Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Biocatalytic Synthesis of Coumarin S-Glycosides: Towards Non-Cytotoxic Probes for Biomedical Imaging and Sensing.

This study unveils an innovative method for synthesizing coumarin S-glycosides, employing original biocatalysts able to graft diverse carbohydrate structures onto 7-mercapto-4-methyl-coumarin in one-pot reactions. The fluorescence properties of the generated thio-derivatives were assessed, providing valuable insights into their potential applications in biological imaging or sensing. In addition, the synthesized compounds exhibited no cytotoxicity across various human cell lines. This research presents a promising avenue for the development of coumarin S-glycosides, paving the way for their application in diverse biomedical research areas.

"Mix and match" auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity.

Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Lectin Analysis of SARS-CoV-2-Positive Nasopharyngeal Samples Using GLYcoPROFILE® Technology Platform.

Nasopharyngeal samples are currently accepted as the standard diagnostic samples for nucleic acid amplification testing and antigenic testing for the SARS-CoV-2 virus. In addition to the diagnostic capacity of SARS-CoV-2-positive crude nasopharyngeal samples, their qualitative potential for direct glycan-specific analysis, in order to uncover unique glycol profiles, was assessed. In this study we provide glycan characterization of SARS-CoV-2-positive and -negative nasopharyngeal samples directly from lectin interactions. Although with limited throughput, this study evaluated the clinical sensitivity and specificity of the GLYcoPROFILE® technology platformon45crude nasopharyngeal samples collected between November 2020 and April 2022. Each GLYcoPROFILE® of 39 SARS-CoV-2-positive samples was compared toglycoprofiling on a panel of 10 selected lectins and the results were paralleled with SARS-CoV-2-negative samples' results. The GLYcoPROFILE® showed a clear distinction between positive and negative samples with WFA, GSL-II, PHA-L (GlcNAc-specific) and BPA (GalNAc-specific) highlighted as relevant lectins in SARS-CoV-2-positive samples. In addition, a significant, positive statistical correlation was found for these lectins (p < 0.01).

Synthesis, Anticancer Activities and Molecular Docking Studies of a Novel Class of 2-Phenyl-5,6,7,8-tetrahydroimidazo [1,2-b]pyridazine Derivatives Bearing Sulfonamides.

In the present study, new 2-phenyl-5,6,7,8-tetrahydroimidazo [1,2-b]pyridazines bearing sulfonamides were synthesized, characterized and evaluated for their anticancer activities. The structures of these derivatives were elucidated by 1H NMR, 13C NMR, infrared and high-resolution mass spectrometry for further validation of the target compound structures. The anticancer activities of the new molecules were evaluated against five human cancer cell lines, including A-549, Hs-683, MCF-7, SK-MEL-28 and B16-F10 cell lines using 5-fluorouracil and etoposide as the reference drugs. Among the tested compounds, 4e and 4f exhibited excellent activities in the same range of the positive controls, 5-fluorouracil and etoposide, against MCF-7 and SK-MEL-28 cancer cell lines, with IC50 values ranging from 1 to 10 µM. The molecular docking studies of 4e and 4f showed a strong binding with some kinases, which are linked to MCF-7 and SK-MEL-28 cancer cell lines.

Design, synthesis, and evaluation of cytotoxic activities of arylnaphthalene lignans and aza-analogs.

A concise and versatile synthetic strategy for the total synthesis of arylnaphthalene lignans and aza-analogs was developed. The main objective was to develop synthetic tactics for the creation of the lactone and lactam unit that would give access to an array of synthetic, natural, and/or bioactive compounds through rather simple chemical manipulation. The flexibility and potentiality of these new processes were further illustrated by the total synthesis of retrojusticidin B (13b), justicidin C (14b), and methoxy-vitedoamine A (22a). In this study, a series of novel aryl-naphthalene lignans and aza-analogs were synthesized, and the cytotoxic activities of all compounds on cancer cell growth were evaluated. The target compounds were structurally characterized by 1 H NMR (nuclear magnetic resonance), 13 C NMR, infrared, high-resolution mass spectrometry, and X-ray crystallography. The IC50 values of these compounds on five tumor cell lines (A549, HS683, MCF-7, SK-MEL-28, and B16-F1) were obtained by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Five of the compounds exhibited excellent activity compared to 5-fluorouracil and etoposide against the five cell lines tested, with IC50 values ranging from 1 to 10 µM.

