Copper catalyzed cycloaddition for the synthesis of non isomerisable 2' and 3'-regioisomers of arg-tRNAarg.

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2'- or 3'-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2'- or 3'-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5'-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5'-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2' and 3') which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

In Situ Bioconjugation of a Maleimide-Functionalized Ruthenium-Based Photosensitizer to Albumin for Photodynamic Therapy.

Maleimide-containing prodrugs can quickly and selectively react with circulating serum albumin following their injection in the bloodstream. The drug-albumin complex then benefits from longer blood circulation times and better tumor accumulation. Herein, we have applied this strategy to a previously reported highly phototoxic Ru polypyridyl complex-based photosensitizer to increase its accumulation at the tumor, reduce off-target cytotoxicity, and therefore improve its pharmacological profile. Specifically, two complexes were synthesized bearing a maleimide group: one complex with the maleimide directly incorporated into the bipyridyl ligand, and the other has a hydrophilic linker between the ligand and the maleimide group. Their interaction with albumin was studied in-depth, revealing their ability to efficiently bind both covalently and noncovalently to the plasma protein. A crucial finding is that the maleimide-functionalized complexes exhibited significantly lower cytotoxicity in noncancerous cells under dark conditions compared to the nonfunctionalized complex, which is a highly desirable property for a photosensitizer. The binding to albumin also led to a decrease in the phototoxicity of the Ru bioconjugates in comparison to the nonfunctionalized complex, probably due to a decreased cellular uptake. Unfortunately, this decrease in phototoxicity was not compensated by a dramatic increase in tumor accumulation, as was demonstrated in a tumor-bearing mouse model using inductively coupled plasma mass spectrometry (ICP-MS) studies. Consequently, this study provides valuable insight into the future design of in situ albumin-binding complexes for photodynamic therapy in order to maximize their effectiveness and realize their full potential.

N 2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing.

Modified nucleotides in non-coding RNAs, such as tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last 'orphan' modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing.

A Novel Near-IR Absorbing Ruthenium(II) Complex as Photosensitizer for Photodynamic Therapy and its Cetuximab Bioconjugates.

A novel Ru(II) cyclometalated photosensitizer (PS), Ru-NH2 , for photodynamic therapy (PDT) of formula [Ru(appy)(bphen)2 ]PF6 (where appy=4-amino-2-phenylpyridine and bphen=bathophenanthroline) and its cetuximab (CTX) bioconjugates, Ru-Mal-CTX and Ru-BAA-CTX (where Mal=maleimide and BAA=benzoylacrylic acid) were synthesised and characterised. The photophysical properties of Ru-NH2 revealed absorption maxima around 580 nm with an absorption up to 725 nm. The generation of singlet oxygen (1 O2 ) upon light irradiation was confirmed with a 1 O2 quantum yield of 0.19 in acetonitrile. Preliminary in vitro experiments revealed the Ru-NH2 was nontoxic in the dark in CT-26 and SQ20B cell lines but showed outstanding phototoxicity when irradiated, reaching interesting phototoxicity indexes (PI) >370 at 670 nm, and >150 at 740 nm for CT-26 cells and >50 with NIR light in SQ20B cells. The antibody CTX was successfully attached to the complexes in view of the selective delivery of the PS to cancer cells. Up to four ruthenium fragments were anchored to the antibody (Ab), as confirmed by MALDI-TOF mass spectrometry. Nonetheless, the bioconjugates were not as photoactive as the Ru-NH2 complex.

An enzymatic activation of formaldehyde for nucleotide methylation.

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

Drimane Derivatives as the First Examples of Covalent BH3 Mimetics that Target MCL-1.

Drimane sesquiterpenoid dialdehydes are natural compounds with antiproliferative properties. Nevertheless, their mode of action has not yet been discovered. Herein, we demonstrate that various drimanes are potent inhibitors of MCL-1 and BCL-xL, two proteins of the BCL-2 family that are overexpressed in various cancers, including lymphoid malignancies. Subtle changes in their structure significantly modified their activity on the target proteins. The two most active compounds are MCL-1 selective and bind in the BH3 binding groove of the protein. Complementary studies by NMR spectroscopy and mass spectrometry analyses, but also synthesis, showed that they covalently inhibit MCL-1 though the formation of a pyrrole adduct. In addition, cytotoxic assays revealed that these two compounds show a cytotoxic selectivity for BL2, a MCL-1/BCL-xL-dependent cell line and induce apoptosis.

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions.

Structural and functional insights into Archaeoglobus fulgidus m2G10 tRNA methyltransferase Trm11 and its Trm112 activator.

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.

Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver.

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host.

Ruthenium-initiated polymerization of lactide: a route to remarkable cellular uptake for photodynamic therapy of cancer.

Ruthenium complexes have attracted a lot of attention as potential photosensitizers (PSs) for photodynamic therapy (PDT). However, some of these PSs are unsuitable for PDT applications due to their low cellular uptake, which is possibly the consequence of their relatively low degree of lipophilicity, which prevents them from penetrating into tumor cells. Here, we report the simple one-pot synthesis of ruthenium-containing nanoconjugates from a non-cell-penetrating, non-phototoxic ruthenium(ii) polypyridyl complex (RuOH), by a drug-initiated ring-opening polymerization of lactide through the formation of a zinc initiator. These conjugates were then formulated into nanoparticles by nanoprecipitation and characterized by means of nuclear magnetic resonance spectroscopy (NMR), matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and dynamic light scattering (DLS). Finally, their photo-therapeutic activity (λ exc = 480 nm, 3.21 J cm-2) in cancerous human cervical carcinoma (HeLa) and non-cancerous retinal pigment epithelium (RPE-1) cells was tested alongside that of RuOH and their cellular uptake in HeLa cells was assessed by confocal microscopy and inductively coupled plasma - mass spectrometry (ICP-MS). All nanoparticles showed improved photophysical properties including luminescence and singlet oxygen generation, enhanced cellular uptake and, capitalizing on this, an improved photo-toxicity. Overall, this study demonstrates how it is possible to transform a non-phototoxic PDT PS into an active PS using an easy, versatile polymerization technique.

