Please cite this article in press as: Tsai et al., Targeting Two-Component Systems Uncovers a Small-Molecule Inhibitor of Salmonella Virulence, Cell
The Singer RoToR HDA was used to inoculate assay plates containing 30 mL/well LPM alone or supplemented with dephostatin
(64 mg/ml), colistin (8 mg/ml), or dephostatin and colistin (64 and 8 mg/ml, respectively). Optical density at 600 nm (OD600) was
measured with a Tecan M1000 Infinite Pro plate reader at the time of inoculation (T0) and after ꢀ20 h (T20) of incubation at 37ꢂC.
Growth was calculated by subtracting reads at T0 from those at T20. Experiments were performed in duplicate.
Suppressor Isolation and Variant Analysis
Spontaneous resistant mutants were selected for by serial passage in liquid culture. A single colony of wild-type SL1344 was
grown overnight in LPM at 37ꢂC. The susceptibility of this strain to colistin was repeatedly tested by diluting bacteria 1:2000 into
200 mL LPM per well of 96-well microtiter plates, containing two-fold serial dilutions of colistin, beginning at 256 mg/ml. Every other
day, the highest well with observable growth was sub-cultured and grown overnight in LPM containing 8 mg/ml dephostatin and the
corresponding colistin concentration at which growth was observed. Serial passaging was done in duplicate. When strains displayed
resistance to >256 mg/ml colistin, genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen). Samples were sequenced on
a MiSeq 2x250 platform with paired-end reads. Raw reads were processed with FastQC and trimmed with Cutadapt (Martin, 2011) to
remove Nextera transposase sequences. Sequencing data was aligned against the reference genome for Salmonella (NC_016810)
Treatment of Infected Animals
Mice were infected intraperitoneally (i.p.) with ꢀ105 CFU Salmonella (SL1344) in 0.1 M Hepes (pH 8.0) with 0.9% NaCl. Groups of mice
were administered treatments every 24 h by i.p. injection beginning at the time of infection. 5% DMSO in water was given to vehicle
control groups of mice, dephostatin solubilized in 5% DMSO in water was administered at 2.5 or 5 mg/kg, and colistin solubilized in
water was administered at 5 or 10 mg/kg. Experimental endpoint was defined as 60 h post-infection for CFU determination, at which
Salmonella-infected mice exhibited ꢀ10-12% weight loss and displayed signs of clinical illness. For these experiments, mice were
euthanized, and the spleen and liver were homogenized in cold PBS, serially diluted, and plated on LB medium containing strepto-
mycin for determination of total CFU. Clinical endpoint in survival experiments was determined using body condition scoring
analyzing weight loss, reduced motility, and hunched posture.
Synthesis of Dephostatin
Dephostatin (compound 5, 3,4-Dihydroxy-N-methyl-N-nitrosoaniline) was synthesized from 4-nitrocatechol (compound 1) via the in-
termediate compounds 2 (3,4-Bis[(tert-butyldimethylsilyl)oxy]nitrobenzene), 3 (3,4-Bis[(tert-butyldimethylsilyl)oxy]-N-methylaniline),
and 4 (3,4-Bis[(tert-butyldimethylsilyl)oxy]-N-methyl-N-nitrosoaniline), using the route shown in Figure S6. For the following descrip-
tion of synthesis procedures, all chemical shifts in 1H NMR and 13C NMR spectra are reported in parts per million (ppm) relative to
tetramethylsilane (TMS), with calibration of the residual solvent peaks according to values previously reported (Gottlieb et al., 1997)
(chloroform: dH 7.26, dC 77.16; acetone: dH 2.05, dC 29.84, 206.26). When peak multiplicities are given, the following abbreviations are
used: s, singlet; d, doublet; t, triplet; q, quartet; sept., septet; dd, doublet of doublets; m, multiplet; br, broad; app., apparent; gem,
geminal. 1H NMR and 13C spectra were recorded at 700 MHz and 175 MHz, respectively, with a digital resolution (Bruker parameter:
€
FIDRES) of 0.15 Hz/point and coupling constants reported herein therefore have uncertainties of 0.3 Hz. Reactions were carried out
at room temperature (rt) if temperature is not specified. Unless otherwise noted, an automated flash chromatography system (Tele-
dyne CombiFlash Rf 200) was used for the purification of compounds on silica gel (either 40–60 mM or 20–40 mM particle size). Low
Resolution Mass Spectrometry (LRMS) measurements were recorded on an Advion Expression CMS Compact Mass Spectrometer.
