Patricia Busca

Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemXWv is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemXWv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1–C72 and G2–C71 base pairs of the acceptor stem as critical for FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues.

A novel tyrosine kinase inhibitor restores chondrocyte differentiation and promotes bone growth in a gain-of-function Fgfr3 mouse model

Activating germline fibroblast growth factor receptor 3 (FGFR3) mutations cause achondroplasia (ACH), the most common form of human dwarfism and a spectrum of skeletal dysplasias. FGFR3 is a tyrosine kinase receptor and constitutive FGFR3 activation impairs endochondral ossification and triggers severe disorganization of the cartilage with shortening of long bones. To decipher the role of FGFR3 in endochondral ossification, we analyzed the impact of a novel tyrosine kinase inhibitor (TKI), A31, on both human and mouse mutant FGFR3-expressing cells and on the skeleton of Fgfr3(Y367C/+) dwarf mice. We found that A31 inhibited constitutive FGFR3 phosphorylation and restored the size of embryonic dwarf femurs using an ex vivo culture system. The increase in length of the treated mutant femurs was 2.6 times more than for the wild-type. Premature cell cycle exit and defective chondrocyte differentiation were observed in the Fgfr3(Y367C/+) growth plate. A31 restored normal expression of cell cycle regulators (proliferating cell nuclear antigen, KI67, cyclin D1 and p57) and allowed pre-hypertrophic chondrocytes to properly differentiate into hypertrophic chondocytes. Our data reveal a specific role for FGFR3 in the cell cycle and chondrocyte differentiation and support the development of TKIs for the treatment of FGFR3-related chondrodysplasias.

Decoding the Logic of the tRNA Regiospecificity of Nonribosomal FemXWv Aminoacyl Transferase

Aminoacyl-tRNAs are key intermediates in protein synthesis. They act as adapters between the codons of mRNA and the growing polypeptide chain in the ribosome. The vicinal hydroxy groups at the 2’and 3’-positions of the terminal nucleotide (A) of tRNA have pivotal roles in the function of these molecules. The tRNA molecules are esterified by aminoacyl-tRNA synthetases, which catalyze the transfer of a specific aminoacyl residue from an adenylate to the 2’or 3’-hydroxy group of A (Scheme 1). Transesterification between the 2’and 3’-positions occurs in the absence of an enzyme with a rate and thermodynamic equilibrium of the order of 5 s 1 and 1, respectively. The A site of the ribosome is specific for the 3’-O-aminoacyl isomer, and the 3’ linkage to the tRNA is conserved in the product of the peptidyl-transfer reaction. The 2’-hydroxy group of the peptidyl-tRNA is thought to assist catalysis of this reaction. Besides their role in protein synthesis, aminoacyl-tRNAs participate in various metabolic pathways, such as the synthesis of cyclodipeptides or the aminoacylation of proteins and membrane phosphatidylglycerol. Transferases of the Fem family catalyze the incorporation of amino acids into peptidoglycan precursors to form a side chain that contains the amino group used as an acyl acceptor in the final cross-linking step of cell-wall synthesis (Scheme 1). The specificity of these enzymes is essential for bacteria, since misincorporated amino acids can act as chain terminators and block peptidoglycan polymerization. Because of their key role in peptidoglycan metabolism, Fem transferases are considered attractive targets for the development of novel antibiotics. We previously used chemical acylation of RNA helices with natural and nonproteinogenic amino acids to gain insight into the specificity of FemXWv of Weissella viridescens, [12, 13] a model enzyme of the Fem family. A combination of modifications in the RNA and aminoacyl moieties of the substrate revealed that unfavorable interactions of FemXWv with the acceptor arm of tRNA and with l-Ser or larger residues quantitatively account for the preferential transfer of l-Ala observed with complete aminoacyl-tRNAs. 13] The main FemXWv identity determinant of Ala-tRNA Ala was found to be the penultimate base pair, G–C, which is replaced with C–G in tRNA isoacceptors. 13] In this study, we synthesized nonisomerizable mimics of Ala-tRNA that contained 2’-deoxyadenosine or 3’-deoxyadenosine to lock the amino acid in the 3’and 2’-position, respectively (Scheme 2). We also synthesized nonisomerizable aminoacyl-tRNA analogues by replacing the ester bond connecting the amino acid residue to the terminal nucleotide with a triazole ring (Scheme 3). We synthesized these molecules to determine the regiospecificity of FemXWv for the 3’ and 2’ isomers and to evaluate the role of the adjacent hydroxy group in the transfer reaction. Ala-tRNA analogues containing a terminal 2’or 3’deoxyadenosine residue and a 24 nucleotide (nt) helix mimicking the acceptor arm of the tRNA (Figure 1) were obtained by semisynthesis (see the Supporting Information and Scheme 2) and assayed as substrates of FemXWv

