Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

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.

Mapping and characterization of G-quadruplexes in the genome of the social amoeba Dictyostelium discoideum.

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.

Ionic Polypeptide Polymers with Unusual ß-Sheet Stability.

Incorporating charged amino acid side chains in polypeptide polymer backbones to improve solubility usually leads to reduced secondary structuring. Here we show that highly water soluble (>15 mg.mL-1) ß-sheets can be obtained via nucleotide monophosphate grafting onto simple poly(γ-propargyl- L-glutamate) backbone. This synthetic methodology has been applied to the synthesis of thymidine-based nucleopolypeptides presenting stable ß-sheet conformation in aqueous solutions with pH values comprised between 4 and 8. These polymeric analogues of nucleoproteins exhibited selective interaction with simple DNA sequences displaying adenine.

Ethionamide biomimetic activation and an unprecedented mechanism for its conversion into active and non-active metabolites.

Ethionamide (ETH), a second-line anti-tubercular drug that is regaining a lot of interest due to the increasing cases of drug-resistant tuberculosis, is a pro-drug that requires an enzymatic activation step to become active and to exert its therapeutic effect. The enzyme responsible for ETH bioactivation in Mycobacterium tuberculosis is a monooxygenase (EthA) that uses flavin adenine dinucleotide (FAD) as a cofactor and is NADPH- and O2-dependant to exert its catalytic activity. In this work, we investigated the activation of ETH by various oxygen-donor oxidants and the first biomimetic ETH activation methods were developed (KHSO5, H2O2, and m-CPBA). These simple oxidative systems, in the presence of ETH and NAD+, allowed the production of short-lived radical species and the first non-enzymatic formation of active and non-active ETH metabolites. The intermediates and the final compounds of the activation pathway were well characterized. Based on these results, we postulated a consistent mechanism for ETH activation, not involving sulfinic acid as a precursor of the iminoyl radical, as proposed so far, but putting forward a novel reactivity for the S-oxide ethionamide intermediate. We proposed that ETH is first oxidized into S-oxide ethionamide, which then behaves as a "ketene-like" compound via a formal [2 + 2] cycloaddition reaction with peroxide to give a dioxetane intermediate. This unstable 4-membered intermediate in equilibrium with its open tautomeric form decomposes through different pathways, which would explain the formation of the iminoyl radical and also that of different metabolites observed for ETH oxidation, including the ETH-NAD active adduct. The elucidation of this unprecedented ETH activation mechanism was supported by the application of isotopic labelling experiments.

Crystal structure of the enoyl-ACP reductase of Mycobacterium tuberculosis (InhA) in the apo-form and in complex with the active metabolite of isoniazid pre-formed by a biomimetic approach.

InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80Å resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40Å. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD(+), NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect.

The nickel(II) complex of guanidinium phenyl porphyrin, a specific G-quadruplex ligand, targets telomeres and leads to POT1 mislocalization in culture cells.

With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells. Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.

Guanosine in a single stranded region of anticodon stem-loop tRNA models is prone to oxidatively generated damage resulting in dehydroguanidinohydantoin and spiroiminodihydantoin lesions.

Oxidation of RNA hairpin models corresponding to anticodon stem-loop (ASL) of transfer RNA led to RNA damage consisting solely of a unique loop guanine oxidation. Manganese porphyrin/oxone treatment of RNA resulted in dehydroguanidinohydantoin (DGh; major) and/or spiroiminodihydantoin (Sp) lesions. Ribose damage was not observed. This two-electron transfer oxidation reaction allowed the identification of guanine oxidation products for further study of RNA species carrying a unique lesion at a single G to investigate their biological impact.

Cationic Porphyrin-Anionic Surfactant Mixtures for the Promotion of Self-Organized 1:4 Ion Pairs in Water with Strong Aggregation Properties.

We performed a systematic study on the spectroscopic and aggregation properties of stoichiometric mixtures (1:4) of the tetracationic meso-tetrakis(4-N-methylpyridinium)porphyrin (H2 TMPyP) and three sodium alkylsulfate surfactants (tetradecyl, hexadecyl, and octadecylsulfate) in an aqueous solution. The objective was to build a supramolecular aggregate, which would favor the internalization of tetracationic porphyrins in cells without chemical modification of the structure of the porphyrin. We show that stoichiometric H2 TMPyP/alkylsulfate (1:4) mixtures lead to the formation of large hollow spherical aggregates (60-160 nm). The TEM images show that the membrane of these aggregates are composed of smaller aggregates, which are probably rod-like micelles. These rod-like micelles have a hydrophobic core composed of the alkyl chains of the alkylsulfate surfactant, whereas the charged surface corresponds to the tetracationic porphyrins.

