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Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective anti-microbial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. To address this, we developed a flow-controlled multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells, compressed inside separate microchambers by a soft hydro-pneumatic membrane. With this platform, we implemented a dynamic single-cell screening for compounds that induce a phenotypic change while decreasing cell-to-cell variation, aiming to undermine the bacterial population, making it more vulnerable to other drugs. We first applied this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our top hit impairsMycobacterium tuberculosisvia a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that pheno-tuning compounds represent a successful approach to tackle pathogens that are increasingly difficult to treat.

International audience; 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.

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; Association of the initiation factor eIF4E with the mRNA cap structure is a key step for translation. Trypanosomatids present six eIF4E homologues, showing a low conservation and also differing significantly from the IF4Es of multicellular eukaryotes. On the mRNA side, while in most eukaryotes the mRNA contains cap-0 (7-methyl-GTP), the trypanosomatid mRNA features a cap-4, which is formed by a cap-0, followed by the AACU sequence containing 2'-O-ribose methylations and base methylations on nucleotides 1 and 4. The studies on eIF4E-cap-4 interaction have been hindered by the difficulty to synthesize this rather elaborated cap-4 sequence. To overcome this problem, we applied a liquid-phase oligonucleotide synthesis strategy and describe for the first time the crystal structure of a trypanosomatid eIF4E (T. cruzi EIF4E5) in complex with cap-4. The TcEIF4E5-cap-4 structure allowed a detailed description of the binding mechanism, revealing the interaction mode for the AACU sequence, with the bases packed in a parallel stacking conformation and involved, together with the methyl groups, in hydrophobic contacts with the protein. This binding mechanism evidences a distinct cap interaction mode in comparison with previously described eIF4E structures and may account for the difference of TcEIF4E5-cap-4 dissociation constant in comparison with other eIF4E homologues.

The structurally unique “fleximer” nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor–ligand recognition and function. Recently they have been shown to have low‐to‐sub‐micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex‐bases) has been successfully coupled to both ribose and 2′‐deoxyribose sugars by using the N‐deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno‐expanded tricyclic heterocyclic base, as well as several distal and proximal flex‐bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein., A thieno‐expanded tricyclic base and several distal and proximal flex‐bases have been converted into their corresponding fleximer nucleosides by enzymatic transglycosylation reactions catalyzed by N‐deoxyribosyltransferase from L. leichmannii (LlNDT) or E. coli purine nucleoside phosphorylase (PNP).

International audience; Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now.

International audience; A versatile two-step synthesis of C4- and C5-arylated 2′-deoxyribosylimidazoles was elaborated using enzymatic N-transglycosylation followed by microwave-assisted Pd-catalysed arylation reactions. We report herein the reaction conditions that permit managing regioselectivity (N3 versus N1-isomers) in the enzymatic glycosylation of 4-iodoimidazole using the nucleoside N-deoxyribosyltransferase from L. leichmannii. Regiocontrolled glycosylation was also observed among several other imidazole derivatives studied, providing simple access to isomers not readily accessible by chemical routes. Finally, a series of flexible nucleosides was obtained in one step from 4- or 5-iodo-imidazole nucleosides by the Suzuki–Miyaura cross-coupling reaction with (hetero)aryl-boronic acids in aqueous media. Moreover, this chemoenzymatic approach is compatible with a one-pot two-step process affording a straightforward access to a broad array of potential anticancer and antiviral drugs as well as new DNA building blocks.

International audience; We developed a versatile access to a series of 4-substituted imidazole 2'-deoxynucleoside triphosphate bearing functionalized phenyl or pyrimidinyl rings. 4-Iodo-1H-imidazole was enzymatically converted into the corresponding 2'-deoxynucleoside, which was then chemically derived into its 5'-triphosphate, followed by 4-arylation via Suzuki-Miyaura coupling using (hetero)arylboronic acids. Both KF (exo-) and Deep Vent (exo-) DNA polymerases incorporated these modified nucleotides in primer-extension assays, adenine being the preferred pairing partner in the template. The 4-(3-aminophenyl)imidazole derivative (3APh) was the most efficiently inserted opposite A by KF (exo-) with only a 37-fold lower efficiency (Vmax/KM) than that of the correct dTTP. No further extension occurred after the incorporation of a single aryl-imidazole nucleotide. Interestingly, the aryl-imidazole dNTPs were found to undergo successive incorporation by calf thymus terminal deoxynucleotidyl transferase with different tailing efficiencies among this series and with a marked preference for 2APyr polymerization.

