Respiratory syncytial virus infection (RSV) is a major global health concern, particularly in infants and elderly populations. In this work, we have screened and identified 3 double-stapled peptides derived from a minimal domain of the RSV F heptad repeat, namely 3/4i, 3/4m, and 4/4g, which are potent inhibitors of RSV fusion and remain active against viral escape mutants resistant to small-molecule fusion inhibitors. Our structural activity relationship (SAR) analysis demonstrates that combining a limited set of staples is sufficient to achieve high antiviral potency. X-ray crystallography revealed that the enhanced potency of 3/4i and 3/4m primarily arises from strong hydrophobic interactions between the N-terminal staple and the trimeric HR1 coiled coil of RSV F. In vivo pharmacokinetic, imaging, and feasibility studies in RSV-infected Balb/c mice further support intranasal administration as a promising route for delivering these stapled peptides to the lung, highlighting their potential as therapeutics against RSV.

Background: The diagnosis of metastatic clear cell renal cell carcinoma (mccRCC) remains challenging due to the tumor's molecular heterogeneity, often resulting in low sensitivity and a high false-positive rate. In this study, we introduce and validate a new imaging modality for mccRCC based on the first radioligand targeting the type 2 vasopressin receptor (V2R) suitable for positron emission tomography (PET). V2R is ectopically expressed in mccRCC. This imaging approach utilizes [18F]F-MQ232, a radiolabeled peptide derived from snake venom which exhibits high in vivo selectivity for V2R. Methods: The V2R-selective peptide MQ232 conjugated with either a cyanine 5 (Cy5) or a fluorine 18 (18F) group were chemically synthetized. V2R mRNA was quantified and protein expression assessed by flow cytometry using Cy5-MQ232. The selectivity and tumor targeting ability of the modified MQ232 peptides were assessed using in vivo fluorescence imagery in tumor-bearing mice using CHO-V2R tumors with graded expression. Metabolic stability and PET pharmacokinetics of [18F]F-MQ232 were assessed in rodents. Specific tumor targeting and imaging contrast were validated in vivo using V2R-expressing tumors. Results: [18F]F-MQ232 is a highly relevant radioligand whose tumor uptake directly correlates with V2R expression levels in tissues, demonstrating its specificity to V2R-expressing tumors. Replacing the peptide moiety by an isoform unable to interact with V2R leads to a drastic decrease in the radioligand's tumor uptake, highlighting its origin in a specific, ligand/receptor type interaction between the MQ232 moiety and V2R. PET/CT imaging of Caki-1 xenografted mice demonstrated the ability of [18F]F-MQ232 to allow specific detection of the tumor compartment associated with high tumor-to-background contrast. RT-qPCR screening of metastatic and non-metastatic ccRCC biopsies from patients confirms V2R expression. Conclusions: This work validates the V2R-targeting strategy in mccRCC using [18F]F-MQ232 and demonstrates that human mccRCC tissues express V2R, confirming the suitability of this specific imaging technique for metastasis extension assessment.

BACKGROUND:Vasopressin, a hormone regulating water metabolism, has been poorly studied in patients on peritoneal dialysis (PD). Vasopressin measurement is challenging and not routinely available in clinical practice. This study aimed to evaluate whether copeptin, a stable surrogate marker of vasopressin, could be used to assess vasopressin levels in patients on PD and to determine if vasopressin maintains its antidiuretic effect in this population. METHODS:We included 34 PD patients from three French nephrology centers. Plasma vasopressin was measured using radioimmunoassay, while copeptin was quantified with a non-competitive immunofluorescence assay. Urine osmolality and 24-hour urine output were assessed, and peritoneal adequacy tests were performed. Associations between copeptin, vasopressin, and clinical parameters were analyzed using Spearman correlations and mixed-effect models. Healthy controls were included for comparison. RESULTS:Copeptin levels were strongly correlated with vasopressin levels (Spearman's rho = 0.62, P < 0.001), confirming its reliability as a biomarker of vasopreassin. Higher copeptin levels were associated with increased urine osmolality (β = 3.63, P = 0.008) and decreased 24-hour urine output (β = -0.53, P = 0.008), indicating that vasopressin retains its antidiuretic activity in PD patients. Compared to healthy controls, PD patients had lower urine osmolality and required higher copeptin levels to achieve similar urine concentration, suggesting vasopressin resistance. Copeptin levels were also associated with lower residual kidney function and higher brain natriuretic peptide levels but were not influenced by blood pressure, plasma sodium, or PD characteristics. CONCLUSIONS:This study provides evidence that vasopressin maintains an antidiuretic effect in PD patients, and supports the use of copeptin as a robust biomarker for vasopressin in this population.

