identified as eukaryotic targets for MarA (1), myosin for PBP (2) and PCP (3),9−11 and human lipoxygenases 15-hLO and 12-hLO for 2.12 However, the conclusion that 1 is a selective Mcl-1 inhibitor has been called into question.13,14 Various reports detailing the deleterious biological effects of 1–3 in mammalian cells,15 plants,16 phytoplankton,17 viruses,18 and parasitic protozoans19−21 continue to be disclosed. Although the original reports of these unusual compounds were tied to their antibacterial activity, where they uniformly showed potent activity against Gram-positive pathogens and little or no activity against Gram-negative pathogens,22,23 little work regarding their antibacterial mechanism of action has been reported.

Figure 1.

Figure 1

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Structures of marinopyrrole A (1), pentabromopseudilin (2), and pentachloropseudilin (3), including the original bacterial producer strain, reported biological activities and reported cellular targets. Mcl-1 = myeloid cell leukemia 1; IspD = 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase.

We therefore initiated a project with the aim of uncovering the antibacterial mechanism of action of 1–3. In 2013 it was proposed that PCP’s mechanism of action involved protein synthesis inhibition, perhaps via inhibition of a bacterial ATPase, using high-content bacterial imaging.24 Since then, several reports with structurally similar, densely halogenated pyrrole- and phenol-containing compounds, such as the pyrrolomycins,25 mindapyrroles,26 and armeniaspirol,27 have strongly indicated that 1–3 may act as protonophores. Nonetheless, a study that specifically addresses this question in live bacterial cells, one that is ideally in agreement with several papers examining the structure–activity relationships of the compounds,16,28−30 is lacking.

Results and Discussion

Antibacterial Activity

First, we assessed the antibacterial activity of (±)-1–3 against an extensive panel of bacterial species including both Gram-positive and Gram-negative pathogens at the Centre for High-Throughput Chemical Biology at Simon Fraser University (Supporting Information Tables S1 and S2). In general, the compounds showed broad but weak activity against Gram-negative pathogens (e.g., Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Vibrio cholerae). However, against Gram-positive pathogens (e.g., Staphylococcus aureus and Streptococcus pneumoniae) the natural products showed mostly sub-μM minimum inhibitory concentrations (MICs) (Supplementary Table S1), which is consistent with previous studies assessing the antibacterial activities of these compounds.1,22,23 We followed up on these results by determining MICs against two model strains, S. aureus NCTC8325 and Bacillus subtilis 168, to be used in our mechanism of action studies (Figure 2). Here, MarA (1) showed MIC values of 0.25 and 0.125 μg mL–1 against the respective bacterial strains. Pentabromopseudilin (2) and pentachloropseudilin (3) were particularly potent; the former gave MICs of 0.016 μg mL–1 against both strains and the latter MICs of 0.004 μg mL–1. The activity was not specific to prokaryotes, as 1–3 also showed significant cytotoxicity (Figure 2) against the cancer cell lines HeLa (human cervix epithelial carcinoma, IC50 0.63–4.20 μg mL–1) and A549 (human lung epithelial carcinoma, IC50 4.17–13.63 μg mL–1) as well as the noncancerous MRC5 (human fetal lung fibroblast, IC50 0.15–1.69 μg mL–1) cell line. The overall trend in cytotoxicity (PCP (3) > PCB (2) > MarA (1)) mirrored the observed trend in antibacterial activity, which might suggest a common mechanism of action for both cell types.

Figure 2.

Figure 2

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Antibacterial and cytotoxic activities of compounds 1–10. (A) MICs of 1–10 against S. aureus NCTC8325 and B. subtilis 168 and half maximal inhibitory concentrations (IC50s) against HeLa, A549, and MRC5 eukaryotic cells. Control = vancomycin (MIC) for the antibacterial assay and mitoxanthrone (IC50) for the cytotoxicity assay. The section in gray highlights the potent antibacterial activity of the natural products (1–3). (B) Structures of 4–10. MarA = marinopyrrole A; PBP = pentabromopseudilin; PCP = pentachloropseudilin; and NT = not tested.

We explored the structure–activity relationship using several synthetic compounds, focusing on the presence and absence of the halogen atoms and the polar N–H and O–H functional groups. Although monodeoxypyoluteorin (4), which corresponds to the monomer of MarA, showed antibacterial activity against S. aureus NCTC8325 and B. subtilis 168 (MIC 2 and 16 μg mL–1), the nonhalogenated synthetic compounds 5, 6, and 7 were devoid of notable activity. Contemplating a specific interaction with a protein target, this result could point toward a key role for the halogen atoms with regard to target affinity and antibacterial activity such as electrostatic halogen bonding interactions. O,O′-Dimethyl MarA (9) displayed greatly diminished antibacterial activity compared to the natural product, while N-methyl MarA (8) and N,O,O′-trimethyl MarA (10) showed no measurable antibacterial activity. This result could indicate key hydrogen-bonding interactions between MarA and its bacterial target(s), perhaps highlighting the particular importance of the N–H pyrrole hydrogen bond donor in MarA.

Cellular Localization of Coumarin Probes and Membrane Dyes

In order to elucidate the cellular mechanism of natural products 1–3, we first explored their localization in bacterial cells. HATU-mediated ester coupling of 1–7 with 6-heptynoic acid produced alkynes that were subsequently appended to a fluorescent coumarin azide via a copper-catalyzed click reaction. The coumarin itself was prepared via amide coupling of 3-azido-1-propanamine and 2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetic acid. The corresponding coumarins 1C–7C were strictly characterized by 1H and 13C NMR spectroscopy, including HSQC and HMBC experiments to determine the exact position of the fluorescent tag (Figure 3A) (Supplementary Tables S3, S4 and Supplementary Figures S13–S43). Against B. subtilis 168, the coumarin derivatives were 6–60 times less active than the unmodified natural products and some maintained considerable antibacterial activity (1C: 1 μg mL–1; 2C: 0.25 μg mL–1; 3C: 0.25 μg mL–1). We then prepared MarA-BODIPY (1B) in a manner similar to 1C via copper-catalyzed click reaction of the corresponding alkyne with azide-BDP-FL and also characterized this probe by NMR spectroscopy (Supplementary Table S5 and Supplementary Figures S47–S51). Against B. subtilis 168, 1B maintained considerable antibacterial activity with an MIC of 0.25 μg mL–1. As expected, control coumarin 11 and control BODIPY 12 displayed no antibacterial activity against B. subtilis 168 (MIC > 64 μg mL–1).

Figure 3.

Figure 3

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Cellular localization of coumarin probes, BODIPY probes, and membrane dye FM 5–95. (A) Structures of 1C–7C, 11, 1B, and 12. (B) B. subtilis 168 cells incubated with 1C (1 μg mL–1, 1× MIC), 2C (0.25 μg mL–1, 1× MIC), 3C (0.25 μg mL–1, 1× MIC) or 1B (0.5 μg mL–1, 2× MIC) for 10 min. Coumarin 11 (8 μg mL–1, MIC > 64 μg mL–1) and BODIPY 12 (8 μg mL–1, MIC > 64 μg mL–1) were used as negative controls. The top row shows the fluorescence channel, and the bottom row shows phase-contrast images. The micrographs display representative images of three biological replicates. Images for the coumarin probes were adjusted to the same microscopy settings for direct qualitative comparison; images for the BODIPY probes were also adjusted to the same microscopy settings, but these were different than the settings for coumarin imaging. Scale bar: 5 μm. (C) B. subtilis 168 cells incubated with 1 (0.125 μg mL–1, 1× MIC), 2 (0.016 μg mL–1, 1× MIC) and 3 (0.004 μg mL–1, 1× MIC) for 15 min and then stained with the membrane dye FM5–95 (20 μg mL–1). DMSO (1%) was used as a negative control and CCCP (100 μM) as a positive control. The top row shows the fluorescence channel, and the bottom row shows the phase-contrast images. The images displayed are representative of two biological replicates and are all adjusted to the same setting for qualitative comparison. Scale bar, 5 μm. CCCP = carbonyl cyanide m-chlorophenyl hydrazone.

Fluorescence images of B. subtilis 168 cells treated with 1C–3C and 1B showed different fluorescence intensities but a similar nonuniform localization with bright, irregular signals in the cell periphery and the septal cell area (Figure 3B). Control compounds 11 and 12 showed no labeling of the cells. This phenotype, shared by all three labeled natural products, was reminiscent of the distribution of widely used membrane dyes in cells exposed to membrane-active agents.31,32 Indeed, when B. subtilis 168 cells were treated with the (unlabeled) natural products 1–3 followed by staining with membrane dye FM 5–95, the images showed the same phenotype as the ones generated with 1C–3C and 1B (Figure 3C). The aggregated, inhomogeneous, “spotty” localization of the membrane dye FM 5–95 can also be generated by exposure of B. subtilis to the protonophore m-chlorophenyl hydrazone (CCCP), which we selected for comparison (Figure 3C). This phenotype provided the first hint that the bacterial membrane is the target of 1–3.

Protonophore Activity

In addition to their possible role in target affinity, the removal of halogen atoms and the derivatization of N–H and O–H groups, which had a significant effect on antibacterial activity, both serve to decrease the overall acidity of the phenolic compounds (Figure 2). Hydrophobic compounds bearing acidic functionality can act simply as protonophores and disrupt the proton gradient across cell membranes by active, protein-decoupled transport of protons across the membrane.31,33 The lower pH directly outside the cell membrane is essential for cell viability, as the passage of protons into the cell is coupled to important cellular processes like ATP synthesis and active transport of certain molecules across the membrane.

In order to probe for possible protonophoric activity of compounds 1–3 in live S. aureus cells, the potential-sensitive dye 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] was first utilized. In healthy cells with polarized membranes, a significant proportion of the fluorescent green dye forms aggregates that exhibit red fluorescence, and the ratio of red–green fluorescence is relatively high. When the membrane is depolarized, red fluorescence is reduced in favor of green fluorescence, and the ratio of red–green fluorescence is decreased. Similar to the prototypical protonophore CCCP, the addition of compounds 1–3 produced clear MIC-dependent membrane depolarization within minutes, as signified by a decrease in the red–green fluorescence ratio (Figure 4A). As often seen for ionophores, the depolarization starts at a fraction of the MIC and is already strong at the MIC, indicating that this mechanism is indeed relevant for bacterial killing.34 Monodeoxypyoluteorin (4) also caused membrane depolarization in an MIC-dependent manner, but, in accordance with the higher MIC, an 8-fold higher concentration than MarA (1) was needed to elicit the same strength of the effect (Supplementary Figure S55). Mirroring their weak antibacterial activity, O,O′-dimethyl MarA (9) gave some slight membrane depolarization, while N-methyl MarA, (8) and N,O,O′-trimethyl MarA (10) produced no measurable membrane depolarization, even at high concentrations (Supplementary Figure S55). Importantly, potent antibacterial activity against S. aureus NCTC8325 was retained for PBP-coumarin (2C, MIC 0.25 μg mL–1) and PCP-coumarin (3C, MIC 0.125 μg mL–1), and, as for the natural products themselves, concentration-dependent membrane depolarization occurred around the MIC, indicating that these fluorescent probes mimic the natural products PBP (2) and PCP (3) in both activity and mechanism of action (Supplementary Figure S56). In contrast, the control coumarin 11, which showed no antibacterial activity against S. aureus NCTC 8325 (MIC > 64 μg mL–1), did not cause membrane depolarization. The inherent fluorescence of MarA-coumarin (1C) interfered with the fluorescence-based assay, such that its ability to depolarize the bacterial membrane could not be accurately assessed.

Figure 4.

Figure 4

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Compounds 1–3 act as protonophores. (A) Time-resolved effect of 1–3 on the membrane potential in S. aureus NCTC8325 cells based on 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] staining at concentrations around the respective MICs. Cells were treated with compounds 1–3 at 4×, 2×, 1×, 0.5×, and 0.25× MIC in the presence of DiOC2(3). DMSO (1%) was used as a negative control and CCCP (5 μM) as a positive control. Error bars indicate the standard deviation (SD) of two biological replicates. The arrow shows the time point of the addition of test compounds. (B) Time-resolved effect of 1–3 on the intracellular pH in S. aureus NCTC8325 cells based on 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) staining in relation to the MICs. Cells were treated with compounds 1–3 at 4×, 2×, 1×, 0.5×, and 0.25× MIC in the presence of BCECF AM. DMSO (1%) was used as a negative control and CCCP (50 μM) as a positive control. Error bars indicate the standard deviation (SD) of the three biological replicates. The first arrow shows the time point of test compound addition; the second arrow shows the time point for adding nigericin (20 μM), an H+/K+ antiporter further reducing the intracellular proton concentration and equilibrating it to the external environment. (C) GFP-MinD distribution in growing B. subtilis 168 cells after compound exposure. Cells were incubated for 10 min with compounds 1–3 at a 1× MIC. DMSO (1%) was used as a negative control, and CCCP (100 μM) as a positive control. The micrographs (top row) show representative images of the fluorescence channel which were all adjusted to the same microscopy settings for direct qualitative comparison. The fluorescence heatmaps (bottom row) display the spatial distribution of the median fluorescent GFP-MinD signal quantified from at least N ≥ 100 cells per experiment from three independent biological replicates. Warmer colors indicate stronger localization in this position. (D) Membrane integrity of S. aureus NCTC8325 after 35 min treatment with compounds 1–3 at 4× MIC visualized by SYTO9 (green) and propidium iodide (PI, red) costaining. DMSO (1%) and CCCP (50 μM) were used as negative controls and nisin (100 μg mL–1) as a positive control. The images shown are an overlay of the brightfield, SYTO9, and PI channels. (E) Brightfield visualization of the formation of blebs in B. subtilis 168 cells after 10 min of treatment with compounds 1–3 at 4× MIC and fixation by acetic acid/methanol. Blebs are formed by the cytoplasmic membrane bulging through breaches in the peptidoglycan. DMSO (1%) was used as a negative control, and vancomycin (25 μg mL–1) was used as a positive control. CCCP = carbonyl cyanide m-chlorophenyl hydrazone. Scale bars, 5 μm.

To further determine whether the membrane depolarization observed upon treatment of S. aureus cells with compounds 1–3 can be specifically attributed to protonophore activity, the ratiometric pH indicator dye 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed. Upon addition of compounds 1–3, the intracellular pH dropped significantly in an MIC-dependent manner within minutes (Figure 4B). For MarA (1) at 1× MIC, the pH dropped from 7.8 to 7.5 (Δ = 0.3 pH units) in 10 min; for PBP (2) and PCB (3) at 1× MIC, the pH dropped from 7.8 to 7.6 (Δ = 0.2 pH units) in 10 min. At 4× MIC all three compounds produced an intracellular reduction in pH from 7.8 to 7.4 (Δ = 0.4 pH units). A PCP (3) concentration of 48 nM (4× MIC) yielded a similar intracellular pH reduction as our positive control CCCP concentration at 50 μM (16× MIC). Again, monodeoxypyoluteorin (4) exhibited MIC-dependent protonophore activity at relatively high concentrations in accordance with its higher MIC. Very weak or no protonophore activity was observed for methylated MarA derivatives 9 and 10 (Supplementary Figure S57A).

Our last method to investigate the membrane depolarization effected by compounds 1–3 in live B. subtilis cells involved the cell division protein MinD, which normally localizes to the polar and septal regions of the cell. Strahl and Hamoen have shown that the loss in transmembrane potential brought about by the presence of a protonophore disrupts bacterial cell division and, more specifically, causes a dramatic delocalization of several conserved cell division proteins including MinD.35 Therefore, we monitored the cellular localization of GFP-MinD in B. subtilis cells after treatment with 1–3 in order to confirm by an independent assay the hypothesis that these compounds are, indeed, protonophores. In untreated B. subtilis cells, a strong fluorescence signal from GFP-MinD can be seen in the polar region as well as the septal region (Figure 4C). After a short treatment with the natural products and positive control CCCP, the fluorescence signal of GFP-MinD became diffuse and spotty, confirming depolarization in vivo. The effect correlated very well with the respective MIC concentrations of the different compounds and the structure–activity relationship observed in the membrane depolarization assay. Accordingly, the less active monodeoxypyoluterin (4) and O,O′-dimethyl MarA (9) showed spotty fluorescence only at their elevated MICs, and N,O,O′-trimethyl MarA (10) did not affect the localization of GFP-MinD (Supplementary Figure S57B). Interestingly, the fluorescence of the delocalized GFP-MinD was significantly brighter in cells treated with natural products 1–3 compared to cells treated with CCCP.

Finally, in order to determine whether the membrane depolarization observed upon treatment with compounds 1–3 involved a disruption of the integrity of the cytoplasmic membrane, commonly termed “pore formation”, we collected fluorescence microscope images of treated cells exposed to a mixture of two dyes, the membrane-permeant green-fluorescing SYTO9 and the red-fluorescing propidium iodide, which cannot cross an intact cytoplasmic membrane. Even after exposure to MarA (1) and PCP (3) at 4× MIC, S. aureus cells showed only green fluorescence after SYTO9/propidium iodide costaining, a result pointing toward cell membrane integrity and the absence of pores (Figure 4D). Like the protonophore CCCP, 1–3 markedly reduced the intensity of the green fluorescence compared with the DMSO control. By contrast, cells treated in parallel with the prototypical pore-forming antibacterial nisin showed a clear red fluorescence indicative of pore formation. Furthermore, treatment of B. subtilis cells with 1–3 did not cause the blebbing phenotype that is associated with a weakening of the peptidoglycan sacculus, a telltale signature of exposure to peptidoglycan-damaging agents. Here, vancomycin served as a positive control (Figure 4E).

The conclusion that the antibacterial mechanism of action for MarA (1), PBP (2), and PCP (3) is tied to their protonophoric activity is supported by further evidence. First, the natural (−)-(M)-MarA atropisomer and the unnatural (+)-(P)-MarA atropisomer showed equal potency against S. aureus,1 a finding that points toward a mechanism of action that does not involve binding to a chiral protein. Second, synthetic derivatives of 1–3 that showed enhanced antibacterial activity (lower MIC) possess greater overall acidity (low pKa) and hydrophobicity (high log D); the presence of an acidic phenol O–H or pyrrole N–H proton and a high degree of halogenation are critical for activity. For example, MarA (1), which showed strong protonophore activity and antibacterial activity against S. aureus, has a calculated pKa of 6.9 and a calculated logD (pH 7.4) of 5.8 (PhysChem Suite, Percepta software, ACD/Laboratories). Dipyrrole 7, a nonhalogenated version of MarA, is less acidic (pKa = 7.5), less hydrophobic (log D = 3.8), and showed significantly weaker antibacterial activity against S. aureus (see Figure 2). In contrast, MarA derivatives prepared by adding chlorine, fluorine, and trifluoromethyl substituents onto the marinopyrrole core structure are more acidic (pKa = 5.7–6.9), more hydrophobic (log D = 5.5–6.3), and showed greater antibacterial activity against S. aureus.23,28−30 Presumably, PBP (2) and PCP (3) are much stronger protonophores and antibacterials than MarA (1) because they are stronger acids, although this distinction is not borne out by calculations.

One model to explain the protonophore activity of MarA, PBP, and PCP specifies that the natural products act as proton shuttles that bind protons in the acidic extracellular microenvironment directly outside the cell, translocate protons across the lipid bilayer, and then release protons into the neutral cytoplasm. According to the Henderson–Hasselbach equation, stronger acids would dissociate to a greater extent inside the cell and more readily dissipate the proton motive force (PMF) and decrease the membrane potential (ΔΨ), and this might explain why stronger marinopyrrole-based acids exhibit greater antibacterial activity. The hydrophobicity of this compound class ensures a strong affinity for and preferential positioning in the membrane environment, and free migration seems plausible given their small size. An alternative model places the hydrophobic natural products inside the membrane within a more static structure, where they associate with the hydrophobic lipid bilayer and facilitate proton translocation and PMF dissipation through an unknown mechanism.