Compound 2: A solution of 4-nitrocatechol (compound 1) (0.994 g, 6.407 mmol), tert-butylchlorodimethylsilane (TBS-Cl, 2.414 g,
16.02 mmol), imidazole (1.090 g, 16.02 mmol) and dimethylaminopyridine (DMAP, 0.020 g, 0.164 mmol) in DMF (12.5 ml) was stirred
at rt for 16 h. The reaction was poured into water (70 ml) and extracted with EtOAc (3 3 50 ml). The organic layers were combined and
washed with water (3 3 50 ml), dried over Na2SO4, and concentrated in vacuo to an orange oil. Flash chromatography (0:100/10:90
EtOAc/Hex) provided the TBS-protected catechol compound 2 as a pale yellow solid (2.34 g, 6.09 mmol, 95%). Rf = 0.69 (EtOAc/Hex
10:90). 1H NMR (700 MHz, CDCl3): d 0.25 (s, 6H, one of Si(CH3)2), 0.26 (s, 6H, one of Si(CH3)2), 0.99 (s, 9H, one of SiC(CH3)3), 1.00 (s,
9H, one of SiC(CH3)3), 6.87 (d, J = 8.9 Hz, 1H, ArH), 7.71 (d, J = 2.8 Hz, 1H, ArH), 7.77 (dd, J = 8.9 Hz, J = 2.8 Hz, 1H, ArH). 13C NMR
(175 MHz, CDCl3): d ꢁ3.9 (2C), ꢁ4.0 (2C), 18.6, 18.7, 25.9 (3C), 26.0 (3C), 116.4, 117.9, 120.1, 141.8, 147.2, 153.8. LRMS (APCI) m/z:
384.2 calcd for C18H34NO4Si2 (M + H); 384.3 obsd. All data were in accordance with the literature (Li et al., 2012). Compound 3: 2-
Propanol (14 ml) was added to a two-neck flask containing 10% Pd/C (0.307 g, 2.89 mmol). A solution of ammonium formate (0.994 g,
15.8 mmol) in water (1.39 ml) was transferred to the same flask. The reaction mixture was stirred for 2 mins to activate Pd/C. Next, the
nitroarene compound 2 (0.403 g, 1.050 mmol) and HCHO (37% solution in water, 0.83 ml, 10.0 mmol) were added. The reaction
mixture was degassed to remove oxygen, and stirred under nitrogen flow overnight at rt. The Pd/C catalyst was then removed by
filtration through Celite and the filtrate concentrated by rotary evaporation. The resulting mixture was diluted with CH2Cl2 (20 ml),
washed with brine (20 ml), dried over Na2SO4, and concentrated in vacuo to a dark brown oil. Flash chromatography (0:100/
10:90 EtOAc/Hex) provided the N-methylaniline derivative 3 as a brown oil (67 mg, 0.26 mmol, 26%). Rf = 0.25 (EtOAc/Hex
10:90). 1H NMR (700 MHz, CDCl3): d 0.15 (s, 6H, one of Si(CH3)2), 0.19 (s, 6H, one of Si(CH3)2), 0.97 (s, 9H, one of SiC(CH3)3),
0.98 (s, 9H, one of SiC(CH3)3), 2.76 (s, 3H, NHCH3), 6.12 (dd, J = 8.6 Hz, J = 2.8 Hz, 1H, ArH), 6.17 (d, J = 2.8 Hz, 1H, ArH), 6.67
(d, J = 8.6 Hz, 1H, ArH). LRMS (APCI) m/z: 368.2 calcd for C19H38NO2Si2 (M + H); 368.5 obsd. All spectral data were in accordance
with the literature (Byun et al., 2007). Compound 4: To a solution of aniline (compound 3) (0.401 g, 1.091 mmol) in THF (42 ml) were
Cell Chemical Biology 27, 1–13.e1–e7, July 16, 2020 e6