There is an urgent need for efficient and innovative therapies to treat brain disorders such as psychiatric and neurodegenerative diseases. Immunotherapies have proved to be efficient in many medical areas, but have not been considered to treat brain diseases due to the poor brain penetration of immunoglobulins1,2. Here we developed a bivalent biparatopic antibody, made of two camelid heavy-chain antibodies (called nanobodies)3, one binding to, and the other potentiating the activity of, homodimeric metabotropic glutamate receptor 2. We show that this bivalent nanobody, given peripherally, reaches the brain and corrects cognitive deficits in two preclinical mouse models with endophenotypes resulting from NMDA receptor hypofunction. Notably, these in vivo effects last for at least 7 days after a single intraperitoneal injection and are maintained after subchronic treatment. Our results establish a proof of concept that nanobodies can target brain receptors, and pave the way for nanobody-based therapeutic strategies for the treatment of brain disorders.

Poxviruses are double-stranded DNA viruses including relevant zoonotic pathogens with high morbidity and potential biological warfare threats. Although African swine fever virus belongs to the Asfarviridae family and is not strictly classified as a Poxviridae member, both fall within the same class of Pokkesviricetes that replicate in the cytoplasm. Among compounds targeting these viruses, acyclic nucleoside phosphonate (ANP) prodrugs are promising inhibitors of viral DNA polymerases. However, some limitations related to their toxicity and the rapid emergence of resistance highlight the need for new antiviral molecules. In this study, we tested a new ANP called LAVR-289. This product effectively inhibits viral replication by targeting a specific domain in the poxvirus DNA polymerase. Using monkeypox virus models, the subcutaneous or oral administration of LAVR-289 demonstrated protective efficacy in infected animals without toxicity. Its in vivo half-life, long on-the-shelf stability and broad-spectrum efficacy make LAVR-289 a promising candidate for further development and stockpiling as a medical countermeasure against dsDNA virus outbreaks. LAVR-289 can be positioned in the context of recurrent viral epidemics, bioterrorism risk, and the emergence of resistant strains in the population.

Preeclampsia (PE) is the most threatening pathology of human pregnancy. Placenta from PE patients releases harmful factors that contribute to the exacerbation of the disease. Among these factors is the prokineticin1 (PROK1) and its receptor, PROKR2 that we identified as a mediators of PE. Here we tested the effects of PKRA, an antagonist of PROKR2, on the attenuation of PE symptoms. We used the genetic PE mouse model, STOX1 that overexpresses Stox1 gene in a heterozygosis manner in the placenta. This model allowed exploiting two genotypes of the offspring, those that overexpress the Stox1 gene, and the WT that grow in a PE environment (STE). We characterised the effect PKRA (1 μM) on the attenuation of PE symptoms and compared its effects on STOX1 and STE placentas. We also used STOX1 overexpressing trophoblast cells to decipher the PROK1-underlying mechanism. We demonstrated that (i) antagonisation of PROKR2 attenuated PE-mediated hypertension and proteinuria, (ii) STE placentas and foetuses exhibited better outcomes in response to PKRA, (iii) the secretome of STOX1-trophoblasts impacted the integrity of the fetal vasculature that was attenuated by PKRA treatment. This study demonstrates the direct involvement of the PROK1 in PE and identifies PKRA as a promising therapy for PE.

Aminopeptidase A (APA) is a membrane-bound zinc metallopeptidase involved in the production of angiotensin III, one effector peptide of the brain renin-angiotensin system, making brain APA a relevant pharmacological target for the development of novel therapeutic treatments against hypertension and heart failure. The structure-based design of new APA inhibitors is described, based on previously developed thiol-containing inhibitors and APA crystal structure. Chemical synthesis, in vitro assessment against APA activity, pharmacological and pharmacokinetic profiling were performed, ultimately leading to a potent and selective APA inhibitor.

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in new-borns, with all children being infected before the age of two. Reinfections are very common throughout life, and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture, and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.

Key PointsMQ232, a disulfide-bond reticulated peptide derived from a natural snake toxin, was optimized as a new aquaretic drug candidate.MQ232 showed very low acute and chronic toxicity in rat and a biodistribution in mice strongly in favor of the kidney organs.MQ232 induced a sole aquaretic effect and demonstrated high in vivo activities on hyponatremia and polycystic kidney disease models.BackgroundVaptans were developed at the end of the previous century as vasopressin type 2 receptor antagonists. Tolvaptan is the most prescribed vaptan for hyponatremia and autosomal dominant polycystic kidney disease (ADPKD). However, its use is not as widespread as it should be due to price issues, a narrow therapeutic window, and some side effects. With the aim of discovering new efficient and safer vasopressin type 2 receptor antagonists, we screened animal venoms and identified several peptide toxins. Among them, mambaquaretin 1 (MQ1) displayed unique biological properties in that regard that it was the starting point for the development of a potential drug candidate.MethodsHuman T-cell assays and bioinformatics were used to mitigate MQ1 immunogenicity risk. MQ232 biodistribution in mice was performed by positron emission tomography. Pharmacodynamics, pharmacokinetics, and acute and chronic toxicity tests were performed on control rats. A rat experimental model of desmopressin-induced hyponatremia, ex vivo mice model of kidney cysts, and mice orthologous model of ADPKD were used to validate MQ232 efficacy in these pathologies.ResultsThree mutations were introduced in MQ1 to mitigate its immunogenicity risk. A fourth gain-of-function mutation was added to generate MQ232. MQ232's safety was demonstrated by a first toxic dose as high as 3000 nmol/kg and a strong kidney organ selectivity by positron emission tomography imaging, while showing almost no interaction with the liver. MQ232's efficacy was first demonstrated with an effective dose of 3 nmol/kg in a hyponatremic model and then in polycystic kidney models, on which MQ232 significantly reduced cyst growth.ConclusionsWe demonstrated, using diverse translational techniques and minimizing animal use, MQ232's safety and efficacy in several rodent models of hyponatremia and ADPKD.