Polymerization of rac-Lactide Using Achiral Iron Complexes: Access to Thermally Stable Stereocomplexes.

Enantiopure poly(lactic acid) (PLA) can form stereocomplexes when enantiomeric PLA chains are mixed in equivalent amounts. Such materials provide interesting features that might be suitable for numerous applications. Despite several advantages, the main drawback of PLA is its narrow window of processing, thus limiting its use for industrial applications. Reported herein are achiral iron complexes, that are highly active, productive, and stereoselective under mild reaction conditions for the ring-opening polymerization of lactide. The corresponding catalytic systems enable the production of stereoblock polymers with high molecular weights, allowing the formation of thermally stable and industrially relevant stereocomplexes.

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells.

Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible.

Inhibition of p53-Murine Double Minute 2 (MDM2) Interactions with 3,3'-Spirocyclopentene Oxindole Derivatives.

3,3'-Spirocyclopentene oxindoles structurally related to Wang's spiropyrrolidine oxindoles have been highlighted as a new class of antiproliferative agents against cancer cell lines with wild-type p53 status (IC50 up to 0.96 µM on SJSA-1 and 2.9 µM in HCT116 p53-wt). Inhibition of the MDM2-p53 interactions has been demonstrated through in vitro HTRF assays (IC50 up to 3.1 nM), while Western blot analysis showed activation of p53 selectively in HCT116 cancer cell lines with wild-type p53.

Unveiling structural and functional divergences of bacterial tRNA dihydrouridine synthases: perspectives on the evolution scenario.

Post-transcriptional base modifications are important to the maturation process of transfer RNAs (tRNAs). Certain modifications are abundant and present at several positions in tRNA as for example the dihydrouridine, a modified base found in the three domains of life. Even though the function of dihydrourine is not well understood, its high content in tRNAs from psychrophilic bacteria or cancer cells obviously emphasizes a central role in cell adaptation. The reduction of uridine to dihydrouridine is catalyzed by a large family of flavoenzymes named dihydrouridine synthases (Dus). Prokaryotes have three Dus (A, B and C) wherein DusB is considered as an ancestral protein from which the two others derived via gene duplications. Here, we unequivocally established the complete substrate specificities of the three Escherichia coli Dus and solved the crystal structure of DusB, enabling for the first time an exhaustive structural comparison between these bacterial flavoenzymes. Based on our results, we propose an evolutionary scenario explaining how substrate specificities has been diversified from a single structural fold.

Mechanistic Aspects of the Polymerization of Lactide Using a Highly Efficient Aluminum(III) Catalytic System.

We report here a unique example of an in situ generated aluminum initiator stabilized by a C2-symmetric salen ligand which shows a hitherto unknown high activity for the ROP of rac-lactide at room temperature. Using a simple and robust catalyst system, which is prepared from a salen complex and an onium salt, this convenient route employs readily available reagents that afford polylactide in good yields with narrow polydispersity indices, without the need for time-consuming and expensive processes that are typically required for catalyst preparation and purification. In line with the experimental evidence, DFT studies reveal that initiation and propagation proceed via an external alkoxide attack on the coordinated monomer.

An extended dsRBD is required for post-transcriptional modification in human tRNAs.

In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upregulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved α-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast complementation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the α-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs.

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase.

In most organisms, the widely conserved 1-methyl-adenosine58 (m1A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m1A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m1A58 formation triggers RNA release. A model of the protein-tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.

Nepheliosyne B, a new polyacetylenic acid from the new caledonian marine sponge Niphates sp.

A new C47 polyoxygenated acetylenic acid, nepheliosyne B (2), along with the previously described nepheliosyne A (1), have been isolated from the New Caledonian marine sponge Niphates sp. Their structures have been elucidated on the basis of extensive spectroscopic analyses. These metabolites exhibited a moderate cytotoxicity against K562, U266, SKM1, and Kasumi cancer cell lines.

The human tRNA m (5) C methyltransferase Misu is multisite-specific.

The human tRNA m ( 5) C methyltransferase Misu is a novel downstream target of the proto-oncogene Myc that participates in controlling cell division and proliferation. Misu catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosines in tRNAs. It was previously shown to catalyze in vitro the intron-dependent formation of m ( 5) C at the first position of the anticodon (position 34) within the human pre-tRNA (Leu) (CAA). In addition, it was recently reported that C48 and C49 are methylated in vivo by Misu. We report here the expression of hMisu in Escherichia coli and its purification to homogeneity. We show that this enzyme methylates position 48 in tRNA (Leu) (CAA) with or without intron and positions 48, 49 and 50 in tRNA (Gly2) (GCC) in vitro. Therefore, hMisu is the enzyme responsible for the methylation of at least four cytosines in human tRNAs. By comparison, the orthologous yeast enzyme Trm4 catalyzes the methylation of carbon 5 of cytosine at positions 34, 40, 48 or 49 depending on the tRNAs.

A new hydroxylated nonaprenylhydroquinone from the Mediterranean marine sponge Sarcotragus spinosulus.

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated.