Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

Glycolysis as a target for the design of new anti-trypanosome drugs.

Glycolysis is perceived as a promising target for new drugs against parasitic trypanosomatid protozoa because this pathway plays an essential role in their ATP supply. Trypanosomatid glycolysis is unique in that it is compartmentalized, and many of its enzymes display unique structural and kinetic features. Structure- and catalytic mechanism-based approaches are applied to design compounds that inhibit the glycolytic enzymes of the parasites without affecting the corresponding proteins of the human host. For some trypanosomatid enzymes, potent and selective inhibitors have already been developed that affect only the growth of cultured trypanosomatids, and not mammalian cells.

Chemical and enzymatic synthesis of fructose analogues as probes for import studies by the hexose transporter in parasites.

Various D-fructose analogues modified at C-1 or C-6 positions were synthesized from D-glucose by taking advantage of the Amadori rearrangement or using the aldol condensation between dihydroxyacetone phosphate and appropriate aldehyde catalyzed by fructose 1,6-diphosphate aldolase from rabbit muscle. The affinities of the analogues for the glucose transporter expressed in the mammalian form of Trypanosoma brucei were determined by inhibition of radiolabelled 2-deoxy-D-glucose (2-DOG) transport using zero-trans kinetic analysis. Interestingly, the analogues bearing an aromatic group (i.e. a fluorescence marker) at C-1 or C-6 positions present comparable apparent affinities to D-fructose for the transporter. This result could find applications for hexose transport studies and also provides criteria for the design of glucose import inhibitors.

Synthesis of phosphono analogues of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

The present paper describes the synthetic routes of six phosphono analogues of dihydroxyacetone phosphate and five phosphono analogues of glyceraldehyde 3-phosphate through alpha-, beta- and gamma-hydroxyphosphonate esters precursors containing a protected carbonyl group. In some situations, depending on the sequence used for the deprotection of the phosphonate and carbonyl groups, the aldol/ketol rearrangement allowed the synthesis of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate analogues from the same precursors. All these analogues are of interest both as active-site probes and as potential substrates for glycolytic enzymes such as fructose 1,6-diphosphate aldolases (EC 4.1.2.13).

Interaction of phosphonomethyl analog of dihydroxyacetone phosphate with rabbit muscle aldolase.

Aldolase presents the same binding affinity for dihydroxyacetone phosphate and its phosphonomethyl analog, but the partition coefficient between the intermediates from the Michaelis complex to the eneamine is different. The effects of the structural modification of the triose phosphate substrate on the interaction with rabbit muscle aldolase are discussed in connection with the mechanistic pathway and the three-dimensional structure of the enzyme.

Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds.

The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2, 4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1, 6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146.

Slow reversible inhibitions of rabbit muscle aldolase with substrate analogues: synthesis, enzymatic kinetics and UV difference spectroscopy studies.

Various dihydroxyacetone-phosphate (DHAP) analogues bearing an aromatic ring or beta-dicarbonyl structures were synthesized. Their capacity to form a stabilized iminium ion or conjugated enamine in the reaction catalyzed by rabbit muscle aldolase (EC 4.1.2.13) were investigated by enzymatic kinetics and UV difference spectroscopic techniques. Whereas the aromatic derivative led to competitive inhibition without detectable iminium ion formation, slow reversible inhibitions of aldolase by beta-dicarbonyl compounds was shown to have taken place. Conjugated enamine formation at the active site of the enzyme was detected by their specific absorbances close to 317 nm.

Effects of chirality and substituents at carbon 3 in dihydroxyacetone-phosphate analogues on their binding to rabbit muscle aldolase.

A series of dihydroxyacetone-phosphate (DHAP) analogues has been synthesized, differing in their stereochemistry and functionality at C-3. The kinetic effects of these compounds on the enzyme aldolase (EC 4.1.2.13) have been studied and differing modes of action observed. Competitive and time dependent reversible inhibition have been shown to take place both with and without borohydride detected formation of an immonium ion.

[Rational concepts and the study of active molecules against various trypanosomiases]. / Conception rationnelle et étude de molécules actives contre les différentes trypanosomiases.

Several sets of compounds, active against different trypanosomes, Trypanosoma brucei and Trypanosoma cruzi are presented, the lethal doses for some of them being less than the micro-molar concentration. These compounds are designed by taking advantage of two metabolic features of these parasites, glucose metabolism and oxidative stress.

Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes.

