An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis.

De novo purine nucleotide biosynthesis (DNPNB) consists of sequential reactions that are majorly conserved in living organisms. Several regulation events take place to maintain physiological concentrations of adenylate and guanylate nucleotides in cells and to fine-tune the production of purine nucleotides in response to changing cellular demands. Recent years have seen a renewed interest in the DNPNB enzymes, with some being highlighted as promising targets for therapeutic molecules. Herein, a review of two newly revealed modes of regulation of the DNPNB pathway has been carried out: i) the unprecedent allosteric regulation of one of the limiting enzymes of the pathway named inosine 5'-monophosphate dehydrogenase (IMPDH), and ii) the supramolecular assembly of DNPNB enzymes. Moreover, recent advances that revealed the therapeutic potential of DNPNB enzymes in bacteria could open the road for the pharmacological development of novel antibiotics.

Novel Antiviral Molecules against Ebola Virus Infection.

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Insight into the role of the Bateman domain at the molecular and physiological levels through engineered IMP dehydrogenases.

Inosine 5'-monophosphate (IMP) dehydrogenase (IMPDH) is an ubiquitous enzyme that catalyzes the NAD+ -dependent oxidation of inosine 5'-monophosphate into xanthosine 5'-monophosphate. This enzyme is formed of two distinct domains, a core domain where the catalytic reaction occurs, and a less-conserved Bateman domain. Our previous studies gave rise to the classification of bacterial IMPDHs into two classes, according to their oligomeric and kinetic properties. MgATP is a common effector but cause to different effects when it binds within the Bateman domain: it is either an allosteric activator for Class I IMPDHs or a modulator of the oligomeric state for Class II IMPDHs. To get insight into the role of the Bateman domain in the dissimilar properties of the two classes, deleted variants of the Bateman domain and chimeras issued from the interchange of the Bateman domain between the three selected IMPDHs have been generated and characterized using an integrative structural biology approach. Biochemical, biophysical, structural, and physiological studies of these variants unveil the Bateman domain as being the carrier of the molecular behaviors of both classes.

Interaction network among de novo purine nucleotide biosynthesis enzymes in Escherichia coli.

In human cells, de novo purine nucleotide biosynthesis is known to be regulated through the formation of a metabolon called purinosome. Here, we employed a bacterial two-hybrid approach to characterize the protein-protein interactions network among the corresponding enzymes of Escherichia coli. Our study revealed a dense network of binary interactions that connect most purine nucleotide biosynthesis enzymes. Notably, PurK, an exclusive prokaryotic enzyme, appears as one of the central hubs of this network. We further showed that modifications in PurK, which disrupted several interactions in the network, affected the purine nucleotide pools and altered the bacterial fitness. Our data suggest that the bacterial de novo purine nucleotide biosynthesis enzymes can assemble in a supramolecular complex and that proper interactions among the components of this complex can contribute to bacterial fitness.

Discovery of Bis-Imidazoline Derivatives as New CXCR4 Ligands.

The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.

SigH stress response mediates killing of Mycobacterium tuberculosis by activating nitronaphthofuran prodrugs via induction of Mrx2 expression.

The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

2-((3,5-Dinitrobenzyl)thio)quinazolinones: Potent Antimycobacterial Agents Activated by Deazaflavin (F420)-Dependent Nitroreductase (Ddn).

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26. Structure-activity relationship (SAR) studies indicated a crucial role for both meta-nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity (rv3547), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

Screening of potential antiviral molecules against equid herpesvirus-1 using cellular impedance measurement: Dataset of 2,891 compounds.

Data presented in this article are associated with the research article "Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: efficacy of decitabine and valganciclovir alone and in combination" [1]. These data correspond to the in vitro screening of 2,891 potential antiviral compounds against equid herpesvirus-1 (EHV-1) based on impedance measurements using the xCELLigence® RTCA MP System. This dataset includes compounds from three different libraries: i) 1,199 compounds from the Prestwick® Chemical Library, which contains mostly US Food and Drug Administration approved drugs (Prestwick® Chemical, Illkirch, France); ii) 1,651 compounds from the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen, France); iii) 41 compounds (called herein in-house antiviral library) selected for their effects against different human viruses. Compounds effective against EHV-1 were selected using the area under normalised curves (AUCn) and the time required for the Cell Index to decrease by 50% after virus infection (CIT50). The full dataset from the screen is made publicly available for further analyses.

Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination.

Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK) inhibitors into potential antimycobacterial agents.

As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 µM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents.

Synthesis and structure activity relationships of cyanopyridone based anti-tuberculosis agents.

Mycobacterium tuberculosis, the causative agent of tuberculosis, relies on thymidylate kinase (MtbTMPK) for the synthesis of thymidine triphosphates and thus also DNA synthesis. Therefore, this enzyme constitutes a potential Achilles heel of the pathogen. Based on a previously reported MtbTMPK 6-aryl-substituted pyridone inhibitor and guided by two co-crystal structures of MtbTMPK with pyridone- and thymine-based inhibitors, we report the synthesis of a series of aryl-shifted cyanopyridone analogues. These compounds generally lacked significant MtbTMPK inhibitory potency, but some analogues did exhibit promising antitubercular activity. Analogue 11i demonstrated a 10-fold increased antitubercular activity (MIC H37Rv, 1.2 µM) compared to literature compound 5. Many analogues with whole-cell antimycobacterial activity were devoid of significant cytotoxicity.

1-(1-Arylethylpiperidin-4-yl)thymine Analogs as Antimycobacterial TMPK Inhibitors.

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 µM).

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors.

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

1-(Piperidin-3-yl)thymine amides as inhibitors of M. tuberculosis thymidylate kinase.

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable MtbTMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3. Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC50=35 µM). This finding opens avenues for future modifications.