Concept for simultaneous and specific in situ monitoring of amyloid oligomers and fibrils via Förster resonance energy transfer.

Oligomeric species of amyloidogenic peptides or proteins are often considered as the most toxic species in several amyloid disorders, like Alzheimer or Parkinson's diseases, and hence came into the focus of research interest and as a therapeutic target. An easy and specific monitoring of oligomeric species would be of high utility in the field, as it is the case for thioflavin T fluorescence for the fibrillar aggregates. Here, we show proof of concept for a new sensitive method to increase specific detection of oligomers by two extrinsic fluorophores. This is achieved by exploiting a Förster resonance energy transfer (FRET) between the two fluorophores. Thus, a mixture of two extrinsic fluorophores, bis-ANS and a styrylquinoxalin derivative, enabled one to monitor simultaneously and in situ the presence of oligomers and fibrils of amyloidogenic peptides. Thereby, the formation of oligomers and their transformation into fibrils can be followed.

Formation of the carboxamidine precursor of cyanuric acid from guanine oxidative lesion dehydro-guanidinohydantoin.

DNA damage under oxidative stress leads to oxidation of guanine base. The identification of the resulting guanine lesions in cellular DNA is difficult due to the sensitivity of the primary oxidation products to hydrolysis and/or further oxidation. We isolated dehydroguanidino-hydantoin (DGh) (or oxidized guanidinohydantoin), a secondary oxidation product of guanine, and showed that this lesion reacts readily with nucleophiles such as asymmetric peroxides and transforms to 2,4,6-trioxo-1,3,5-triazinane-1-carboxamidine residue. Further hydrolysis of this intermediate leads to cyanuric acid and finally to urea residue. This work demonstrates a new possible pathway for the formation of the well-known carboxamidine precursor of cyanuric acid lesion.

N'-(3-Sulfanyl-idene-3,4-di-hydro-quinoxalin-2-yl)benzohydrazide di-methyl-formamide monosolvate.

The 2-sulfanyl-idene-3,4-di-hydro-quinoxalin-2-yl ring system of the title solvate, C15H12N4OS·C3H7NO, is essentially planar, the maximum deviation from the mean plane being 0.024 (2) Šfor the thione C atom. The mean plane through the fused-ring system is almost perpendicular to the terminal phenyl ring, as indicated by the dihedral angle of 70.05 (8)°. In the crystal, the main and solvent mol-ecules are linked by N-H⋯O hydrogen bonds, forming a layer parallel to (010).

A single nuclease-resistant linkage in DNA as a versatile tool for the characterization of DNA lesions: application to the guanine oxidative lesion "G+34" generated by metalloporphyrin/KHSO(5) reagent.

The oxidation of an oligonucleotide containing a single nuclease-resistant phosphodiester link, a stereoisomerically pure methylphosphonate, by manganese (Mn-TMPyP) or iron (Fe-TMPyP) porphyrin associated to KHSO(5) allowed the isolation and characterization of a guanine lesion corresponding to an increase of mass of 34 amu as compared to guanine ("G+34"), namely, 5-carboxamido-5-formamido-2-iminohydantoin. Enzymatic digestion of the damaged oligonucleotide afforded, apart from the undamaged nucleotide monomer pool, a unique dinucleotide doubly modified with a methylphosphonate and an oxidized guanine base that is suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually analyzed by the easy introduction of a single nuclease-resistant link at the 3'- or 5'-position.

Cross-docking study on InhA inhibitors: a combination of Autodock Vina and PM6-DH2 simulations to retrieve bio-active conformations.

InhA, the NADH-dependent enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis (Mtb) is the proposed main target of the first-line antituberculosis drug isoniazid (INH). INH activity is dependent on activation by the catalase peroxidase KatG, a Mtb enzyme whose mutations are linked to clinical resistance to INH. Other inhibitors of InhA that do not require any preliminary activation are known. The design of such direct potent inhibitors represents a promising approach to circumvent this resistance mechanism. An ensemble-docking process with four known InhA X-ray crystal structures and employing the Autodock Vina software was performed. Five InhA inhibitors whose bioactive conformations are known were sequentially docked in the substrate cavity of each protein. The efficiency of the docking was assessed and validated by comparing the calculated conformations to the crystallographic structures. For a same inhibitor, the docking results differed from one InhA conformation to another; however, docking poses that matched correctly or were very close to the expected bioactive conformations could be identified. The expected conformations were not systematically well ranked by the Autodock Vina scoring function. A post-docking optimization was carried out on all the docked conformations with the AMMP force field implemented on the VEGAZZ software, followed by a single point calculation of the interaction energy, using the MOPAC PM6-DH2 semi-empirical quantum chemistry method. The conformations were subsequently submitted to a PM6-DH2 optimization in partially flexible cavities. The resulting interaction energies combined with the multiple receptor conformations approach allowed us to retrieve the bioactive conformation of each ligand.