Oxygen concentration regulates the expression of nitrogen fixation genes in the symbiotic bacterium Rhizobium meliloti. We demonstrate that two proteins, FixL and FixJ, that belong to the two-component family of regulatory proteins are necessary and sufficient for oxygen-regulated in vitro transcription of the two key regulatory genes, nifA and fixK. We show directly that FixJ is a transcriptional activator, working in conjunction with the RNA polymerase sigma 70 holoenzyme. Addition of FixL122, a soluble form of the sensor FixL protein, to the transcription assay enhanced FixJ transcriptional activity in response to low oxygen concentration. This enhancement of FixJ activity was correlated with FixJ phosphorylation.

Inhibition of the glycolytic enzymes in the trypanosome: an approach in the development of new leads in the therapy of parasitic diseases.

Glycolysis in the trypanosome represents an important target for the development of new therapeutic agents due to the fact that this metabolism is essential for the parasite, glucose being its sole source of energy. In addition, different features of this metabolism and those associated with glycolytic enzymes offer opportunities for the development of efficient and selective compounds. Examples are given in this work of inhibitors directed to the enzymes aldolase and glyceraldehyde-phosphate-dehydrogenase and also of molecules acting specifically on the clusters of basic amino-acids present at the surfaces of the glycolytic enzymes in the parasite.

Antisense effect of oligodeoxynucleotides complementary to the mini-exon sequence of the protozoan parasite Leishmania amazonensis.

We have targeted the mini-exon of Leishmania amazonensis, the sequence present at the 5' end of every mRNA of this protozoan parasite, with a complementary 12-mer, either unmodified (12 Le II) or linked to an acridine derivative (12 Le II Acr). Physical measurements performed either in solution or on nitrocellulose filters showed that the two oligomers exhibited the same affinity for both DNA and RNA target sequences. Furthermore, the two oligomers 12 Le II and 12 Le II Acr inhibited in vitro translation of L amazonensis mRNAs, in a wheat germ extract, to the same extent. Those results indicated that the intercalating agent did not stabilize the duplex formed by the antisense oligomer and its target sequence.

RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity.

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.

Acridine-linked oligonucleotide probes for the short dAT-rich motifs in the 3'-untranslated region of cytokine genes.

We have investigated the use of oligonucleotide probes for identifying cDNA clones containing the short dAT-rich motifs found in the 3'-untranslated region of cytokine genes. To obtain sufficiently stable duplexes between the octameric probes used to identify genes containing the sequence dTATTTATT and its complement, it was necessary to couple an intercalating agent, an acridine derivative (acr), to the 5'-positions of the probes. The resulting octamers 5'-acr-dAATAAATA and, particularly, 5'-acr-dTATTTATT were successfully used to distinguish the complementary sequences in cDNA from internal, single point mismatched sequences. Southern blot analyses of plasmids containing IL-1 beta and IL-8 gave positive results with the 3' degenerate probe, 5'-acr-dTATTTATTN, clearly showing that the very short probe approach can be used in this type of analysis. Subsequently, in slot blot analyses we found that, even without the degenerate nucleotide, N, plasmids bearing cytokine sequences with at least 7 contiguous matched nucleotides could be unambiguously identified with 5'-acr-dTATTTATT. Unfortunately, because of the ubiquity of these dAT-rich sequences in bacterial DNA, it was not possible to use these probes for direct colony screening. In contrast to the results obtained with DNA, at the RNA level, with IL-1 beta mRNA bound to nitrocellulose, the hybrid formed with 5'-acr-dAATAAATA was very unstable, even in 1M LiCl solution at 2 degrees C; however, in the same salt solution the slightly longer acridine-coupled probes 5'-acr-dAATAAATAGGG and 5'-acr-dAAAGAACAA remained hybridized to their complementary sequences up to about 18 degrees C.

Inhibition of reverse transcription by unmodified and modified antisense oligodeoxynucleotides.

We used oligodeoxynucleotides to prevent reverse transcription of beta-globin mRNA by reverse transcriptase of avian myeloblastosis virus. Unmodified oligomers hybridized to the template arrested synthesis of cDNA in a dose dependent manner. The longer the oligomer the more efficient the inhibition, 50% inhibition being achieved at 0.3 and 30 microM of a 17- or a 12-mer, respectively. The use of complementary oligonucleotides with a 3' end blocked either by a dideoxy residue or by a dodecanol group also induced inhibition of cDNA synthesis.

Characterization and sequencing of normal and modified oligonucleotides by 252Cf plasma desorption mass spectrometry.

Plasma desorption (PD) mass spectra of normal deoxyribo-oligonucleotides and of neutral methylphosphonate deoxyribo-oligonucleosides are examined and discussed. Molecular ions of oligonucleotides up to nonamer have been observed for neutral species. It is also shown that PD mass spectra can be used to monitor chemical modifications of oligonucleotides, such as the covalent binding of an organic fluorescent probe, along the synthesis process.