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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 non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the “serinosome”. Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH , the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients’ fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.

Infections caused by Staphylococcus aureus are closely linked to its ability to secure essential nutrients, including iron, which is extracted from the heme of human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[[2-[[5- (1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetyl]amino]benzoate (C35) was recently identified as a new potential antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by specifically targeting the hemophore-driven iron acquisition system. Our findings confirm both the potential of C35 as a first-in-class protein-protein interaction inhibitor with antimicrobial activity, and the effectiveness of targeting hemophores as a strategy to inhibit S. aureus growth. To gain information for drug discovery purposes, the X-ray structure of Hb in the presence of the compound was solved. Unexpectedly, we discovered that, rather than the predicted binding pose, the molecule binds to tetrameric Hb in a cleft between the alpha subunits, stabilizing an R2 relaxed Hb conformation. This triggered further investigation of the effect of C35 on Hb functional properties, which showed a pronounced left-shift activity on oxygen binding curve (i.e., it strongly increases the Hb oxygen affinity). These results highlight C35 as a promising dual-acting compound with both antimicrobial activity and the ability to modulate Hb function through non-covalent stabilization of a high-affinity state. Staphylococcus aureus is a dangerous bacterium that can cause severe infections in humans. To grow and survive it needs iron, which it steals from our red blood cells by taking it from hemoglobin, the protein that carries oxygen in the blood. In this study, we focused on a small molecule, called C35, that blocks the interaction between hemoglobin and a key bacterial protein involved in heme acquisition. We found that C35 strongly inhibits the growth of S. aureus when hemoglobin is the only available source of iron, showing a potential new method to starve the pathogen and consequently fight the infection. Surprisingly, we also found that C35 increases the affinity of hemoglobin for oxygen. This dual action makes C35 a unique molecule for future therapeutic development, with potential applications both as a new antimicrobial agent and in the treatment of diseases related to hemoglobin function.