Comparative study of the phototoxicity of long-wavelength photosensitizers targeted by the MornigaG lectin.

Morniga G is a plant lectin selective for high density of tumor-associated carbohydrate T and Tn antigens on the surface of cells. The interaction of the protein with Tn induces its cell penetration. This property was used for targeting photosensitizers (consisting of the porphyrins TrMPyP and TPPS, the Al(III)-phthalocyanin AlPcS(4), and the chlorin e6) against leukemic Jurkat T cells after covalent coupling to the protein. The control of MornigaG/photosensitizer loading allowed the comparison of the toxicity of the different photosensitizer conjugates. Conjugate including a single AlPcS(4) per protein appeared promising, since it is poorly toxic when irradiated under white light, while it shows a strong phototoxicity (LD(50) = 4 nM) when irradiated in the therapeutic window, it preferentially kills cancerous lymphocytes, and the sugar binding specificity of the lectin part of the molecule remains unaltered.

Chemical synthesis, biological evaluation and structure-activity relationship analysis of azaisoindolinones, a novel class of direct enoyl-ACP reductase inhibitors as potential antimycobacterial agents.

The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relationships concerning the length and the location of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group, the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal nitrogen were also investigated, revealing insightful information and thereby enabling further diversification of the azaisoindolinone scaffold for new antitubercular agents.

Gold(III) porphyrins: Synthesis and interaction with G-quadruplex DNA.

G-quadruplex nucleic acids (G4s) are RNA and DNA secondary structures involved in the regulation of multiple key biological processes. They can be found in telomeres, oncogene promoters, RNAs, but also in viral genomes. Due to their unique structural features, very distinct from the canonical duplexes or single-strands, G4s represent promising pharmacological targets for small molecules, namely G4-ligands. Gold(III) penta-cationic porphyrins, as specific G4 ligands, are able to inhibit HIV-1 infectivity and their antiviral activity correlates with their affinity for G4s. Up to now, one of the best antiviral compounds is meso-5,10,15,20-tetrakis[4-(N-methyl-pyridinium-2-yl)phenyl]porphyrinato gold(III) (1). Starting from this compound, we report a structure/affinity relationship study of gold(III) cationic porphyrins to find out the best porphyrin candidate for functionalization, in order to study the antiviral mechanism of action of these gold(III) porphyrins.

Photolysis and thermolysis of platinum(IV) 2,2'-bipyridine complexes lead to identical platinum(II)-DNA adducts.

Two Pt(IV) and two Pt(II) complexes containing a 2,2'-bipyridine ligand were treated with a short DNA oligonucleotide under light irradiation at 37°C or in the dark at 37 and 50°C. Photolysis and thermolysis of the Pt(IV) complexes led to spontaneous reduction of the Pt(IV) to the corresponding Pt(II) complexes and to binding of Pt(II) 2,2'-bipyridine complexes to N7 of guanine. When the reduction product was [Pt(bpy)Cl(2)], formation of bis-oligonucleotide adducts was observed, whereas [Pt(bpy)(MeNH(2))Cl](+) gave monoadducts, with chloride ligands substituted in both cases. Neither in the dark nor under light irradiation was the reductive elimination process of these Pt(IV) complexes accompanied by oxidative DNA damage. This work raises the question of the stability of photoactivatable Pt(IV) complexes toward moderate heating conditions.

Interaction of cationic nickel and manganese porphyrins with the minor groove of DNA.

The capacity of a series of new cationic nickel and manganese metalloporphyrins to bind in the minor groove of DNA was evaluated by binding competition experiments with manganese(III)-bis-aqua-meso-tetrakis(4-N-methylpyridiniumyl)porphyrin, Mn-TMPyP, a powerful artificial nuclease when associated with KHSO(5). The four N-methylpyridiniumyl substituents on this porphyrin macrocycle are responsible for a strong binding affinity for the minor groove of AT-rich DNA. The inhibition of DNA cleavage mediated by Mn-TMPyP/KHSO(5) by the various tested porphyrins correlated with their competitive occupancy of the minor groove site of Mn-TMPyP. Introduction of long and flexible cationic substituents at the periphery of the porphyrin macrocycle precluded the interaction of the porphyrin derivative in the minor groove and resulted in low affinity for DNA. On the other hand, introduction of phenylpyridiniumyl substituents on the porphyrin macrocycle surprisingly conferred the new porphyrin derivative with a tight binding in the minor groove of a six consecutive AT base pairs sequence. These data on structural requirements for minor groove DNA binding will help the rational design of porphyrin derivatives for selective targeting of quadruplex DNA versus double-stranded DNA.

New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi.

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.