Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein–protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

The stability of the IsdB:Hb complex upon plate washing was assessed using 2 pmol StrepTag®II-Y165A IsdB attached on the bottom of the microplate, 30 μM oxyHb solution and 1:1,000 diluted Ab.The number of washings is reported for each step of the procedure: after IsdB functionalization, after Hb incubation for 1 h and after Ab incubation.The reference is the procedure with three washings for each step.

N-Carbamoyl-4-chlorobutanamide (11) and 4-chloro-N-(methylcarbamoyl)butanamide (11a).
To a suspension of urea or 1-methylurea (33.33 mmol) in benzene (2.5 mL) and 2 drops of concentrated H2SO4, 4-chlorobutanoyl chloride (16.67 mmol) was added and the mixture was refluxed for 3 h, after which the reaction mixture was allowed to reach room temperature and was stirred for 12 h.Upon completion, the formation of a precipitate was observed.The solid was filtered and dried in vacuum to obtain the desired compounds 11 (80% yield) or 11a (84 %).

1-(4-Carboxyphenyl)-4-(2-hydroxyethyl)-5-oxo-2,5-dihydro-1H-pyrazole-3-carboxylic acid (C58).
To a solution of C53 (0.100 g, 0.299 mmol) in MeOH (8 mL) 1 M NaOH (2 mL) was added and the resultant mixture was stirred at room temperature for 12 h.Then the MeOH was removed under reduced pressure.The residue was diluted with H2O (15 mL) and acidified to pH 1 with 1 M HCl.The light orange precipitate was filtered, washed with H2O and CH2Cl2, collected, and dried under vacuum (0.077 g, 88%).9, main text).Raw data for ITC titration are shown in the upper panels, the binding isotherm of the integrated titration curve are reported in the bottom panels.The dilution heat from the control experiment was subtracted from the titration in the binding isotherm.Experiments were carried out at 25 °C in 50 mM HEPES buffer, pH 7.6.The fitting for experiment in panel A gave the following parameters: KD = 0.48 ± 0.07 µM; n = 2.31 ± 0.02; ΔH = -73.0± 0.78 kJ/mol.The fitting for experiment in panel B gave the following parameters: KD = 0.68 ± 0.05 µM; n = 2.72 ± 0.01; ΔH = -66.1 ± 0.39 kJ/mol.C. control experiment in which the reaction cell was filled with the buffer solution with 0.5% DMSO only, while the syringe was filled with 500 µM C35*.

3 Figure
Figure S2.IsdB N1 (A) and IsdH N2 (B) binding to Hba (PDB ID 5VMM and 4XS0, respectively).Proteins are shown as cartoon, the groove used for virtual screening as green contour.Residues of IsdB N1 or IsdH N2 at the interface are shown as sticks and labelled according to their PDB numbering.IsdB residues are labelled in blue, IsdH residues are labelled in yellow, Hba residues are labelled in magenta, and Hbb residues are labelled in teal.Y165 in IsdB is highlighted in lilac, while Y366 in IsdH is highlighted in orange.

Figure S3 .
Figure S3.Superposition of Hp:Hb complex (PDB ID 4X0L) to IsdB:Hb complex (PDB ID 5VMM).The proteins are shown in cartoons (IsdB: blue; Hbα: pink; Hbβ: light blue, Hp: orange), the targeted pocket as light green-yellow contour and the heme groups in yellow capped sticks.

Figure S4 .
Figure S4.Visible spectra of commercial compounds at 1 mM concentration.The yellow line is a reference at 450 nm.

Figure S5 .
Figure S5.Spectral comparison of Hb Q bands before (grey lines) and after (red lines) incubation with each commercial compound.The spectra of Hb after incubation were subtracted by the signal of the compound alone in solution.

Figure S6 .
Figure S6.STD-NMR spectra (top, for each compound) and off-resonance spectra (bottom, for each compound) for the commercial compounds C35, C41, C44, and C53.Blue arrows indicate the positions of the peaks in the STD spectra; blue circles show the corresponding protons in the structures.