Crystal structure of Dictyoglomus thermophilum ß-d-xylosidase DtXyl unravels the structural determinants for efficient notoginsenoside R1 hydrolysis.

Dictyoglomus thermophilum ß-d-xylosidase DtXyl is attractive as a potential thermostable biocatalyst able to produce biologically active ginsenosides intermediates from ß-(1,2)-D-xylosylated compounds, including Notoginsenoside-R1. DtXyl was expressed as an active N-terminal His-tagged protein, and its crystal structure was solved in presence or absence of d-xylose product. Modelling of notoginsenoside R1 in DtXyl active site led to the identification of several hydrophobic residues interacting in close contact to the substrate hydrophobic core. Unlike other residues involved in substrate binding, these residues are not conserved among GH39 xylosidase family, and their physico-chemical properties can be correlated to the efficient binding and subsequent hydrolysis of Notoginsenoside R1.

Synthesis of an STnThr analogue, structurally based on a TnThr antigen mimetic.

The monosaccharide Tn and the disaccharide STn are tumor antigens with similar structures and common biosynthetic pathways. Both are always over-expressed simultaneously on tumor cell surfaces. We report herein the efficient synthesis of the STnThr antigen analogue 2, featuring the immunogenic TnThr mimetic 1 aglycon. Analogously to the native STn, 2 is recognized by the influenza N1 neuraminidase. A model of the N1·2 complex showed the sialyl moiety of 2 well nested in the active site pocket, with docking unaffected by the rigid aglycon. The analogue 2 is, therefore, in association with mimetic 1, a good determinant for the design of new multiantigen cancer vaccines.

Thioglycoligation of aromatic thiols using a natural glucuronide donor.

The ß-d-glucuronidase DtGlcA from Dictyoglomus thermophilum was engineered to generate an active thioglycoligase that is able to catalyse the formation of numerous S-glucuronides. Its X-ray structure analysis indicated the ability of the biocatalyst to bind aromatic thiol acceptors for S-glycosylation. Noteworthily, the DtGlcA mutant was found to be the first thioligase that is able to use a natural sugar donor different from the widely used synthetic para-nitrophenyl glycosides.

Galactofuranose-Related Enzymes: Challenges and Hopes.

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we recently characterized as an efficient recombinant biocatalyst.

Biochemical Characterization of the α-l-Rhamnosidase DtRha from Dictyoglomus thermophilum: Application to the Selective Derhamnosylation of Natural Flavonoids.

α-l-Rhamnosidases are catalysts of industrial tremendous interest, but their uses are still somewhat limited by their poor thermal stabilities and selectivities. The thermophilic DtRha from Dictyoglomus thermophilum was cloned, and the recombinant protein was easily purified to homogeneity to afford 4.5 mg/L culture of biocatalyst. Michaelis-Menten parameters demonstrated it to be fully specific for α-l-rhamnose. Most significantly, DtRha demonstrated to have a stronger preference for α(1 → 2) linkage rather than α(1 → 6) linkage when removing rhamnosyl moiety from natural flavonoids. This selectivity was fully explained by the difference of binding of the corresponding substrates in the active site of the protein.

Monitoring of phosphorylation using immobilized kinases by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry.

Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40 µL). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [M - H]- ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4 °C in ammonium acetate buffer), after 20 uses and more than 750 min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5 min), efficient (conversion efficiency > 55%), precise and repeatable (CV < 3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).

Galactofuranosidase from JHA 19 Streptomyces sp.: subcloning and biochemical characterization.

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP ß-d-Galf, giving a KM value of 250 µM, and the highest enzymatic efficiency ever observed of 14 mM-1  s-1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry.

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

The LPG1x family from Leishmania major is constituted of rare eukaryotic galactofuranosyltransferases with unprecedented catalytic properties.