Methods

General Experimental Procedure

Compounds 1, 2, and 4–10 were synthesized according to published procedures (Supplementary Figures S1–S10).23,36,37 PCP (3) was synthesized using a modification of the published PBP (2) procedure (Supplementary Figures S11 and S12).37 All other reagents and solvents were purchased commercially and were used without further purification. Compounds and reaction mixtures were analyzed on an analytical 1100 Series Agilent Technologies HPLC system coupled to UV/vis (210, 254, and 360 nm) and an evaporative light-scattering detector (ELSD) using a Phenomenex Luna reversed-phase C18(2) column (100 × 4.6 mm, 5 μm, 100 Å) with a 10 or 20 min gradient from 10–100% CH3CN in water containing 0.1% formic acid and a 1.0 or 0.7 mL min–1 flow rate. Using the same column and a 20 min gradient from 10–100% CH3CN in water containing 0.1% formic acid and a 0.7 mL min–1 flow rate, HPLC-ESI-QTOF-MS/MS was performed on an analytical Agilent 1260 Infinity Series LC system coupled to a 6530 Series QTOF mass spectrometer. Column chromatography was performed on a Teledyne CombiFlash Rf+ Lumen flash chromatography system. Preparative HPLC was performed using a Millipore Waters 600E solvent delivery system with a Phenomenex Luna C18(2) or C8(2) column (250 × 21.2 or 250 × 10 mm, 5 μm, 100 Å) or Phenomenex Kinetex C18 (250 × 21.2 mm, 5 μm, 100 Å) and a 13 or 3 mL min–1 flow rate. Compounds were detected with a single-wavelength Knauer UV detector at 254 or 360 nm. 1H and 2D NMR spectra were recorded on a Jeol 500 MHz spectrometer in CD3OD (3.31 ppm) or a Bruker Avance III 400 MHz spectrometer in CDCl3 (7.26 ppm). 13C NMR spectra were recorded on the same instruments at 125 MHz in CD3OD (49.0 ppm) or CDCl3 (77.2 ppm). The following abbreviations are used to indicate the multiplicity in 1H NMR spectra: s = singlet, d = doublet, t = triplet, dd = doublet of doublets, ddd = doublet of doublets of doublets, td = triplet of doublets, q = quartet, p = quintet, m = multiplet, br s = broad singlet.

General Procedure for 6-Heptynoic Acid Coupling

Phenols 1–7 were dissolved in DMF (1 mL). EDC (7 equiv) and DMAP (0.1 equiv) were then added, followed by 6-heptynoic acid (1.2–3.0 equiv) and Et3N (2 equiv). The reaction mixtures were stirred for 2–23 h at RT. The progress of the reactions was monitored using HPLC and TLC. The reactions were quenched by adding a saturated NH4Cl solution (3 mL), and the aqueous layers were then extracted with EtOAc (3 × 3 mL). The combined EtOAc extracts were washed with brine (1 mL), dried over Na2SO4, filtered, and concentrated to dryness. The products were purified using preparative reversed-phase HPLC.

General Procedure for the Click Reaction

Click reaction was conducted under standard conditions using alkyne (1 equiv), coumarin azide or BODIPY azide (1.5 equiv), CuSO4 (0.1 equiv), and sodium ascorbate (0.2 equiv) in DMF:water (1:1). The reaction mixtures were directly purified using preparative reversed-phase HPLC.

1C: the compound was purified via HPLC [C8(2) Luna, 250 × 10 mm, 65% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 15 min]. See Table S3 for the NMR data. HRESIMS m/z [M + Na]+ 968.1514, calcd for C45H39Cl4N7O8Na, 968.1512.

2C: the compound was purified via HPLC [C8(2) Luna, 250 × 10 mm, 70% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 13 min]. See Table S4 for the NMR data. HRESIMS m/z [M + Na]+ 1008.8168, calcd for C33H31Br5N6O5Na, 1008.8171.

3C: the compound was purified via HPLC [C8(2) Luna, 250 × 10 mm, 65% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 18 min]. See Table S4 for NMR data. HRESIMS m/z [M + Na]+ 789.0692, calcd for C33H31Cl5N6O5Na, 789.0696.

4C: the compound was purified via HPLC [C8(2) Luna, 250 × 10 mm, 50% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 20 min]. 1H NMR (500 MHz, CD3OD): δ 8.30 (t, J = 5.4 Hz, 1H), 7.70 (s, 1H), 7.58 (overlap, 1H), 7.55 (overlap, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.73 (dt, J = 9.0, 2.5 Hz, 1H), 6.54 (s, 1H), 6.54 (overlap, 1H), 6.04 (s, 1H), 4.36 (t, J = 6.9, 2H), 3.68 (s, 2H), 3.22 (m, 2H), 3.04 (s, 6H), 2.68 (t, J = 2.7 Hz, 2H), 2.47 (t, J = 6.6 Hz, 2H), 2.09 (quint, J = 6.8 Hz, 2H), 1.62 (m, 4H). 13C NMR (125 MHz, CD3OD): δ 183.2, 173.4, 171.5, 164.4, 157.3, 155.0, 152.9, 149.8, 133.3, 132.6, 130.8, 130.5, 127.1, 127.0, 124.7, 123.6, 123.1, 120.0, 120.0, 112.3, 110.7, 110.7, 109.9, 98.9, 40.4, 37.9, 34.6, 31.2, 29.8, 26.0, 25.1. HRESIMS m/z [M + Na]+ 715.1804, calcd for C34H34Cl2N6O6Na, 715.1815.

5C: the compound was purified via HPLC [C8(2) Luna, 250 mm × 10 mm, 45% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 14 min]. 1H NMR (500 MHz, CD3OD): δ 8.29 (t, J = 5.4 Hz, 1H), 7.69 (s, 1H), 7.56 (td, J = 7.4, 1.6 Hz, 1H), 7.54 (d, J = 7.6, 1.7 Hz, 1H), 7.36 (td, J = 7.6, 1.0 Hz, 1H), 7.20 (dd, J = 8.0, 0.6 Hz, 1H), 7.13 (m, 1H), 6.74 (dd, J = 9.0, 2.5 Hz, 1H), 6.62 (dd, J = 3.9, 1.4 Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 6.23 (dd, J = 3.9, 2.4 Hz, 1H), 6.04 (s, 1H), 4.36 (t, J = 7.0, 2H), 3.68 (s, 2H), 3.35 (s, 1H), 3.21 (m, 2H), 3.04 (s, 6H), 2.65 (t, J = 7.1 Hz, 2H), 2.42 (t, J = 6.8 Hz, 2H), 2.08 (quint, J = 6.9 Hz, 2H), 1.59 (m, 4H). 13C NMR (125 MHz, CD3OD): δ 184.6, 173.4, 171.5, 164.4, 157.4, 155.0, 152.9, 149.7, 148.9, 134.0, 132.9, 132.7, 130.8, 128.2, 127.0, 126.9, 124.6, 123.7, 122.1, 111.8, 111.8, 110.8, 110.7, 110.7, 109.9, 98.9, 40.4, 37.9, 34.6, 31.1, 29.7, 26.0, 25.1. HRESIMS m/z [M + Na]+ 647.2584, calcd for C34H36N6O6Na, 647.2594.

6C: the compound was purified via HPLC [C18(2) Luna, 250 × 10 mm, 50% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 16 min]. See Table S4 for NMR data. HRESIMS m/z [M + Na]+ 619.2636, calcd for C33H36N6O5Na, 619.2645.

7C: the compound was purified via HPLC [C8(2) Luna, 250 × 10 mm, 55% MeCN in water (0.1% TFA), 360 nm, 3 mL min–1, tR = 18 min]. See Table S3 for NMR data. HRESIMS m/z [M + Na]+ 832.3067, calcd for C45H43N7O8Na, 832.3071.

11: the compound was purified via HPLC [C18(2) Luna, 250 × 21.2 mm, 40% MeCN in water (0.1% TFA), 360 nm, 13 mL min–1, tR = 11 min]. 1H NMR (500 MHz, CD3OD): δ 8.31 (t, J = 5.5 Hz, 1H), 7.72 (s, 1H), 7.57 (d, J = 8.9 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.05 (s, 1H), 4.36 (t, J = 7.0, 2H), 3.70 (s, 2H), 3.64 (s, 3H), 3.23 (m, 2H), 3.06 (s, 6H), 2.70 (t, J = 7.1 Hz, 2H), 2.35 (t, J = 7.0 Hz, 2H), 2.09 (quint, J = 6.8 Hz, 2H), 1.66 (m, 2H), 1.66 (m, 2H). 13C NMR (125 MHz, CD3OD): δ 175.7, 171.3, 164.3, 157.2, 154.9, 152.7, 126.9, 123.4, 110.6, 110.5, 109.8, 98.8, 49.5, 49.3, 52.0, 40.2, 37.7, 34.4, 31.0, 29.9, 25.9, 25.4. HRESIMS m/z [M + Na]+ 492.2219, calcd for C24H31N5O5Na, 492.2223.

1B: the compound was purified via HPLC [C18 Kinetex, 250 mm × 21.2 mm, 60% MeCN in water (0.1% FA), 254 nm, 13 mL min–1, tR = 6 min]. See Table S5 for NMR data. HRESIMS m/z [M + H]+ 991.2040, calcd for C46H42BCl4F2N8O6, 991.2043.

12: the compound was purified via HPLC [C18 Kinetex, 250 mm × 21.2 mm, 75% MeCN in water (0.1% FA), 254 nm, 13 mL min–1, tR = 17 min]. 1H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 7.10 (s, 1H), 6.87 (d, J = 4.0 Hz, 1H), 6.30 (d, J = 4.0 Hz, 1H), 6.14 (s, 1H), 6.05 (m, 1H), 4.20 (t, J = 6.7 Hz, 2H), 3.66 (s, 3H), 3.27 (t, J = 7.3 Hz, 2H), 3.20 (q, J = 6.2 Hz, 2H), 2.75 (t, J = 7.12 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H), 2.57 (s, 3H), 2.35 (t, J = 7.0 Hz, 2H), 2.26 (s, 3H), 2.00 (p, J = 6.6 Hz, 2H), 1.70 (m, 4H). 13C NMR (101 MHz, CDCl3) δ: 174.1, 172.4, 161.0, 156.7, 147.2, 144.6, 135.5, 133.4, 128.3, 124.1, 122.0, 120.9, 117.4, 51.7, 47.8, 36.3, 35.9, 33.8, 30.4, 28.8, 25.1, 25.0, 24.5, 15.1, 11.5. HRESIMS m/z [M + H]+ 515.2751, calcd for C25H34BF2N6O3, 515.2754.

