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Biomphalaria glabratais a fresh-water mollusk that serves as obligatory intermediate host toSchistosoma mansoni, agent of the neglected tropical disease schistosomiasis that affects roughly 250 Mio people. One of the ways to control the pathogenic agent is to interrupt the life cycle by eliminating the intermediate snail host though foal treatment of water bodies with molluscicides. Currently recommended molluscicides were developed in the 1950ths and lack sufficient specificity, e.g., they are toxic to fish. To provide new lead compounds for the development of a new type of molluscicides we used a rational approach based on the hypotheses that interfering with an important epigenetic mark, DNA methylation, would impede development of the snail host. We present here the compound 29, analogues-based compound that mimic substrates of DNA methyltransferases. We show that compound 29 has (i) low cytotoxicity for human cells, (ii) it inhibits DNA methylation, and (iii) it decreases fecundity inB.glabrata. It is therefore conceivable to produce compounds that act as specific epigenetic molluscicides.

Malaria eradication requires the development of new drugs to combat drug-resistant parasites. The search for new chemical scaffolds that target novel pathways of the human malaria parasite Plasmodium falciparum is of highest priority. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus. (trilobine derivatives) as active in the nanomolar range against P. falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed us to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC50 ranging from 35-88nM). Efforts were made to obtain compounds with significantly improved preclinical properties. Out of those, compound 125 delays the onset of parasitemia in P. berghei infected mice and inhibits P. falciparum transmission stages in vitro (culture assays) and in vivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P. falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, we used a chemical pull-down strategy to identify potential protein targets of this chemical family. Mass spectrometry analysis identified the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that have not been previously targeted by antimalarial drugs or implicated in drug-resistance mechanisms.

International audience; Background: Post-translational modifications of histones constitute a dynamic process impacting gene expression. A well-studied modification is lysine methylation. Among the lysine histone methyltransferases, DOT1L is implicated in various diseases, making it a very interesting target for drug discovery. DOT1L has two substrates, the SAM cofactor that gives the methyl group and the lysine H3K79 substrate. Results: Using molecular docking, the authors explored new bisubstrate analogs to enlarge the chemical landscape of DOT1L inhibitors. The authors showed that quinazoline can successfully replace the adenine in the design of bisubstrate inhibitors of DOT1L, showing similar activity compared with the adenine derivative but with diminished cytotoxicity. Conclusion: The docking model is validated together with the use of quinazoline in the design of bisubstrate inhibitors.

International audience; Epigenetics has received much attention in the past decade. Many insights on epigenetic (dys)regulation in diseases have been obtained, and clinical therapies targeting them are in place. However, the readers of the epigenetic marks are lacking enlightenment behind this revolution, and it is poorly understood how DNA methylation is being read and translated to chromatin function and cellular responses. Chemical probes targeting the methyl-CpG readers, such as the methyl-CpG binding domain proteins (MBDs), could be used to study this mechanism. We have designed analogues of 5-methylcytosine to probe the MBD domain of human MBD2. By setting up a protein thermal shift assay and an AlphaScreen-based test, we were able to identify three fragments that bind MBD2 alone and disrupt the MBD2-methylated DNA interactions. Two-dimensional NMR experiments and virtual docking gave valuable insights into the interaction of the ligands with the protein showing that the compounds interact with residues that are important for DNA recognition. These constitute the starting point for the design of potent chemical probes for MBD proteins.

International audience; Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

International audience; Epigenetic regulation is a dynamic and reversible process that controls gene expression. Abnormal function results in human diseases such as cancer, thus the enzymes that establish epigenetic marks, such as histone methyltransferases (HMTs), are potentially therapeutic targets. Noteworthily, HMTs form multiprotein complexes that in concert regulate gene expression. To probe epigenetic protein complexes regulation in cells, we developed a reliable chemical biology high-content imaging strategy to screen compound libraries simultaneously on multiple histone marks inside cells. By this approach, we identified that compound 4, a published CARM1 inhibitor, inhibits both histone mark H3R2me2a, regulated also by CARM1, and H3K79me2, regulated only by DOT1L, pointing out a crosstalk between CARM1 and DOT1L. Based on this interaction, we combined compound 4 and DOT1L inhibitor EPZ-5676 resulting in a stronger inhibition of cell proliferation and increase in apoptosis, indicating that our approach identifies possible effective synergistic drug combinations.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future. The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

International audience; ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC50 of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies.

International audience; Over the last 20 years, natural peptides playing a key role in defense mechanisms and innate immunity have been isolated from unicellular organisms. Amphibian skin secretes dermaseptins, 24–34 amino acids in length that have a wide antimicrobial spectrum incorporating yeast, fungi, protozoa, bacteria and enveloped viruses. The anti-rabies virus (RABV) activity of dermaseptins S3 (30aa) and S4 (28aa) from Phyllomedusa sauvagei has been investigated, and further dissected its molecular basis by comparing punctual mutation or deletion of S4 analogues. The results showed that: (1) S4 is more active than S3 against RABV infection, 89% versus 38% inhibition at 7.5 lM; (2) the 5 NH2-aa of S4 are crucial for its inhibitory potential (S46–28 lost any inhibition) but the COOH terminus stabilizes the inhibitory potential (S41–16 showed only 23% inhibition at 7.5 lM); (3) there is a correlation between viral inhibition and dermaseptin cytotoxicity, which remains however moderated for BSR cells (12% at 10 lM). A single mutation in position 4 (S4M4K) slightly reduced cytotoxicity while keeping its antiviral activity, 97% at 7.5 lM. S4 and S4M4K showed an antiviral activity in vitro when provided 1 h after infection. In vivo experiments in mice by intramuscular injection of non-toxic doses of dermaseptin S4M4K 1 h post-infection by a lethal dose of RABV at the same site allowed more than 50% improvement in mice survival. This study highlights the potential interest of dermaseptins as non-expansive alternatives to rabies immunoglobulins for the treatment of rabies that continues to claim about 60,000 human lives per year worldwide, almost exclusively in developing countries.

International audience; Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Although rabies is reputed to be 100% fatal once the symptomatic phase is reached, in recent years an increased interest has arisen for therapeutic approaches. The “Milwaukee” protocol successfully applied to a rabid patient in 2005 has revitalized interest (although highly criticized by some investigators) in the rabies community. Most approaches still remain at a very early and conceptual stage of development. Rabies and its long incubation period is obviously an excellent target to develop an antiviral, even if its status as a neglected disease and its neurotropism are two economical and physiological obstacles, respectively, from this perspective. This review presents the empirical arsenal of substances having shown “some” anti-rabies viral (RABV) activity, then describes two complementary strategies that are currently elaborated for the rationale development of antivirals against RABV and other lyssaviruses: (1) a cognitive approach by destabilization of the known functional interactions between viral components, and (2) a random approach by high-throughput screening of highly diverse libraries of molecules using functional tests.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r = 0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Responsiveness of T cells (RTC) was studied in BALB/c mice intramuscularly infected with various lyssaviruses. After infection by this peripheral route, two types of viruses could be classified according to their effects: 1) pathogenic viruses, including fixed rabies Pasteur virus (serogenotype 1) and wild viruses belonging to serogenotype 1 (from a rabid fox in France and from a cow infected by a vampire bat in Brazil) or to serogenotype 5 (European bat lyssavirus 1); and 2) non-pathogenic viruses, including Mokola virus (serogenotype 3). RTC was tested by analysing in vitro the capacity of splenic T cells from infected BALB/c mice to produce cytokines after antigenic (purified lyssavirus antigens) or polyclonal stimulation (concanavalin A). Cytokine production was followed by assaying the biological activity of interleukin-2 and by testing for interleukin-2, interleukin-4 and interferon-gamma (IL2, IL4 and IFN gamma ) messenger RNAs (mRNA) by transcription into complementary DNA and amplification by the polymerase chain reaction. The initial biologically active IL2 and cytokine mRNA production was observed in mice infected with pathogenic or non-pathogenic lyssaviruses. Only mice with symptoms (infected with pathogenic viruses) lost the capacity to produce cytokines in vitro after antigen-specific stimulation. No such loss was observed after polyclonal stimulation. In mice peripherally infected with non-pathogenic viruses, no loss was observed after stimulation with lyssavirus antigens. Thus, infection with pathogenic lyssaviruses by the peripheral route induces in BALB/c mice a loss of T-cell responsiveness after antigen activation, but not after polyclonal activation.

International audience; Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4(+) and CD8(+) lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH 2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations. The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2–5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be effective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-effective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the first studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control.

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1: genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. Thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.

The aetiologic agents of rabies encephalomyelitis are classified into six genotypes within the Lyssavirus genus (1). It is generally accepted that the classical rabies vaccine strains (genotype 1) are inefficient in protecting against distantly rabies-related Lyssaviruses (genotypes 2 to 6). In an attempt to enlarge the protective activity of rabies vaccines, we have expressed the two principal viral antigens as recombinant proteins using a baculovirus expression system: 1° the glycoprotein (G) which constitutes viral spikes and induces virus-neutralizing antibody (VNAb); 2° the nucleoprotein (N) which is the major constituant of the ribonucleoprotein complex (RNP) involved in the T helper cell response. This work was performed with the Pasteur virus strain (PV: genotype 1).