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The charge of paddlewheel diruthenium complexes has a major role in defining their interaction with proteins: negatively charged complexes bind proteins non-covalently, while cationic complexes form adducts where the Ru 2 core binds to Asp side chains at the equatorial sites, or to the main chain carbonyl groups or the side chains of His, Arg or Lys residues at the axial sites. Here we study the interactions of the neutral compound [Ru 2 (D-p-FPhF)(O 2 CCH 3 ) 2 (O 2 CO)]·3H 2 O (D-p-FPhF -  = N,N'-bis(4-fluorophenyl)formamidinate), a very rare example of a paddlewheel diruthenium compound with three different equatorial ligands, with the model protein bovine pancreatic ribonuclease (RNase A) by means of UV-vis absorption spectroscopy, circular dichroism, electrospray ionization mass spectrometry (ESI-MS) and X-ray crystallography. It is the first attempt to investigate the binding of a neutral diruthenium compound to a protein. ESI-MS data indicate that, in solution, under the investigated experimental conditions, the diruthenium compound binds the protein upon the loss of an acetate ligand. The crystallographic results indicate the replacement of an acetate by two water molecules and the coordination of the [Ru 2 (D-p-FPhF)(O 2 CCH 3 ) 2 (O 2 CO)(OH 2 ) 2 ] + ion, that is expected to be a highly reactive species in the absence of the protein, to the imidazole ring of His105 at the axial site. The side chains of Glu9 and His119 are also identified as possible diruthenium binding sites. The binding significantly affects the protein ability to form dimers and higher-order oligomers, without significantly altering its secondary structure content and thermal stability. These data show that: i) Glu side chain has to be considered as a possible alternative binding site for diruthenium compounds, ii) diruthenium containing fragments that would be unstable in solution can be formed upon reaction of diruthenium compounds with a protein, iii) diruthenium compounds could be used as modulators of protein aggregation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2024. Published by Elsevier B.V.)

Typhoid and Paratyphoid fever cause a global health burden, especially for the children of Southern Asia. The impact of the disease is further exacerbated by the dramatic increase of antimicrobial resistance. While vaccines against Salmonella Typhi have been developed and successfully introduced, an effective vaccine targeting S. Paratyphi A is still lacking. Several efforts are currently ongoing to develop vaccines targeting both S. Typhi and S. Paratyphi A. In order to analyze the immune response induced by vaccination and in sero-epidemiological studies, easy to perform and high throughput immunoassays are needed. Here we present the setup and characterization of a customized ELISA assay and of a luminescent-based serum bactericidal assay (L-SBA) to measure the quantity of S. Paratyphi O antigen specific antibodies and their functional activity against S. Paratyphi A. Robust quality control criteria have been put in place both for ELISA and SBA and assays have been fully characterized in terms of quantitation limit, limit of blanks, specificity, linearity and precision. Assays are being employed to analyze samples from clinical trials, enabling the assessment of immunogenicity during clinical vaccine development., Competing Interests: This work was sponsored by GlaxoSmithKline Biologicals SA. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. MC, LR, LM, MI, FBS, SR and OR are employees of the GSK group of companies. EL, BS, PP, EM, SG are employees of Vismederi Srl. FBS, MI, SR, MC, and OR report ownership of GSK shares/share options. AJP is chair of the UK Department of Health and Social Care’s Joint Committee on Vaccination and Immunisation, is chair of WHOs technical advisory group on Salmonella vaccines and was a member of WHO’s SAGE until 2022. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Carducci, Massai, Lari, Semplici, Grappi, Maria, Jones, Conti, Piu, Scorza, Iturriza-Gómara, Montomoli, Pollard, Rondini and Rossi.)

Alum is the most used vaccine adjuvant, due to its safety, low cost and adjuvanticity to various antigens. However, the mechanism of action of alum is complex and not yet fully understood, and the immune responses elicited can be weak and antigen-dependent. While several antigens rapidly desorb from alum upon exposure to serum, phosphorylated proteins remain tightly bound through a ligand-exchange reaction with surface hydroxyls on alum. Here, bacterial proteins and glycoconjugates have been modified with phosphoserines, aiming at enhancing the binding to alum and prolonging their bioavailability. Tetanus toxoid protein and Salmonella Typhi fragmented Vi-CRM conjugate were used. Both antigens rapidly and completely desorbed from alum after incubation with serum, verified via a competitive ELISA assay, and set up to rapidly evaluate in vitro antigen desorption from alum. After antigen modification with phosphoserines, desorption from alum was slowed down, and modified antigens demonstrated more prolonged retention at the injection sites through in vivo optical imaging in mice. Both modified antigens elicited stronger immune responses in mice, after a single injection only, compared to unmodified antigens. A stronger binding to alum could result in potent single-dose vaccine candidates and opens the possibility to design novel carrier proteins for glycoconjugates and improved versions of bacterial recombinant proteins.

