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.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

Genetically Encoded Biosensors for the Fluorescence Detection of O2 and Reactive O2 Species.

The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

Inhibitors of O-Acetylserine Sulfhydrylase with a Cyclopropane-Carboxylic Acid Scaffold Are Effective Colistin Adjuvants in Gram Negative Bacteria.

Antibacterial adjuvants are of great significance, since they allow one to downscale the therapeutic dose of conventional antibiotics and reduce the insurgence of antibacterial resistance. Herein, we report that O-acetylserine sulfhydrylase (OASS) inhibitors could be used as colistin adjuvants to treat infections caused by critical pathogens spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. Starting from a hit compound endowed with a nanomolar dissociation constant, we have rationally designed and synthesized a series of derivatives to be tested against S. Typhimurium OASS isoenzymes, StOASS-A and StOASS-B. All acidic derivatives have shown good activities in the nanomolar range against both OASS isoforms in vitro. Minimal Inhibitory Concentrations (MICs) were then evaluated, as well as compounds' toxicity. The compounds endowed with good activity in vitro and low cytotoxicity have been challenged as a potential colistin adjuvant against pathogenic bacteria in vitro and the fractional inhibitory concentration (FIC) index has been calculated to define additive or synergistic effects. Finally, the target engagement inside the S. Typhimurium cells was confirmed by using a mutant strain in which the OASS enzymes were inactivated. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants.

Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction.

Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the ß-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/ß dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.

Enzyme Replacement Therapy for Genetic Disorders Associated with Enzyme Deficiency.

Mutations in human genes might lead to the loss of functional proteins, causing diseases. Among these genetic disorders, a large class is associated with the deficiency in metabolic enzymes, resulting in both an increase in the concentration of substrates and a loss in the metabolites produced by the catalyzed reactions. The identification of therapeutic actions based on small molecules represents a challenge to medicinal chemists because the target is missing. Alternative approaches are biology-based, ranging from gene and stem cell therapy, CRISPR/Cas9 technology, distinct types of RNAs, and enzyme replacement therapy (ERT). This review will focus on the latter approach that since the 1990s has been successfully applied to cure many rare diseases, most of them being lysosomal storage diseases or metabolic diseases. So far, a dozen enzymes have been approved by FDA/EMA for lysosome storage disorders and only a few for metabolic diseases. Enzymes for replacement therapy are mainly produced in mammalian cells and some in plant cells and yeasts and are further processed to obtain active, highly bioavailable, less degradable products. Issues still under investigation for the increase in ERT efficacy are the optimization of the interaction of the enzymes with cell membrane and internalization, the reduction in immunogenicity, and the overcoming of blood-brain barrier limitations when neuronal cells need to be targeted. Overall, ERT has demonstrated its efficacy and safety in the treatment of many genetic rare diseases, both saving newborn lives and improving patients' life quality, and represents a very successful example of targeted biologics.

Revealing the Dynamic Allosteric Changes Required for Formation of the Cysteine Synthase Complex by Hydrogen-Deuterium Exchange MS.

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design.

Investigational Studies on a Hit Compound Cyclopropane-Carboxylic Acid Derivative Targeting O-Acetylserine Sulfhydrylase as a Colistin Adjuvant.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure-activity relationship studies.

Iron Metabolism at the Interface between Host and Pathogen: From Nutritional Immunity to Antibacterial Development.

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery.

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight.

Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and ß subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein-protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle.

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States.

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase-the enzyme responsible for allantoin hydrolization in plants and lower organisms-for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase.

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: Differential oxidative stress in a Guinea pig transfusion model.

Hemoglobin-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto-transfusion control group. Particularly, tissue 4-hydroxynonenal adducts, tissue malondialdehyde adducts and plasma 8-oxo-2'-deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with the high-affinity HBOC, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high-affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Diatom Allantoin Synthase Provides Structural Insights into Natural Fusion Protein Therapeutics.

Humans have lost the ability to convert urate into the more soluble allantoin with the evolutionary inactivation of three enzymes of the uricolytic pathway. Restoration of this function through enzyme replacement therapy can treat severe hyperuricemia and Lesch-Nyhan disease. Through a genomic exploration of natural gene fusions, we found that plants and diatoms independently evolved a fusion protein (allantoin synthase) complementing two human pseudogenes. The 1.85-Å-resolution crystal structure of allantoin synthase from the diatom Phaeodactylum tricornutum provides a rationale for the domain combinations observed in the metabolic pathway, suggesting that quaternary structure is key to the evolutionary success of protein domain fusions. Polyethylene glycol (PEG) conjugation experiments indicate that a PEG-modified form of the natural fusion protein provides advantages over separate enzymes in terms of activity maintenance and manufacturing of the bioconjugate. These results suggest that the combination of different activities in a single molecular unit can simplify the production and chemical modification of recombinant proteins for multifunctional enzyme therapy.

The Energy Landscape of Human Serine Racemase.

Human serine racemase is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg2+, Ca2+, anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

PURPOSE: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.