Antimicrobial Assays

Minimal inhibitory concentrations were determined by the broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI).38 Two-fold dilutions of the test compounds were prepared in cation-adjusted Mueller-Hinton broth (BD Difco) in 96-well round-bottom polystyrene plates (Sarstedt) with each well containing 50 μL of test compound solution at twice the final concentration. The wells were then inoculated with 50 μL of the bacterial suspension of the test strains, prepared by the direct colony suspension method, to a final inoculum of 5 × 105 colony-forming units (CFU) mL–1 in 100 μL final volume. The plates were incubated at 37 °C for 20 h, and the minimal inhibitory concentration (MIC) was determined as the lowest concentration of the test compound that inhibited visible bacterial growth.

Cytotoxicity Assays

Cytotoxicity evaluation was performed using a 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin) assay in RPMI cell culture medium (Gibco Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Gibco Life Technologies) for the HeLa cell line and Dulbecco’s Modified Eagle’s Medium (DMEM) cell culture medium (Gibco Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum for the A549 and MRC5 cell lines. A 2-fold serial dilution of the test compounds was prepared in a 96-well polystyrene microtiter plate and seeded with trypsinized HeLa, A549, or MRC5 cells to a final cell concentration of 1 × 104 cells per well. After 24 h of incubation at 37 °C, 5% CO2, and 95% relative humidity, resazurin was added to a final concentration of 200 μM, and cells were again incubated overnight. Cell viability was assessed by determining the reduction of resazurin to resorufin by measuring the fluorescence in a Tecan Infinite M200 Pro reader at an excitation wavelength of 560 nm and an emission wavelength of 600 nm in relation to an untreated control.

Fluorescence Microscopy

In order to monitor the localization of the coumarin derivatives, B. subtilis cells were grown in LB overnight at 37 °C with agitation (180 rpm), diluted 1:100 in fresh medium, and grown at 37 °C to an OD600 of 0.3–0.4. Cells were incubated with 1C (1 μg mL–1, 1× MIC), 2C (0.25 μg mL–1, 1× MIC), 3C (0.25 μg mL–1, 1× MIC) or 1B (0.5 μg mL–1, 2× MIC) for 10 min and then washed at least four times with PBS. Coumarin 11 (8 μg mL–1) or BODIPY 12 (8 μg mL–1) were used as negative controls. Subsequently, the cells were placed on microscopy slides covered with a thin layer of agarose (1.2% in PBS) and visualized by brightfield and fluorescence microscopy in a Zeiss Axio Observer Z1 LSM800 at λex 353 nm/λem 465 nm for the coumarin probes and λex 488 nm/λem 509 nm for the BODIPY probes. Images were acquired with an Orca Flash 4.0 V2 camera (Hamamatsu) and an α Plan-Apo 100x/1.46 Oil Ph3 objective (Zeiss). Image processing was performed in FIJI.39 All the labeling experiments were repeated three times.

FM 5–95 labeling after antibiotic exposure was conducted as described previously.40 Briefly, B. subtilis cells were grown in LB medium at 37 °C, 200 rpm to an OD600 of 0.3. Cells were treated with either 1 (0.125 μg mL–1, 1× MIC), 2 (0.016 μg mL–1, 1× MIC), 3 (0.004 μg mL–1, 1× MIC), 4 (16 μg mL–1, 1× MIC), 8 (32 μg mL–1, 1× MIC), 9 (128 μg mL–1) or DMSO (1%) as a negative control and incubated at 37 °C for 15 min (9 for 30 min). Samples were labeled with 20 μg mL–1 of N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 5–95, Molecular Probes) and visualized as described above at λex 587 nm/λem 610 nm.

In order to visualize the cellular localization of GFP-MinD in B. subtilis cells, a strain harboring a GFP-minD construct was grown in LB overnight at 37 °C with agitation (180 rpm).35 The culture was then diluted 1:100 in fresh medium, supplemented with 0.1% xylose (w/v) for GFP-MinD expression, and grown at 37 °C to an OD600 value of 0.6–0.8. Cells were incubated for 10 min with compounds 1–4, 8, and 9 at 1× MIC. CCCP (100 μM) was used as a positive and DMSO (1%) as a negative control. Cells were visualized as described above at λex = 488 nm/λem = 509 nm. To quantitatively assess the GFP-MinD localization, fluorescence heatmaps, presenting the spatial distribution of the median fluorescence of the GFP-MinD signal, were created with MicrobeJ41 from N ≥ 100 cells per experiment from three independent biological replicates.

