Isatin

All chemical substances have been used as bought from Sigma-Aldrich and Merck Companies. Tetraethyl orthosilicate (tetraethoxysilane) (98% w/w), ethanol (99.5% w/w), (3-aminopropyl) triethoxysilane (95% w/w), HNO₃ (65% w/w), and silver nitrate (≥ 99.0%), potassium persulphate (99.99%), hydrazine hydrate (24–26% in H₂O (RT)), isatin (97%), acenaphthene quinone (97%), malononitrile (98%), ethyl cyanoacetate (98%), dimedone (95%), barbituric acid ethyl acetoacetate (99%), 4-hydroxycoumarin (98%), three-methyl-1H-pyrazole-five (4H)-one (98%), α-naphthol (99%), β-naphthol (99%), n-hexane (99%), ethyl acetic acid (99%), and desired derivations have been provided from the Sigma-Aldrich Company. The stem and roots of the Spear Thistle were purchased from a local shop in Tehran (in Iran). The leaves were purchased from a local shop in Tehran. The plant we used in this work is a plant that is found in abundance in local shops and is not wild and endangered. This study complies with relevant institutional, national, and international guidelines and legislation.

The nitroketene dithioacetals, cysteamine hydrochloride, isatin, malononitrile, ethyl cyanoacetate, methyl cyanoacetate, cyanoacetohydrazide, barbituric acid, derivatives of isatin, triethylamine and solvents were obtained from Sigma Aldrich and used without further purification. Nano-silica (CAB-O-SIL® M5) was obtained from Cabot Co. IR spectra: Bruker Tensor 27 spectrometer. NMR spectra: Bruker DRX-300 Avance instrument (300 MHz for ¹H and 75.4 MHz for ¹³C) with DMSO-d₆ as solvents. Chemical shifts are expressed in parts per million (ppm), and coupling constant (J) are reported in hertz (Hz). Mass spectra: Agilent 5975C VL MSD with Triple-Axis detector operating at an ionization potential of 70 eV. Elemental analyses for C, H and N: Heraeus CHNO-Rapid analyzer. Melting points: electrothermal 9100 apparatus.

C. acnes KCCM 41747 (ATCC 6919) (isolated from human facial acne), methicillin-sensitive S. aureus ATCC 6538, and fluconazole-resistant C. albicans strain DAY185 were used in this study. The C. acnes strain was cultured on Reinforced Clostridium Media (RCM)-agar plates for colony preparation and in liquid RCM at 37°C under anaerobic conditions (BD GasPak EZ Gas Generating Anaerobic Pouch Systems; Fisher Scientific, Pittsburgh, PA, USA) for all other experiments. The S. aureus strain was cultured in LB medium at 37°C, and the C. albicans strain was cultured in Potato Dextrose Broth (PDB) medium at 37°C.
Twenty indoles, namely, 7-azaindole, 7-benzyloxyindole, 3,3′-diindolylmethane (DIM), 7-formylindole, 7-hydroxyindole, indole, indole-3-acetamide, indole-3-acetic acid, indole-3-acetonitrile, indole-3-butyric acid, indole-3-carbinol, indole-3-carboxyaldehyde, indole-7-carboxylic acid, indole-3-propionic acid, isatin, 7-methoxyindole, 7-methylindole, methyl indole-7-carboxylate, 7-nitroindole, and 2-oxindole were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako Chemicals Inc. (Richmond, VA, USA) or Combi-Blocks, Inc. (San Diego, CA, USA). Dimethyl sulfoxide (DMSO) was used to dissolve all indoles, and 0.1% (vol/vol) DMSO was used as the negative control; at this concentration, DMSO did not affect bacterial growth or biofilm formation.

Hyaluronan sample of high molar mass (M_w = 1.53 MDa; M_w/M_n = 1.76) was purchased from Lifecore Biomedical Inc., Chaska, MN, USA. l-Ascorbic acid and K₂S₂O₈ p.a. were the products of Merck KGaA, Darmstadt, Germany. NaCl and CuCl₂·2H₂O p.a. were purchased from Slavus Ltd., Bratislava, Slovakia. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; purum, >99%) was from Fluka, Steinheim, Germany. 2,2-Diphenyl-1-picrylhydrazyl radical (DPP^•) and methanol were purchased from Sigma-Aldrich, Steinheim, Germany. Deionized water of high-purity (conductivity of ≤0.055 µS/cm), was made by using the TKA water purification system (Water Purification Systems GmbH, Niederelbert, Germany). Isatin was purchased from Sigma-Aldrich, Prague, Czech Republic. Cemtirestat was a product of Akos Consulting & Solutions, Wurttemberg, Germany. Stobadine·2HCl, SM1M3EC2·HCl, and SME1i-ProC2·HCl (cf. Figure 1) were prepared at the Institute of Experimental Pharmacology and Toxicology, Bratislava, Slovakia.