Effective vaccines against Salmonella Typhi, targeting the Vi capsular polysaccharide, have been developed and are being introduced into routine immunization in endemic countries. Vi conjugated vaccines are also being tested in new multi-component vaccine formulations. Simple, high-throughput and cost-effective assays to quantify Vi-specific IgG in clinical sera are needed. In this study we present the development and qualification of a new anti-Vi ELISA with continuous readout, which expresses results as ELISA Unit/mL (EU/mL). We have qualified the assay in terms of precision, linearity and specificity, demonstrating performance in line with a commercially available anti-Vi ELISA. We have also calibrated the assay against the 16/138 anti-Vi international standard and established conversion factor between EU/mL and international units/mL, to allow comparability of results across studies. In summary, this new assay met all the suitability criteria and is being used to evaluate anti-Vi responses in clinical studies., Competing Interests: This work was sponsored by GlaxoSmithKline Biologicals SA. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. MC, LM, MS, DS, MI-G, FS, SR, RC and O.R. are employees of the GSK group of companies. FS, MI-G, RC, SR, MC, and OR report ownership of GSK shares/share options. EL, BS, PP, EM, SG are employees of Vismederi Srl. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission., (Copyright © 2024 Carducci, Massai, Lari, Semplici, Aruta, De Simone, Piu, Montomoli, Berlanda Scorza, Grappi, Iturriza-Gómara, Canals, Rondini and Rossi.)

Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provided a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Introduction: Auranofin (AF) is a well-established, FDA-approved, antiarthritic gold drug that is currently being reevaluated for a variety of therapeutic indications through drug repurposing. AF has shown great promise as a potential anticancer agent and has been approved for a few clinical trials in cancer. The renewed interest in AF has led to extensive research into the design, preparation and biological evaluation of auranofin analogs, which may have an even better pharmacological profile than the parent drug., Areas Covered: This article reviews the strategies for chemical modification of the AF scaffold. Several auranofin analogs have been prepared and characterized for medical application in the field of cancer treatment over the last 20 years. Some emerging structure-function relationships are proposed and discussed., Expert Opinion: The chemical modification of the AF scaffold has been the subject of intense activity in recent years and this strategy has led to the preparation and evaluation of several AF analogs. The case of iodauranofin is a particularly promising example. The availability of homogeneous biological data for a group of AF derivatives allows some initial structure-function relationships to be proposed, which may inspire the design and synthesis of new and better AF analogs for cancer treatment.

Spin qubits defined by valence band hole states are attractive for quantum information processing due to their inherent coupling to electric fields, enabling fast and scalable qubit control. Heavy holes in germanium are particularly promising, with recent demonstrations of fast and high-fidelity qubit operations. However, the mechanisms and anisotropies that underlie qubit driving and decoherence remain mostly unclear. Here we report the highly anisotropic heavy-hole g-tensor and its dependence on electric fields, revealing how qubit driving and decoherence originate from electric modulations of the g-tensor. Furthermore, we confirm the predicted Ising-type hyperfine interaction and show that qubit coherence is ultimately limited by 1/f charge noise, where f is the frequency. Finally, operating the qubit at low magnetic field, we measure a dephasing time of T 2 *  = 17.6 μs, maintaining single-qubit gate fidelities well above 99% even at elevated temperatures of T > 1 K. This understanding of qubit driving and decoherence mechanisms is key towards realizing scalable and highly coherent hole qubit arrays., (© 2024. The Author(s).)

The reactivity of the anticancer drug picoplatin ( cis -amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies., Competing Interests: Declaration of competing interest This work was performed with funds from CARB-X, and with internal funding form GlaxoSmithKline Biologicals SA, who is the sponsor of the study.The work was also supported by funding from the New Zealand Ministry of Health (Te Whatu Ora) as part of an initiative to accelerate Strep A vaccine development for Aotearoa New Zealand. The external funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. M.C., L.R., L.M, M.I, F.B.S., D.G.M. and O.R. are employees of the GSK group of companies. FBS, MI, DGM, MC, and OR report ownership of GSK shares/share options., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)