International audience; A straightforward route to original imidazole-based nucleosides that makes use of an enzymatic N-transglycosylation step is reported in both the ribo- and deoxyribo-series. To illustrate the scope of this approach, a diverse set of 4-aryl and 4-heteroaryl-1H-imidazoles featuring variable sizes and hydrogen-bonding patterns was prepared using a microwave-assisted Suzuki-Miyaura cross-coupling reaction. These imidazole derivatives were examined as possible substrates for the nucleoside 2'-deoxyribosyltransferase from L. leichmannii and the purine nucleoside phosphorylase from E. coli. The optimum transglycosylation conditions, including the use of co-adjuvants to address solubility issues, were defined. Enzymatic conversion of 4-(hetero)arylimidazoles to 2'-deoxyribo- or ribo-nucleosides proceeded in good to high conversion yields, except bulky hydrophobic imidazole derivatives. Nucleoside deoxyribosyltransferase of class II was found to convert the widest range of functionalized imidazoles into 2'-deoxyribonucleosides and was even capable of bis-glycosylating certain heterocyclic substrates. Our findings should enable chemoenzymatic access to a large diversity of flexible nucleoside analogues as molecular probes, drug candidates and original building blocks for synthetic biology.

In vivo targeting of C-type lectin receptors is an effective strategy for increasing antigen uptake and presentation by dendritic cells (DCs). To induce efficient immune response, glycosylated tumor-associated Tn antigens were used to target DCs through binding to macrophage galactose-type lectin (MGL). The capacity of Tn-glycosylated antigens—and the multiple antigenic glycopeptide Tn3 therapeutic candidate vaccine—to target mouse and human MGL+ DCs are demonstrated, especially regarding dermal DCs. In mice, MGL+ CD103− dermal DCs efficiently captured and processed glycosylated Tn antigen in vivo, inducing a potent major histocompatibility complex (MHC) class II–restricted T-cell response. Intradermal immunization with Tn-glycopeptides induced high levels of Th2 cytokines—even in the presence of unmethylated cytosine-phosphate-guanosine—and was associated with increased expansion of the germinal center B-cell population. Therefore, MGL acts as an efficient endocytic antigen receptor on dermal DCs in vivo, able to prime Tn-specific T- and B-cell responses. Moreover, even in the absence of adjuvant, immunization with this glycosidic Tn-based vaccine induced high levels of anti-Tn antibody responses, recognizing human tumor cells. In vivo DC-targeting strategies, based on Tn-MGL interactions, constitute a promising strategy for enhancing antigen presentation and inducing potent antibody response.

Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant's broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.

Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor-associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte-associated antigens (HLA). In order to stimulate a T-cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B-cell epitope (Tn antigen: alpha-GalNAc-Ser) covalently linked to peptides corresponding to the Pan DR 'universal' T-helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA-DR1, and HLA-DR4). A strong T-cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.

Isolation of genes encoding catalysts for defined chemical reactions should be facilitated by selection of proteins for catalysis from molecular repertoires. Display of proteins on phage allows the coupling between a protein and its gene. Furthermore, if the reaction product can be linked to the phage enzyme catalyzing the reaction, affinity chromatography for the product yields the protein catalyzing the reaction and its gene. One main advantage of this selection method is that it can be in principle generalized to most chemical reactions.Here, iterative in vitro selections for polymerase activity were used to isolate a single phage-polymerase among more than 108 phage particles. Three to five selection cycles were required depending on the fraction of infectious phage-polymerases in the initial phage population. This is the first report quantifying the enrichment over iterative selection rounds for the isolation of rare catalysts displayed on phage.

International audience; A simple and rapid access to a variety of 4(5)-arylated imidazoles via palladium-catalyzed Suzuki–Miyaura cross-coupling reaction is described. Coupling parameters were screened for efficient C-4 arylation of N-unprotected 4-iodoimidazole with a broad range of boronic acids under microwave irradiation. Twenty-one imidazole derivatives were synthesized in modest to excellent yields in short reaction times.

Two contiguous pairs of enantiomeric 2′-deoxy-l-ribonucleotides have been incorporated in a 5-methyl-CG alternating decadeoxyribonucleotide duplex. Data from circular dichroism and UV melting experiments indicate that the two base-pair long enantio-domain within the duplex was able to stabilize a Z-DNA-like conformation.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.

