Mélanie Etheve-Quelquejeu

Inactivation of Mycobacterium tuberculosis l,d-Transpeptidase LdtMt1 by Carbapenems and Cephalosporins

ABSTRACT The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase LdtMt1 by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of LdtMt1, which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited LdtMt1, with a binding rate constant of 0.08 μM−1 min−1. The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min−1) in comparison to the acylation rate constant (3.1 min−1). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10−3 min−1. Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient LdtMt1 inactivator. Cephalosporins also formed covalent adducts with LdtMt1, although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

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.

Synthesis of Stable Aminoacyl-tRNA Analogues Containing Triazole as a Bioisoster of Esters

Aminoacyl-tRNAs have important roles in a variety of biological processes, including protein synthesis by ribosomes, targeting of proteins for degradation by the proteasome, and bacterial cell wall synthesis. Here we describe the synthesis of stable aminoacyl-tRNA analogues containing 1,4- and 1,5-substituted 1,2,3-triazole rings. The procedure involves i) Cu- and Ru-catalysed cycloadditions of 3-azidoadenosine and alkynes, which produced the 1,4 and 1,5 regioisomers of the triazoles, respectively, ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and iii) the enzymatic ligation of the substituted dinucleotides with a 22 nt RNA microhelix that mimics the acceptor arm of tRNA. Nucleoside and nucleotide compounds were characterized by MS spectrometry and (1)H, (31)P and (13)C NMR spectroscopy and were assayed for inhibition of FemX(Wv), an alanyltransferase essential for the formation of the peptidoglycan network of gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 microM) indicate that the five-membered triazole rings acted as bioisosters of esters and can be used for the design of stable aminoacyl-tRNA analogues.

Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemXWv. Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemXWv with the acceptor arm of tRNAGly and with l-Ser or larger residues quantitatively accounts for the preferential transfer of l-Ala observed with complete aminoacyl-tRNAs. The main FemXWv identity determinant was identified as the penultimate base pair (G2-C71) of the acceptor arm instead of G3•U70 for the alanyl-tRNA synthetase. FemXWv tolerated a configuration inversion of the Cα of l-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemXWv is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNAGly from the active site.

Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity.

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldtfm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldtfm does not confer resistance to β-lactams of the carbapenem class (imipenem MICu200a=u200a0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MICu200a=u200a128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldtfm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldtfm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldtfm acylation was followed by rupture of the C5–C6 bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldtfm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

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

Kinetic Analysis of Enterococcus faecium l,d-Transpeptidase Inactivation by Carbapenems

ABSTRACT Bypass of classical penicillin-binding proteins by the l,d-transpeptidase of Enterococcus faecium (Ldtfm) leads to high-level ampicillin resistance in E. faecium mutants, whereas carbapenems remain the lone highly active β-lactams. Kinetics of Ldtfm inactivation was determined for four commercial carbapenems and a derivative obtained by introducing a minimal ethyl group at position 2. We show that the bulky side chains of commercial carbapenems have both positive and negative effects in preventing hydrolysis of the acyl enzyme and impairing drug binding.

Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemX(Wv) aminoacyl transferase.

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2-azido RNA helix starts from 2-azido-2-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.

gem‐Difluorocarbadisaccharides: Restoring the exo‐Anomeric Effect

Molecular mimicry is an essential part of the development of drugs and molecular probes. In the chemical glycobiology field, although many glycomimetics have been developed in the past years, it has been considered that many failures in their use are related to the lack of the anomeric effects in these analogues. Additionally, the origin of the anomeric effects is still the subject of virulent scientific debates. Herein, by combining chemical synthesis, NMR methods, and theoretical calculations, we show that it is possible to restore the anomeric effect for an acetal when replacing one of the oxygen atoms by a CF2 group. This result provides key findings in chemical sciences. On the one hand, it strongly suggests the key relevance of the stereoelectronic component of the anomeric effect. On the other hand, the CF2 analogue adopts the natural glycoside conformation, which might provide new avenues for sugar-based drug design.

Synthesis and crystal structure of a “double picket fence” 5,10,15,20-tetrakis-(2',6'-diacrylamido-4'-tert-butylphenyl)Zn porphyrin

Metalation of 5,10,15,20-tetrakis-(2,6-diacrylamido-4-tert-butylphenyl)porphyrin with Zn(OAc)2·2H2O afforded the expected Zn derivative whose X-ray crystal structure reveals a Zn atom hexacoordinated by the four pyrrolic nitrogen atoms and two molecules of H2O.