Normal human adult keratinocytes (Lonza) were seeded into 48-well culture plates and cultured to passage three with an approximate density of 70–80%. Keratinocytes were stimulated with IL-17A (100 ng/mL) (R&D Systems, Inc., Minneapolis, MN) and treated with varying concentrations of isatin, tryptanthrin, and indirubin (Sigma-Aldrich, St. Louis, MO), and a control compound, PD 0325901 [33 (link)]. PD 0325901 is a mitogen-activated protein kinase kinase (MEK) inhibitor; a similar compound has been demonstrated to inhibit IL-17-induced cytokine release [34 (link)]. Cell viability was confirmed using the PrestoBlue® assay (Thermo Fisher Scientific, Waltham, MA). tryptanthrin showed no impact on cell viability at all testing concentrations. Tissue culture supernatants were analyzed for pro-inflammatory cytokines IL-6 and IL-8 using MesoScale Discovery V-plex assay (MesoScale Discovery, Rockville, MD). Data were analyzed using Graphpad Prism v5.0 (http://www.graphpad.com).

Escherichia coli DH5α and BL21 (DE3) strains were used as cloning and protein expression hosts, respectively. E. coli strains carrying plasmids were cultivated in a Luria-Bertani (LB) medium supplemented with antibiotic (50 μg/mL kanamycin). An LB medium was purchased from Huankai Microbial (China). M9 media salts were purchased from Sangon Biotech (China). Plasmid DNA was isolated using a Tiangen plasmid miniprep kit (TIANGEN, Ltd. China). 6-Bromo-Tryptophan and 6-chloro-Tryptophan were purchased from GL Biochem (China). Tryptophan and NADH were purchased from Sigma (United States). Indole, 6-bromo-Indole, indigo, FAD, and isatin were purchased from Sigma (United States), Bidepharm (China), and Yuanye Biotech (China), respectively. Indirubin was purchased from TCI (Japan). Chemically synthesized 6, 6′-dibromoindigo was synthesized from Abace Biotech (China). Gene and oligomer synthesis and sequencing were carried out in BGI (China), Sangon (China), and Rui Biotech (China), respectively. Enzymes involved in restriction reaction, ligation, and PCR were purchased from New England Biolabs (United States), Vazyme (China), and TAKARA (Japan), respectively. All other chemicals used in this study were of analytical grade. All media and reagent solutions were prepared with Milli-Q water (Merck Millipore).

Boltorn H40
(98%) was purchased from Perstorp AB, Sweden. Isatin, methyl bromoacetate,
potassium carbonate (K₂CO₃), 1-ethyl-3-(3′-dimethylaminopropyl)
carbodiimide·HCl (EDC·HCl), 4-dimethylaminopyridine (DMAP),
indole-3-acetic acid (2), N-methylindole-3-acetic
acid, and methylindole-3-acetate were purchased from Sigma-Aldrich.
Tetrahydrofuran (THF), N,N-dimethylformamide
(DMF), N,N-dimethylacetamide (DMAc),
1,4-dioxane, chloroform, dichloromethane (DCM), dimethyl sulfoxide
(DMSO), HCl, NaOH, ethanol, methanol, acetone, acetonitrile, ethyl
acetate, diethyl ether, sodium carbonate (Na₂CO₃), NaHCO₃ (ACS, Reag. Ph. Eur.), and NaSO₄ were
purchased from VWR Chemicals. Poly(3-hydroxybutyrate) (PHB) powder
was supplied by BIOMER (Germany), which was washed with 0.001 N aqueous
HCl for 30 min, washed with deionized water, and dried at 50 °C
under vacuum for 2 days before further use. The acid-treated PHB had
a weight-average molar mass (M_w) of 620 000
g mol^–1 and polydispersity index (PDI) of 2.0. PCL
was purchased from Sigma-Aldrich with a weight-average molar mass
(M_w) of 14 000 g mol^–1 and polydispersity index (PDI) of 1.4.