Inhibiting quinolone biosynthesis of Burkholderia†

2-Alkylquinolones are important signalling molecules of Burkholderia species. We developed a substrate-based chemical probe against the central quinolone biosynthesis enzyme HmqD and applied it in competitive profiling experiments to discover the first known HmqD inhibitors. The most potent inhibitors quantitatively blocked quinolone production in Burkholderia cultures with single-digit micromolar efficacy.


Materials
All solvents and chemical reagents for synthesis and LC-MS/MS analysis were purchased from Sigma-Aldrich, VWR or Carl Roth. The LC-MS standards C6-HSL, C8-HSL, C10-HSL, 3-OH-C8-HSL and 3-OH-C10-HSL were obtained from Cayman Chemicals. Flash chromatography was performed using silica gel. NMR spectra were obtained using a Bruker Avance III 400 NMR instrument equipped with a BBFO plus probe and calibrated on the residual solvent peak.
Chemical shifts are reported in ppm and coupling constants (J) are given in Hz. High-resolution mass spectra were recorded by an ESI-TOF MS (Bruker Daltonics amicroTOFII) equipped with a Chromolith FastGaradient Rp18e 50x2 mm column (Merck). Low-resolution mass analysis was performed using an ESI-IT MS (Bruker Daltonics Esquire 3000plus) with a Nucleoshell 50x2 mm RP-18 2.7 µm column (Macherey-Nagel). Quantitation was performed on a Finnigan TSQ  Quantum (Thermo electron corporation) mass spectrometer. For SDS-gel preparation and SDS-PAGE a PeqLab PerfectBlue TM was used. The recording and analysis of the gels was performed with the Fusion-FX7 Advanced of Vilber Lourmat (Eberhardzell, Germany) using the software CaptAdvance. The expression vectors pET-51b(+)/hmqD and E. coli BL21 pET-51b(+)/hmqDmutant (Cys114Ala) were purchased from GeneScript, and the genes were codon optimized for expression in E. coli.

Preparation of overnight cultures
For preparation of overnight cultures, a small amount of a bacterial cryo-stock (15% glycerol, stored at -80 °C) was inoculated in 5 mL LB-(lysogenic broth)-Lennox medium (LB medium) in sterile 13 mL polypropylene tubes (Sarstedt,ref 62.515.028), supplemented with antibiotics if indicated, and grown for 14-16 h at indicated growth conditions (Table S1)

Growth curve analysis of Burkholderia ambifaria during inhibitor treatment
In sterile 15 mL polypropylene centrifuge tubes with screw caps (Roth), 4 µL of a 50 mM DMSO Stock of inhibitor 4, or 4 µL DMSO as vehicle control, was added (0.1 % final DMSO concentration). 4 mL of LB medium and 60 µL of an overnight culture of Burkholderia ambifaria AMMD was added per sample. The caps of the sample tubes were slightly opened and fixed in a defined position by tape to ensure equal oxygen delivery to all samples. For oxygen limiting Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2021 conditions caps were tightly closed. The samples were incubated until indicated time points at 30 °C (240 rpm). Afterwards, 50 µL LB medium and 50 µL of culture were transferred in a cuvette, mixed and the optical density of cells was measured at a wavelength of λ= 600 nm (OD 600 ) with a photometer of Eppendorf (BioPhotometer® plus). The experiment was performed in 3 biological replicates.
In situ labelling of Burkholderia ambifaria with probe CAA 60 µL of an overnight culture of Burkholderia ambifaria DSM 16087 was added to 20 mL LB medium in 50 mL sterile polypropylene centrifugal tubes with screw caps (Roth) and grown for 10 h at 37 °C (180 rpm). Then, 2 mL of cell culture was transferred into a 2 mL Eppendorf tube, centrifuged (4500 g, 5 min, 4 °C), and the cells washed with 1 mL PBS. The remaining cell pellet was carefully resuspended in 100 µL PBS before addition of 1 µL of a 0.1 mM CAA probe stock in DMSO. After incubation for 1h at 37 °C in a shaking incubator (180 rpm) the cells were washed two times with 1 mL PBS. For cell lysis, 100 µL PBS was added and the cells lysed by ultrasound treatment (10 % amplitude, 0.5 sec on, 1.0 sec off, 20 pulses, Branson Digital Sonifier), and the cell debris removed by centrifugation (12000 g, 10 min, 4 °C). The resulting lysates were used for click-chemistry and SDS-PAGE.
Competitive labelling of Burkholderia ambifaria proteome with probe CAA and BA 60 µL of an overnight culture of Burkholderia ambifaria DSM 16087 was added to 15 mL LB medium in 50 mL sterile polypropylene centrifugal tubes with screw caps (Roth) and grown for 10 h at 37 °C (220 rpm). The caps of the tubes were slightly opened and fixed in a defined position to ensure equal oxygen delivery. Then, 2 mL of cell culture was transferred into a 2 mL Eppendorf tube, centrifuged (4500g, 5 min) and the cells were washed with 1 mL PBS.
The remaining cell pellet was carefully resuspended in 100 µL PBS before 1 µL inhibitor stock in DMSO was added to obtain final concentrations of 10, 50, 100 and 200 µM. The cell suspension was incubated for 30 min at 37 °C (180 rpm) and lysed using the FastPrep-24 system (MP biomedicals). Cell debris was removed by centrifugation (13500 rpm, 5min, 4 °C) and the supernatant was sterile filtrated. 100 µL of filtrate were transferred to a 1.5 mL Eppendorf tube, 1 µL of 2 mM CAA or BA probe stock in DMSO was added (final concentration 20 µM) and incubated at room temperature for 30 min. Subsequently, click chemistry and SDS-PAGE analysis were performed.
In situ labelling of E. coli HmqD cells E. coli BL21 cells containing pET-51b(+)/hmqD (wild type hmqD) or pET-51b(+)/hmqDmutant (Cys112Ala mutant) were grown in an overnight culture supplemented with 100 μg/mL carbenicillin. On the next day, the culture was inoculated 1:50 in LB media, supplemented with 100 μg/mL carbenicillin and incubated at 37°C (180 rpm) to an OD 600 of 0.5. Next, 150 µM (final concentration) isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the culture was incubated at 37°C for 30 min (180 rpm). For each sample, 500 µL of induced bacterial culture was transferred into a 1.5 mL Eppendorf tube, the cells pelleted by centrifugation (4500 g, 5 min, 4°C), and the cell pellet washed with 800 µL PBS before re-suspending in 100 µL PBS. 1 μL of inhibitor stock in DMSO or DMSO as vehicle was added for the indicated final concentration, mixed gently and pre-incubated for 15 min at rt. Then, 1 μL of a 100 µM CAA stock in DMSO was added to a final concentration of 1 µM and incubated for 60 min at rt. After incubation, the cells were pelleted by centrifugation (4500 g, 5 min, 4 °C ), washed (2 x 800 µL PBS), the cell pellet was re-suspended in 100 μL PBS, lysed by ultrasound treatment (10 % amplitude, 0.5 sec on, 1.0 sec off, 10 pulses, Branson Digital Sonifier), and the cell debris removed by centrifugation (12000 g, 10 min, 4 °C). The resulting lysates were used for click-chemistry and SDS-PAGE. After fluorescence scanning, Coomassie staining was applied to compare protein concentrations in the gel and thereby validate the experiments ( Figure S11).

In situ determination of IC 50 values
E. coli BL21 cells containing pET-51b(+)/hmqD (wild type HmqD) were grown in an overnight culture supplemented with 100 μg/mL carbenicillin. On the next day, the culture was inoculated 1:50 in LB medium, supplemented with 100 μg/mL carbenicillin and incubated at 37°C (180 rpm) to an OD 600 of 0.5. Next, 150 µM IPTG was added to induce protein expression, and the culture was incubated at 37°C for 120 min (180 rpm). For each sample, 2 mL of induced bacterial culture was transferred in a 2 mL Eppendorf tube, the cells pelleted by centrifugation (4500 g, 5 min, 4 °C), and the cell pellet washed with 1 mL PBS before re-suspending in 100 µL PBS. To this cell suspension, 1 µL of inhibitor stock in DMSO was added at indicated concentrations (n = 3) and incubated for 30 min at 37 °C (180 rpm). Afterwards, the probe CAA was added to give a final concentration of 1 µM and incubated 60 min at rt. The cells were washed (2 x 1 mL PBS), lysed by sonication, cell debris was removed by centrifugation and rhodamine attached via click chemistry. Subsequently, the samples were analyzed by SDS-PAGE and in-gel visualization. Integrated fluorescence intensities of the bands were measured using ImageJ software and IC 50 values calculated from a dose-response curve generated using GraphPad Prism software.

LC-MS/MS analysis of MAQs, MAQNOs and AHLs
Ultra-high performance liquid chromatography was performed on a Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific, Waltham, MA) using a Nucleodur C18 Gravity-SB 100 x 2 mm, 3 µm column (Macherey-Nagel). The flow rate was 0.5 mL/min and the column temperature was set to 40 °C. The injection volume was 5 µL using pick up injection mode.
Eluent A was 0.1% formic acid in water and eluent B was acetonitrile. The gradient was 20-100 % B in 10 min, 100 % B for 2 min, 100-20 % B in 1 min and 20 % B for 2 min. MS/MS analysis was performed by a Finnigan TSQ  Quantum (Thermo electron corporation) mass spectrometer in the SRM (Selected Reaction Monitoring) scan mode. As ion source a heatedelectrospray ionization (HESI-II probe, Thermo scientific) was used. In the optimized conditions the ion spray voltage was 3500 V, vaporizer temperature 300 °C, capillary temperature 380 °C, sheath gas pressure 60 psi, ion sweep gas pressure 2 psi, aux gas 10 psi. The tube lens offset was 80 V and skimmer offset 0. Data was acquired in a mass range of m/z 130-350, and MS/MS acquisition of all compounds was performed in positive mode and fixed collision energy of 30 eV. Dwell time was 0.050 sec for all compounds. The software Quan Browser Thermo Xcalibur 3.1.66.10 was used for quantitative analysis. The standard peak area of the product ions was fitted by linear regression versus the known concentrations to generate a standard curve.  151.2, 140.6, 135.2, 132.7, 126.2, 125.0, 124.6, 124.5, 118.7, 116.7, 36.4, 33.5, 32.8, 30.3, 29.8, 23.7, 14.4, 10.7 A MeOH stock solution of isolated and purified  2 MNQ were prepared at 1 mg/mL in a glass vial and stored at -80 °C up to one month. A series of standard solutions was prepared freshly before LC-MS/MS analysis by serial dilutions of the stock solution in LB medium (standard concentrations and equation see Table S4). For each sample, 2.8 mL of standard containing LB medium was transferred in 10 mL headspace vials, 2 mL ethyl acetate was added and vortexed for 5 sec. Then, 1 mL of organic layer was transferred in a clean 1.5 mL glass vial and the solvent evaporated by a gentle stream of nitrogen. Standards were extracted in 3

AHL standards:
MeOH stock solutions of C6-HSL, C8-HSL, C10-HSL, 3-OH-C8-HSL and 3-OH-C10-HSL were prepared at 0.1 mg/mL in glass vials and stored at -80 °C up to one month. A series of standard solutions was prepared freshly before LC-MS/MS analysis by serial dilutions of the stock solution of each analyte in acetonitrile/water (1:1) (standard concentrations and equations see Table S5).

Determination of MAQ levels after inhibition with inhibitors 4, 5 and 7
In general, quinolone levels were determined in 3 replicates. 4 µL of a respective DMSO stock solution of inhibitors or 4 µL DMSO as vehicle control (0.1 % final DMSO concentration) was added in 15 mL polypropylene centrifuge tubes with screw caps (VWR). Afterwards 4 mL LB medium was added in each tube to obtain final inhibitor concentrations of 5, 20 and 50 µM.
Then, 60 µL of a Burkholderia ambifaria AMMD overnight culture was added. Caps of the sample tubes were tightly screwed. Samples were incubated for 24 h at 28 °C (240 rpm).
Afterwards, samples were centrifuged, supernatants sterile filtered, metabolites extracted and quantified by LC-MS/MS. The experiment was performed in 3 biological replicates.
Quantification of MAQ and AHL levels in dose dependence with inhibitor 4 4 µL of a respective DMSO stock solution of inhibitor 4 or DMSO as vehicle control was added in 15 mL polypropylene centrifugal tubes with screw caps (VWR). Then, 4 mL LB medium was added in each tube to obtain final inhibitor concentrations of 1, 5, 10, 20 and 50 µM.
Subsequently, 60 µL of a Burkholderia ambifaria DSM 16087 overnight culture was added and samples incubated for 11 h at 37 °C (220 rpm). Caps of the tubes were slightly opened and fixed in a defined position to ensure equal oxygen delivery. Afterwards, samples were centrifuged (4500 rpm, 10 min) and supernatants sterile filtrated. 2.8 mL filtrate was transferred to a 10 mL headspace vial, 2 mL ethyl acetate were added and vortexed for 5 seconds. Then, 1 mL of organic layer was transferred to a clean 1.5 mL glass vial and the solvent evaporated under a gentle stream of nitrogen. For LC-MS/MS analysis, 25 µL acetonitrile/water (1:1) was added to each sample. The experiment was performed in 3 biological replicates.

Extraction of MAQs and AHLs from supernatant
2.8 mL of sterile supernatant was transferred in 10 mL headspace vials (VWR, VWRI548-0237), 2 mL ethyl acetate was added, closed with a magnetic screw cap (silicone/PTFE, LABSOLUTE, WC/07621126) and vortexed thoroughly for 5 sec. Then, 1 mL of the organic layer was transferred in a clean 1.5 mL glass vial (in case of poor layer separation vials were centrifuged at low speed). The solvent evaporated by a gentle stream of nitrogen. The extract was stored at -80 °C until measurement. For analysis, 25 µL acetonitrile/water (1:1) was added to each sample.

Proteomic analysis of HmqD after in situ labelling in E. coli cells with probe CAA
For proteomic analysis, in situ labelling of E. coli HmqD cells was performed as described above, and 2 x SDS loading buffer was added directly to the resulting lysates. After SDS-PAGE and Coomassie staining, the corresponding protein band was cut out of the gel. Tryptic Protein samples were centrifuged (18000 x g, 5 min, rt), 500 µL protein solution was transferred in avidin-bead containing eppendorf tubes and incubated for 1h under careful shaking.
Afterwards, beads were washed with 1 mL 0.4% SDS in PBS (3x, 400 x g, rt), 1 mL 6M urea in milliQ water (2x, 400 x g, rt) and 1 mL PBS (3x, 400 x g, rt). For proteomic analysis, proteins on beads were digested by trypsin and analyzed by LC-MS/MS (performed by the Proteomics facility of the University of Konstanz as described above).

Standard procedure to synthesize chloromethyl ketones
In accordance to the literature, 2 ZnCl 2 (4.0 eq.) was dried under high vacuum and under nitrogen atmosphere dry 1,2-dichloroethane was added. Aniline (1.0 eq.) and chloroacetonitrile (3.4 eq.) were added and BCl 3 (1 M in dichloromethane, 3.0 eq.) was added slowly. The mixture was heated to reflux (90°C, oil bath) for 15 h. It was cooled to room temperature, 1 M aq. HCl was added and heated to reflux (100°C, oil bath) for 90 min. After cooling to room temperature, DCM and water were added and the aqueous layer was extracted with DCM (3x). The organic layers were combined, dried over MgSO 4 and the solvent evaporated.

1-(2-Amino-5-(aminomethyl)phenyl)-2-chloroethan-1-one (2)
Synthesis according to the standard procedure to synthesize chloromethyl ketones. Scale: ZnCl 2 (4.46 g, 32.7 mmol, 4 eq), 51 mL dry 1,2-dichloroethan, 4-aminobenzylamine (927 µL, 8.19 mmol, 1 eq), chloroacetonitrile (1.78 mL, 28.2 mmol, 3.44 eq), 1 M BCl 3 in dichloromethane (24.6 mL, 24.6 mmol, 3 eq) and in the second step 51 mL 1 M HCl aq . The suspension was diluted with 50 mL DCM and 50 mL water. The organic phase was separated to neutralise the aqueous phase with Na 2 CO 3 to pH 8. The aqueous phase was diluted with 100 mL water and 200 mL DCM and the suspension filtered. The filter cake was washed with water (100 mL), DCM (250 mL) and EE (250 mL) and the filtered aqueous phase extracted with DCM (5 x 100 mL). The organic phases were combined and dried over MgSO 4 . After solvent evaporation, the crude was taken up in 10 mL cold chloroform by sonification to discard the formed precipitate and evaporate the solvent of the filtrate. This step was repeated three times to receive the product as yellow solid (90 mg, 5.5% yield).
The solution was stirred for 60 min at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for additional two hours. Afterwards, water (5 mL) was added and extracted with DCM (3 x 5 mL). The combined organic layers were dried over anhydrous MgSO 4 and concentrated to yield BA as a colorless oil (169.7 mg, 89.3 %).    100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78   Supporting Figure S1:

MS of peptide AQC*SGGLYGIEIAR (Cys*114) labeled by probe CAA: A Q C* S G L L Y G I E I E I A R
y 4 y 5 y 6 y 7 y 8 y 9 y 10 y 11 y 12 y 13 b 4 b 5 b 6 b 7 b 8 b 9 b 10 b 11 b 12 b 13  Figure S3: Tryptic peptide fragment covalently modified by probe CAA after in situ labelling of E. coli BL21 cells overexpressing HmqD. In the background of whole E. coli cell lysate only peptide fragments with CAA bound to the active site cysteine of HmqD were detected.  Supporting Figure S20. 1 H (a) and 13 C (b) spectra of probe BA in CDCl 3 .