Galactofuranosyltransferases are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. These enzymes are considered as potential targets for therapeutic action. In addition to the only well-characterised prokaryotic GlfT2 from Mycobacterium tuberculosis, four putative genes in Leishmania major were previously described as potential galactofuranosyltransferases. In this study, we have cloned, over-expressed, purified and fully determined the kinetic parameters of these four eukaryotic enzymes, thus demonstrating their unique potency in catalysing the transfer of the galactofuranosyl moiety into acceptors. Their individual promiscuity revealed to be different, as some of them could efficiently use NDP-pyranoses as donor substrates in addition to the natural UDP-galactofuranose. Such results pave the way for the development of chemoenzymatic synthesis of furanosyl-containing glycoconjugates as well as the design of improved drugs against leishmaniasis.

Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides.

Carbohydrate related enzymes, like glycosyltransferases and glycoside hydrolases, are nowadays more easily accessible and are thought to represent powerful and greener alternatives to conventional chemical glycosylation procedures. The knowledge of their corresponding mechanisms has already allowed the development of efficient biocatalysed syntheses of complex O-glycosides. These enzymes can also now be applied to the formation of rare or unnatural glycosidic linkages.

TLC-UV hyphenated with MALDI-TOFMS for the screening of invertase substrates in plant extracts.

In this study, thin-layer chromatography (TLC) hyphenated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the screening of invertase substrates in complex matrices. BfrA, a specific ß-D-fructofuranosidase from Leishmania major, was chosen as a model enzyme to screen biological activity in plant extracts due to its capacity to hydrolyze specific carbohydrates. TLC was considered to be a reliable technique for screening substrates (bioactive molecules) in plant extracts due to its quantitative capabilities whereas MALDI-TOFMS was particularly useful for rapid identification. The first part of this approach consisted of a differential analysis by TLC-densitometry to highlight band under- and over-expressions in plant extract between blank and enzymatic reaction. Zones of interest were then immediately analyzed by TLC-MALDI-TOFMS coupling to identify bioactive molecules. Development of the method presented various challenges: the separation and analysis of isomers (such as glucose and fructose), the high matrix effect (demonstrated by the analysis of products with invertase enzyme naturally present in plant extract), and the analysis of polar molecules with low molecular mass (sugars). Thanks to the separative technique, the specificity of detection, and the high precision of the characterization, this method was shown to be feasible for the analysis of bioactive molecules in complex mixtures containing interfering compounds (e.g. proteins, salts). Overall, this study demonstrates that Thin-layer chromatography hyphenated with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a simple, rapid, precise and efficient method for the analysis of suitable substrates in raw samples.

Is the acid/base catalytic residue mutation in ß-d-mannosidase DtMan from Dictyoglomus thermophilum sufficient enough to provide thioglycoligase activity?

Glycoside hydrolases can be turned into thioglycoligase by mutation of the acid/base catalytic carboxylate residue. These mutants have proven valuable to generate S-glycosides, however, few examples in literature have described efficient thioglycoligase activity, and even fewer the underlying molecular mechanism. DtMan, a GH2 family ß-d-mannosidase from the thermophilic Dictyoglomus thermophilum was cloned and expressed in E. coli. The recombinant protein is highly specific for ß-d-mannosides, and exhibits efficient catalysis constants coupled to thermostability. However, seven variants bearing mutated acid/base residue could not be turned into efficient thioligases. Crystal structure of DtMan Glu425Cys mutant and molecular modeling calculations have demonstrated that unlike other GH2 thioligase reported, active site accessibility of thiol acceptor may be impaired by entrance loop rigidity. This structural feature may explain why DtMan mutants do not exhibit thioglycoligase activity.

Synthetic Modification of 9α- and 9ß-Hydroxyparthenolide by Heck or Acylation Reactions and Evaluation of Cytotoxic Activities.

Motivated by the widely reported anticancer activity of parthenolides and their derivatives, a series of new substituted parthenolides was efficiently synthesized. Structural modifications were performed at the C-9 and C-13 positions of 9α- and 9ß-hydroxyparthenolide, which were isolated from the aerial parts of Anvillea radiata. Twenty-one derivatives were synthesized and evaluated for their in vitro cytotoxic activity against HS-683, SK-MEL-28, A549, and MCF-7 human cancer cell lines using the MTT colorimetric assay. Among the derivatives, seven exhibited excellent activity compared to 5-fluorouracil and etoposide against the four cell lines tested, with IC50 values ranging from 1.1 to 9.4 µM.