Membrane Potential Assay

S. aureus NCTC8325 cells were grown in LB medium supplemented with 0.1% glucose at 37 °C with agitation (200 rpm) to an OD600 of 0.75. Cells were then pelleted and resuspended to an OD600 of 0.5 in PBS and incubated with 30 μM 3,3′-diethyloxacarbocyanine iodide (DiOC2(3), Molecular Probes, Fisher Scientific) for 15 min in the dark. The loaded cells were transferred to a black 96-well flat bottom polystyrene microtiter plate (BRAND), and a baseline measurement was taken for 2 min at an excitation wavelength of 485 nm and two emission wavelengths, 530 nm (green) and 630 nm (red), using a microplate reader (Tecan Spark). A concentration series of the test compounds (in 1% DMSO) was added, and the measurement continued as described above for 15 min. The protonophore CCCP was used as a positive control at a concentration of 5 μM, and DMSO was used as a negative control at a concentration of 1%.

Protonophore Assay

S. aureus NCTC8325 cells were grown in LB medium at 37 °C with agitation (200 rpm) to an OD600 of 0.8. Cells were then pelleted and resuspended in PBS and incubated with 25 μg mL–1 BCECF-AM dye (Molecular Probes, Fisher Scientific) for 30 min in a water bath at 30 °C. Cells were washed twice with PBS buffer containing 25 mM glucose and incubated for another 5 min in a water bath at 37 °C. The treated cells were transferred to a black 96-well flat bottom polystyrene microtiter plate (BRAND), and a baseline measurement was taken for 5 min at two excitation wavelengths, λex 440 and λex 490 nm, and one emission wavelength, λem 530 nm, using a microplate reader (Tecan Spark). A concentration series of the test compounds (in 1% DMSO) was added, and the measurement continued as described above for 10 min. Then, 20 μM nigericin was added to each well, and the measurement was continued for a final 5 min. The protonophore CCCP was used as a positive control at a concentration of 50 μM, and DMSO was used as a negative control at a concentration of 1%. The intracellular pH was calculated based on a calibration curve with nigericin and PBS buffer adjusted for various defined pH values.

Pore-Forming Assay

Pore formation was monitored by using the Live/Dead BacLight bacterial viability kit (Molecular Probes). S. aureus NCTC8325 cells were grown in LB medium at 37 °C to the exponential phase. Aliquots (100 μL) of the cells were treated with 1 (1 μg mL–1, 4× MIC), 2 (0.064 μg mL–1, 4× MIC), 3 (0.016 μg mL–1, 4× MIC), CCCP (50 μM), nisin (100 μg mL–1, Sigma-Aldrich) or DMSO (1%) as a negative control. Samples were incubated for 20 min, and then 0.2 μL of a 1:1 mixture of SYTO9 and propidium iodide (PI) was added per 100 μL of culture. Samples were further incubated for 15 min at RT in the dark before microscopic analysis. Samples were visualized by brightfield and fluorescence microscopy at λex 483 nm/λem 500 nm (SYTO9) and at λex 305 nm/λem 617 nm (PI) on microscope slides covered with a thin film of 1% agarose using a Zeiss Axio Observer Z1 automated microscope. Images were acquired with an Orca Flash 4.0 V2 camera (Hamamatsu) and an alpha Plan-Apochromat 100x/1.46 Oil Ph3 objective (Zeiss). Images were processed by using the Zen software package (Zeiss).

Blebbing Assay

B. subtilis 168 cells were grown in cation-adjusted Mueller–Hinton medium at 37 °C with agitation (200 rpm) to an OD600 of 0.35. Aliquots (100 μL) of the cells were treated with 1 (0.5 μg mL–1, 4× MIC), 2 (0.064 μg mL–1, 4× MIC), 3 (0.016 μg mL–1, 4× MIC), vancomycin (25 μg mL–1) or DMSO (1%) as a negative control. Samples were incubated for 30 min and then 25 μL were added to a fresh tube containing 100 μL of a 1:3 (v:v) mixture of acetic acid and methanol. Samples were visualized by bright-field microscopy on microscope slides covered with a thin film of 1% agarose using a Zeiss Axio Observer Z1 automated microscope. Images were acquired with an Orca Flash 4.0 V2 camera (Hamamatsu) and an alpha Plan-Apochromat 100x/1.46 Oil Ph3 objective (Zeiss). Images were processed using the Zen software package (Zeiss).

Acknowledgments

This work was supported by funding from the Deutsches Zentrum für Infektionsforschung (German Center for Infection Research, DZIF) (H.B.-O. and C.H.) via project TTU 09.826—Precision access to antibiotic compounds and targets (PAACT), from the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) (J.B., J.S., H.B.-O.) via TRR 261 (project ID 398967434), and from the Natural Sciences and Engineering Research Council of Canada Discovery grant program (R.G.L.). H.B.-O. and C.H. acknowledge infrastructural support from the Cluster of Excellence EXC 2124: Controlling Microbes to Fight Infection (CMFI, project ID 390838134).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00773.

  • 1H NMR spectra for compounds 1–10; 1H and 13C NMR spectra for compounds 11–12; 1H, 13C, COSY, HSQC, and HMBC for compounds 1C–7C and 1B. Additional membrane potential data, intracellular pH data, and GFP-MinD images for compounds 4, 8–10, 1C–3C, and 11 (PDF)

The authors declare no competing financial interest.

Supplementary Material

cb3c00773_si_001.pdf (4.6MB, pdf)

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