Laurence Dugué

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

Thymidine monophosphate kinase (TMPK) from Mycobacterium tuberculosis (TMPKmt) is an attractive target for the design of specific inhibitors. This fact is the result of its key role in the thymidine pathway and of unique structural features in the active site observed by X‐ray crystallography, especially in comparison to its human counterpart (TMPKh). Different 5‐modified thymidine derivatives, as well as purine and pyrimidine analogues or C‐nucleosides were tested on TMPKmt and TMPKh, and the results were rationalized by docking studies. 5‐Halogenated 2′‐deoxyuridines are the best inhibitors of TMPKmt found and present the highest selectivity indexes in favor of TMPKmt.

Substrate specificity of vaccinia virus thymidylate kinase

Anti‐poxvirus therapies are currently limited to cidofovir [(S)‐1‐(3‐hydroxy‐2‐phosphonylmethoxypropyl)cytosine], but drug‐resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP‐Mg2+ and dTMP were 0.15 mm and 20 µm, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5‐Halogenated dUMP such as 5‐iodo‐2′‐deoxyuridine 5′‐monophosphate (5I‐dUMP) and 5‐bromo‐2′‐deoxyuridine 5′‐monophosphate (5Br‐dUMP) were also efficient alternative substrates. Using thymidine‐5′‐(4‐N′‐methylanthraniloyl‐aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP‐induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.

Enzymatic synthesis of 1-(2-deoxy-β-D-ribofuranosyl) imidazole-4-carboxamide, a simplified DNA building block

Abstract Coupling of silylated 4-(5)-imidazolecarboxamide (5) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranose (6) in the presence of SnCl4 in CH3CN or condensation of the sodium salt of 5 with the halogenose 6, afforded a mixture of 1-β-glycosylated 4-(5)-imidazolecarboxamide (11) and the corresponding α anomer (12). Using a crude extract of nucleoside N-deoxyribosyltransferases from Lactobacillus leichmannii only one product was formed, the N-1-β-stereoisomer (2), in a good yield.

In Vivo Reshaping the Catalytic Site of Nucleoside 2′-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution

Nucleoside 2′-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2′-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2′,3′-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2′,3′-dideoxyuridine, cytosine, and 2′,3′-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2′,3′-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2′,3′-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G → A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the “adaptation” of the nucleoside 2′-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2′,3′-didehydro-2′,3′-dideoxyribosyltransferase activity.

Imidazole-4-Carboxamide and 1,2,4-Triazole-3-Carboxamide Deoxynucleotides as Simplified DNA Building Blocks with Ambiguous Pairing Capacity

Abstract 2′-Deoxynucleosides of imidazole-4 (or 1,2,4-triazole-3)-carboxamide, ethyl imidazole-4 (or 1,2,4-triazole-3)-carboxylate were synthesized by enzymatic glycosylation using N-deoxyribosyltransferase from a lactobacterium. The base pairing properties of Y and V when placed opposite the natural DNA bases as well as their self were evaluated by thermal denaturation experiments. DNA templates containing imidazole-4-carboxamide base were used in elongation reaction catalysed by Klenow fragment.

Nucleotide binding to human UMP‐CMP kinase using fluorescent derivatives − a screening based on affinity for the UMP‐CMP binding site

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor‐binding and acceptor‐binding sites of human UMP‐CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP‐CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in‐line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor‐binding site of the human UMP‐CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP‐CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP‐CMP kinase. Also, the 5‐halogen‐substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor‐binding site is in agreement with a large substrate‐binding pocket, as shown by the 2.1 Å crystal structure.

Design of Mycobacterium tuberculosis Thymidine Monophosphate Kinase Inhibitors

M. tuberculosis is a major pathogen in the world and infects almost one third of the population. The current chemotherapy, which stands on a few molecules and involves long treatments of the patients, is challenged by emerging multiresistant strains. TMPK belongs to the family of nucleoside monophosphate kinase (NMPK): it catalyses the phophorylation of thymidine monophosphate (dTMP) to thymidine diphosphate (dTDP) utilizing ATP as its preferred phosphoryl donor. TMPK, essential for cell proliferation, was studied intensively the last years for its role in activation of antiviral drugs such AZT. Biochemical and physicochemical characterization of M. tuberculosis TMPK (TMPKmt) revealed distinct features when compared to its counterpart from yeast, human and E. coli, which renders this protein a good target for antituberculosis drugs. Based on the 3D-structure of TMPKmt (Fig. 1,), different positions on the natural substrate (dTMP) were

Ambiguous Base Pairing of 1-(2-Deoxy-β-D-Ribofuranosyl)imidazole-4-carboxamide During PCR

Abstract The use of 5′-triphosphate of 1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide (dYTP) in DNA amplification reaction in place of dATP or dGTP yielded mutations frequencies of 3–4×10−2 per base per amplification. A reasonable proportion of transversions (11–15%) was observed in the absence of deletions and insertions.

OLIGODEOXYNUCLEOTIDES EMBODYING THE AMBIGUOUS BASE Z, 5-AMINO-IMIDAZOLE-4-CARBOXAMIDE

Abstract Short oligomers containing 5-amino-1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide (dZ) were synthesized in solution using the phosphotriester methodology. Usual acyl groups were used for the canonical bases. For the exocyclic amino function of Z residue, the hydrogenolyzable benzyloxycarbonyl group was introduced.

A Parallel Synthesis Scheme for Generating Libraries of DNA Polymerase Substrates and Inhibitors

We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5′‐triphosphate derivative of 1‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐imidazole‐4‐hydrazide (dY  NH 2TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dYNHRTP. Vent (exo−) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dYNHbenTP and, to a lesser extent, dYNHpheTP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.