International audience; Several studies have shown that alpha-oligonucleotides (alpha-ONs) are more resistant to degradation by nucleases than are beta-oligonucleotides (beta-ONs), but a few exceptions have been reported. The present work indicates that the resistance of alpha-ONs to 3'-exonucleases contained in calf or human sera strongly depends on their 3'-terminal sequence. When the 2 last residues were ...A-G, ...C-A, or ...C-T, the degradation rates in tissue culture media with fetal calf serum were similar to those observed for beta-ONs, but stabilization factors up to 200 were observed when the 2 last residues were ...T-C, ...A-C, or ...C-C, and intermediate stabilization factors were found with ...G-A, ...T-A, or ...T-T terminal sequences. Other data confirm that alpha-ONs are significantly more resistant than beta-ONs to purified 5'-exo- and endonucleases as well as to 3'-exonucleases contained in snake venom or CEM cell lysates, but suggest that sequence specificity may also exist in these media. These findings, which are consistent with literature data and explain the behavior of the aforementioned exceptions, also emphasize that the stability observed in the presence of purified nucleases cannot be extrapolated to sera. Other results show that handling, heat inactivation, and storage conditions have little effect on the 3'-exonuclease activity of serum-containing media, but can completely modify the nuclease activities of cell extracts. This study, which was carried out by means of the accurate "on-line ISRP cleaning" HPLC technique, brings new insights to the general problem of oligonucleotide stability.

International audience; Tumor Associated Carbohydrate Antigens (TACAs) constitute powerful tools as tumor markers and as targets for anticancer immunotherapy. In this review, methods of production of glycopeptide-based vaccines, as well as results of preclinical and clinical studies in cancer patients are discussed.

For identification of mutations associated with the broadening of enzyme substrate specificity, three strategies, including directed enzyme evolution, are described for selected examples. Implications concerning enzyme models are highlighted. Applications to the field of biocatalysis are discussed. A bidimensional map for the classification of enzyme activities is suggested so as to improve genome annotations.

We recently developed an efficient strategy based on a fully synthetic dendrimeric carbohydrate display (multiple antigenic glycopeptide; MAG) to induce anticarbohydrate antibody responses for therapeutic vaccination against cancer. Here, we show the superior efficacy of the MAG strategy over the traditional keyhole limpet hemocyanin glycoconjugate to elicit an anticarbohydrate IgG response against the tumor-associated Tn antigen. We highlight the influence of the aglyconic carrier elements of such a tumor antigen for their recognition by the immune system. Finally, we additionally developed the MAG system by introducing promiscuous HLA-restricted T-helper epitopes and performed its immunological evaluation in nonhuman primates. MAG:Tn vaccines induced in all of the animals strong tumor-specific anti-Tn antibodies that can mediate antibody-dependent cell cytotoxicity against human tumor. Therefore, the preclinical evaluation of the MAG:Tn vaccine demonstrates that it represents a safe and highly promising immunotherapeutic molecularly defined tool for targeting breast, colon, and prostate cancers that express the carbohydrate Tn antigen.

International audience; As part of our program on Tn-specific anti-tumor immunotherapy, our aim was to vary the nature of the aglyconic part of the tumor-associated Tn antigen (alpha-d-GalNAc-Ser/Thr). This report describes the synthesis of Fmoc-hSer-(alpha-d-GalNAc)-OH (4) in 19% overall yield from protected aspartic acid. The building block 4 was incorporated as trimeric clusters into a glycopeptide vaccine [MAG:Tn(hSer)3-PV], using solid-phase peptide synthesis. When injected in mice, the resulting MAG induces a strong antibody response, which recognizes native tumor-associated antigens (TAA) at the surface of human tumor cells. This approach may be extended to the use of other nonnatural TAA in order to improve half-life of synthetic anti-cancer vaccines.

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O-linked to a CD4+ T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

International audience; All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.

The Tsuji‐Trost Reaction is a palladium‐catalysed allylation of nucleophiles that consists in the reaction of a nitrogen, carbon or oxygen‐based nucleophiles with an allylic substrate bearing a leaving group. Here we present the use of 2‐nitrosulfonamide derivatives as nucleophile, which are reactive under mild conditions. 2‐nitrosulfonyl groups are well‐known dual protective activator groups easy to introduce in any type of amine substrates. The resulting 2‐nitrosulfonamide derivatives are ideal substrates for the Tsuji‐Trost reaction to afford a convenient and flexible access to primary and dissymmetric secondary allyl amines. The optimised procedure is flexible (for solvent, temperature, functional groups) and has been applied with good to excellent yield to access to a wide range of allyl amine derivatives., A direct method for the allylation of 2‐nitrosulfonamides is described and exemplified. The mild and flexible conditions used as well as the ease to remove the 2‐nitrosulfonamide group are compatible with a large set of chemical moieties.