Two birds with one stone: a novel antibiotic hit blocking Staphylococcus aureus heme uptake with serendipitous hemoglobin left-shifting activity

The compound C35 inhibits <i>Staphylococcus aureus</i> growth by blocking hemophore-mediated iron acquisition through interference with the interaction between the bacterial receptor IsdB and human hemoglobin (Hb). Additionally, C35 binds Hb and stabilizes a high-affinity oxygen-binding state, highlighting its potential as a dual-acting antimicrobial and hemoglobin modulator.The sheets "<b>Growth promotion with Hb and Fe</b>", "<b>Toxicity of C35</b>", and "<b>EDDHA 500 Hb 80+ C35 +Fe 500</b>" report the OD values from individual <i>S. aureus</i> growth experiments. These data support the inhibitory activity of C35 on bacterial growth and demonstrate the absence of non-specific or toxic effects of the compound on the bacterium. The sheet "<b>Hb binding</b>" contains the data used to evaluate the effect of C35 and INN298 on oxygen binding to Hb. It includes the reference spectra used for linear combination analysis, an example of the fitting procedure comparing an experimental spectrum with the corresponding linear fit, and the oxygen binding curves obtained at 37 °C in the absence and presence of the two compounds. The linear combination was performed on Hb spectra collected both in the presence and absence of C35 or INN298, recorded at varying partial oxygen pressures (pO2) inside a specially designed gas-tight cuvette. Both the reference spectra and the example spectra used in the fitting procedure are provided as two-column datasets, where column 1 corresponds to the wavelengths (nm) and column 2 to the absorbance values. The oxygen binding curves are also provided as two-column datasets, with column 1 representing the pO2 and column 2 the fractional saturation of Hb. The "<b>SEC - HbHp</b>" sheet contains the raw data for the size exclusion chromatography (SEC) chromatograms used to rule out any potential destabilization of the Hb–haptoglobin complex. The "<b>ITC</b>" sheet of the Excel file contains the raw ITC data and fitting results. Column 1 contains the injection number, Column 2 contains the time, Column 3 contains the molar ratio, and Column 4 contains the DQ in microJ. The "<b>SEC - Hb</b>" sheet contains the analysis performed to evaluate the ability of C35 to stabilize the tetrameric state of Hb. The sheet includes information on the protein standards used, their chromatogram, and the data points describing the dependence of the estimated molecular weight on Hb concentration in the absence and presence of C35. Both dependencies were measured in duplicate, and all obtained values are explicitly reported.