The accurate quantification of biomarkers is paramount in modern medicine, particularly in cancer where precise diagnosis is imperative for targeted therapy selection. In this paper we described a multiplexed analysis diagnostic approach based on cleavable MS-tagged antibodies. The technology uses MS-tag isotopologues and the sydnonimine-cyclooctyne click-and-release bioorthogonal reaction. In a proof of concept study, we demonstrated the potential of this approach for cancer cell immunoprofiling in culture cells, tissues and in vivo as well, thereby unveiling promising diagnostic avenues.

Research on graphene-based nanomaterials has experienced exponential growth in the last few decades, driven by their unique properties and their future potential impact on our everyday life. With the increasing production and commercialization of these materials, there is significant interest in understanding their fate in vivo. Herein, we investigated the distribution of 14C-few-layer graphene (14C-FLG) flakes (lat. dim. ∼ 500 nm) in mice over a period of one year. Furthermore, we compared the effects of repeated low-dose and acute high-dose exposure by tracheal administration. The results showed that most of the radioactivity was found in the lungs in both cases, with longer elimination times in the case of acute high-dose administration. In order to gain deeper insights into the distribution pattern, we conducted ex vivo investigations using μ-autoradiography on tissue sections, revealing the heterogeneous distribution of the material following administration. For the first time, μ-autoradiography was used to conduct a comprehensive investigation into the distribution and potential presence of FLG within lung cells isolated from the exposed lungs. The presence of radioactivity in lung cells strongly suggests internalization of the 14C-FLG particles. Overall these results show the long-term accumulation of the material in the lungs over one year, regardless of the administration protocol, and the higher biopersistence of FLG in the case of an acute exposure. These findings highlight the importance of the exposure scenario in the context of intratracheal administration, which is of interest in the evaluation of the potential health risks of graphene-based nanomaterials.

Brevetoxins (BTXs) constitute a family of lipid-soluble toxic cyclic polyethers mainly produced by Karenia brevis, which is the main vector for a foodborne syndrome known as neurotoxic shellfish poisoning (NSP) in humans. To prevent health risks associated with the consumption of contaminated shellfish in France, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) recommended assessing the effects of BTXs via an acute oral toxicity study in rodents. Here, we investigated the effect of a single oral administration in both male and female mice with several doses of BTX-3 (100 to 1,500 µg kg−1 bw) during a 48 h observation period in order to provide toxicity data to be used as a starting point for establishing an acute oral reference dose (ARfD). We monitored biological parameters and observed symptomatology, revealing different effects of this toxin depending on the sex. Females were more sensitive than males to the impact of BTX-3 at the lowest doses on weight loss. For both males and females, BTX-3 induced a rapid, transient and dose-dependent decrease in body temperature, and a transient dose-dependent reduced muscle activity. Males were more sensitive to BTX-3 than females with more frequent observations of failures in the grip test, convulsive jaw movements, and tremors. BTX-3’s impacts on symptomatology were rapid, appearing during the 2 h after administration, and were transient, disappearing 24 h after administration. The highest dose of BTX-3 administered in this study, 1,500 µg kg−1 bw, was more toxic to males, leading to the euthanasia of three out of five males only 4 h after administration. BTX-3 had no effect on water intake, and affected neither the plasma chemistry parameters nor the organs’ weight. We identified potential points of departure that could be used to establish an ARfD (decrease in body weight, body temperature, and muscle activity).

In preclinical models, the development and optimization of protein-drug conjugates require accurate determination of the plasma and tissue profiles of both the protein and its conjugated drug. To this aim, we developed a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging. By combining enzymatic and chemical reactions, we obtained homogeneous dual-labeled anti-MMP-14 Fabs (antigen-binding fragments) conjugated to monomethyl auristatin E where the protein scaffold was labeled with carbon-14 (14C) and the conjugated drug with tritium (3H). These antibody-drug conjugates with either a noncleavable or a cleavable linker were then evaluated in vivo. By combining liquid scintillation counting and ex vivo dual-isotope radio-imaging, it was possible not only to monitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulated within the different organs.

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.