Tyrosine kinase inhibitor NVP-BGJ398 functionally improves FGFR3-related dwarfism in mouse model

Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the fibroblast growth factor receptor 3-encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral growth plate and calvaria in organ cultures from embryos of the Fgfr3Y367C/+ mouse model of ACH. Moreover, we demonstrated that a low dose of NVP-BGJ398, injected subcutaneously, was able to penetrate into the growth plate of Fgfr3Y367C/+ mice and modify its organization. Improvements to the axial and appendicular skeletons were noticeable after 10 days of treatment and were more extensive after 15 days of treatment that started from postnatal day 1. Low-dose NVP-BGJ398 treatment reduced intervertebral disc defects of lumbar vertebrae, loss of synchondroses, and foramen-magnum shape anomalies. NVP-BGJ398 inhibited FGFR3 downstream signaling pathways, including MAPK, SOX9, STAT1, and PLCγ, in the growth plates of Fgfr3Y367C/+ mice and in cultured chondrocyte models of ACH. Together, our data demonstrate that NVP-BGJ398 corrects pathological hallmarks of ACH and support TKIs as a potential therapeutic approach for ACH.

A convenient synthesis of α- and β-d-glucosamine-1-phosphate and derivatives

Abstract Derivatives of α- and β- d -glucosamine-1-phosphate and of α- d -galactosamine-1-phosphate, as well as free β- d -glucosamine-1-phosphate, were prepared stereoselectively and in high yield by way of the opening of the 1,2-oxazoline derived from the corresponding 3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-α- d -hexopyranosyl bromide with dibenzyl phosphate.

Synthesis and biological evaluation of new UDP-GalNAc analogues for the study of polypeptide-α-GalNAc-transferases

A series of three O-methylated UDP-GalNAc analogues have been synthesised using a divergent strategy from a 3,6-di-O-pivaloyl GlcNAc derivative. The biological activity of these probes toward polypeptide-α-GalNAc-transferase T1 has been investigated. This study shows that this glycosyltransferase exhibits a very high substrate specificity.

Copper(I)/Copper(II)-Assisted Tandem Catalysis: The Case Study of Ullmann/Chan–Evans–Lam N1,N3-Diarylation of 3-Aminopyrazole

Unprecedented CuI/CuII‐assisted tandem catalysis allowing an Ullmann/Chan–Evans–Lam sequence was achieved. This three‐component, one‐pot reaction triggered by a change in the oxidation state of the metal leads to the selective N1,N3‐diarylation of 3‐aminopyrazole. This new method should be a valuable tool for small‐molecule drug discovery that requires suitable regio‐ and/or chemoselective strategies for the N‐arylation of nitrogen‐containing heterocycles.

Synthesis of purin-2-yl and purin-6-yl-aminoglucitols as C-nucleosidic ATP mimics and biological evaluation as FGFR3 inhibitors.

Two new series of C-nucleosidic ATP mimics have been synthesized using an efficient and versatile synthetic pathway. These compounds were designed as FGFR3 inhibitors using purine as a central scaffold. The two substituents, a polyhydroxylated ribose mimic and a lipophilic moiety, were linked either in position 2 or 6 of the purine ring in order to explore any possible binding mode. All the compounds were able to inhibit FGFR3 kinase activity at a concentration of 50 μM. Unexpectedly, the best inhibitor was found to be one of the synthetic intermediates 13 bearing an iodine atom in position 2. Docking studies have confirmed its location in the ATP binding site and revealed halogen bonding among key interactions.

Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle

Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co2+-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [3H]OSP release from [3H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [3H]OSP release from [3H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.

Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP).

Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [3H]OSP from [3H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.