Chemical stimulation of the innate immune response with ChX710: Toward a new class of antiviral molecules – ChemInnate

To identify compounds capable of stimulating the innate immune response and in particular the production of interferons, we have developed different functional tests based on established cell lines or primary immune cells purified from peripheral blood of healthy donors. These cellular assays were used to screen different libraries of several thousands of natural compounds (metabolites, xenobiotics) or synthetic molecules, thus identifying a number of active candidates, notably ChX710 and DD363. We then study the innate immune response activated by these compounds in different cell types by RT-qPCR methods, reporter gene assays, transcriptomics or bead-based immunoassays to quantify cytokines. In addition, we have different models of viral infections in vitro (measles, chikungunya, dengue, etc.) to evaluate the antiviral properties of these molecules. Finally, if we succeed in optimizing the pharmacological properties of the molecules identified in collaboration with chemists, in vivo studies will be carried out in well established mouse models of infection (West Nile virus or chikungunya virus). These studies will make it possible to evaluate the antiviral effects of these compounds but also the impact that these molecules have on the innate and adaptive immune response to viruses.

The first significant outcome of this project is the demonstration of the potentiating effects of the ChX710 compound on the innate immune response to cytosolic DNA, at least in HEK-293 cells. The response to cytosolic DNA plays a key role in the innate immune response to DNA viruses as well as to various metabolic or genotoxic stresses, and therefore in the elimination of pre-tumor and tumor cells. The identification of a molecule capable of amplifying this response makes it possible to establish an important proof of concept, although the mechanisms involved remain to be characterized and observations extended to different cell types.
The second significant result relates to the compound DD363. We have shown that this molecule is a new inhibitor of the pyrimidine biosynthesis pathway. As such, it amplifies the cellular response to viral RNAs as previously shown for other compounds having this same mode of action. However, the study of this molecule allowed us to go further by showing for the first time that an inhibitor of this metabolic pathway not only amplifies the expression of ISGs in response to viral RNAs, but also interferon secretion. This observation reveals a new mechanism by which a cell would communicate with its neighbors on its metabolic status, especially in the context of a viral infection.

The ChX710 having the property of amplifying the cellular response to cytosolic DNA, we will study the effects of this molecule on the replication of various DNA viruses, in particular adenoviruses and herpesviruses, first in vitro then possibly in vivo depending on the results obtained. Moreover, this molecule could be useful for amplifying the innate immune response to agents used as anticancer therapies: DNA intercalating or alkylating agents and antimetabolites. The mode of action of these molecules relies on the induction of DNA damages, which lead to the release of cellular DNA into the cytosol and the activation of immune sensors to DNA. The ChX710 could thus potentiate the effects of these anticancer treatments. Finally, the study of the mode of action of this molecule is continuing and recent results show that the amplification of the cellular response to DNA by the ChX710 implies the implementation of an oxidative stress and the activation of cellular caspases.
In addition, new screening campaigns have been launched and the first results have led to the identification of a new set of compounds capable of inhibiting the inflammatory response associated to a viral infection. By avoiding a cytokine storm, these molecules might also have an interest in treating some viral infections.

Publication: « Identification of a small molecule that primes the type I interferon response to cytosolic DNA », Khiar S. & al, Scientific Reports. 2017 May 31;7(1):2561.

Seminar/congress:
1. S. Khiar & PO Vidalain, Selected speaker, Congress « Journées Francophones de Virologie » (March 2016)
2. PO Vidalain, Symposium « Viruses, Immunity and innovative therapeutic strategies », Cagliari, Italy (March 2017)
3. PO Vidalain, Seminar « Dompter la réponse immunitaire contre les virus: l’approche chemobiologique », Université Saint Esprit de Kaslik, Lebanon (December 2016)
4. S Khiar, Virology Congress 2017 « Identification of a small molecule that primes the type I interferon response to cytosolic DNA », London, UK, (March 2017)
5. PO Vidalain (Team 1), Invited Speaker, Journée de la Chimiothèque Nationale (May 2017)
6. PO Vidalain, Seminar, « Dompter la réponse immunitaire contre les virus: l’approche chemobiologique », EA7426, HCL-Biomérieux, Lyon (January 2017)
7. PO Vidalain, Invited Speaker, Third Cochin Conference on Infection, Immunity and Inflammation
8. S. Khiar, Selected speaker, Congress « Journées Francophones de Virologie » (March 2017)

Book chapter:
PO Vidalain, Chapter in TDC Journal (« Textes et Documents pour la Classe ») – Réseau Canopé. - « La mémoire sous la peau », TDC N°1103. P58-59.

Although viral infections represent a major burden for public health, our therapeutic arsenal is quite limited. Current antiviral therapies mostly rely on nucleoside analogs, few virus-specific drugs (oseltamivir ; palivizumab), and recombinant IFN-a/ß of limited efficacy in most cases. Despite the success of direct acting antivirals (DAA) against HIV or HCV, viruses often escape drugs targeting components of their replication machinery through mutations. Added to the fact that new pathogenic viruses are emerging permanently, there is a critical need for innovative approaches to fight seasonal epidemics and unexpected outbreaks like Ebola and chikungunya crises.
In this perspective, we are exploring a new concept: broad-spectrum antivirals that, instead of targeting the virus itself, block viral growth by modulating cellular defense mechanisms. To identify new chemical entities that stimulate the expression of the antiviral cluster of interferon-stimulated genes (ISGs), we developed a stable cell-line expressing luciferase under the control of an interferon-stimulated response element that is both activated by IRF1/3/7 transcription factors and type I interferons. This cellular assay was used to screen an unbiased chemical library of 10.000 compounds, and only one referred as ChX710 was found to activate the ISRE promoter in a range similar to IFN-ß. Further analyses by RT-qPCR showed that ChX710 efficiently stimulates the expression of interferon genes and ISGs in different human cell types, in particular primary monocytes and plasmacytoid dendritic cells. Such immunostimulatory properties were never reported before for this chemical series, and its mode of action is totally unknown. To decipher the signaling pathways activated by ChX710, we knocked-down cellular factors involved in the induction of ISGs by pathogen recognition receptors (MAVS, IRF1, IRF3) or type I interferons (TYK2). None of these different factors, except IRF1, seems to be involved in the activation of the ISRE promoter by ChX710. Added to the fact that our ISRE-luciferase reporter cell line does not express TLR7/8/9 or STING, our data suggest that ChX710 is targeting a novel signaling pathway to induce ISGs and the innate antiviral response. Furthermore, this molecule increased the antibody response to ovalbumin in mice, establishing its immunostimulatory properties in vivo. Although the antiviral response and the cellular stress induced by ChX710 eventually lead to cell death in vitro, this compound showed neither local nor systemic toxicity when injected to animals.
Altogether, our data suggest that ChX710 is a potent stimulator of the innate antiviral response that targets some yet uncharacterized cellular pathway. Objectives of this research program are to study the immunostimulatory properties, the mode of action, and the antiviral activity of this molecule. To reach these goals, we will (i) develop chemical analogs of ChX710 that are more active and more stable in vivo, (ii) document the immunostimulatory and antiviral properties of ChX710 both in vitro on primary human cells and in vivo in mouse models of immunization and infection, and (iii) look for the mode of action of this molecule using transcriptomics and proteomics approaches. This interdisciplinary research program will require specific skills in organic chemistry, pharmacology and immuno-virology that are all well covered by the members of our consortium. Main outcomes of this program include the identification of novel cellular pathways that stimulate the host antiviral response, and the development of an innovative immunostimulatory drug of potential therapeutic interest against viral infections, or whenever the stimulation of innate immunity can be beneficial (cancer treatments, vaccine development, etc).