Serge Van Calenbergh

Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

BackgroundTo date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.ResultsOur results indicate that cinnamaldehyde and several substituted derivatives interfere with AI-2 based QS without inhibiting bacterial growth. The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2. Study of the effect in various mutants suggested that the target protein is LuxR. Mobility shift assays revealed a decreased DNA-binding ability of LuxR. The compounds were further shown to (i) inhibit biofilm formation in several Vibrio spp., (ii) result in a reduced ability to survive starvation and antibiotic treatment, (iii) reduce pigment and protease production in Vibrio anguillarum and (iv) protect gnotobiotic Artemia shrimp against virulent Vibrio harveyi BB120.ConclusionCinnamaldehyde and cinnamaldehyde derivatives interfere with AI-2 based QS in various Vibrio spp. by decreasing the DNA-binding ability of LuxR. The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress. Since inhibitors of AI-2 based quorum sensing are rare, and considering the role of AI-2 in several processes these compounds may be useful leads towards antipathogenic drugs.

A plant-derived ligand favoring monomeric glucocorticoid receptor conformation with impaired transactivation potential attenuates collagen-induced arthritis

The glucocorticoid receptor (GR) is a transcription factor regulating its target genes either positively, through direct binding to the promoter of target genes, or negatively by the interference with the activity of transcription factors involved in proinflammatory gene expression. The well-known adverse effects of glucocorticoids are believed to be mainly caused by their GR-mediated gene-activating properties. Although dimerization of GR is thought to be essential for gene-activating properties, no compound has yet been described which selectively imposes GR monomer formation and interference with other transcription factors. In the present study, we report on a GR-binding, plant-derived compound with marked dissociative properties in rheumatoid arthritis fibroblast-like synoviocytes, which are important effector cells in inflammation and matrix degradation in rheumatoid arthritis. In addition, these findings could be extended in vivo in murine collagen-induced arthritis, in which joint inflammation was markedly inhibited without inducing hyperinsulinemia. Therefore, we conclude that GR monomers are sufficient for inhibition of inflammation in vivo.

Electrospun cellulose acetate phthalate fibers for semen induced anti-HIV vaginal drug delivery

Despite many advances in modern medicine, human immunodeficiency virus (HIV) still affects the health of millions of people world-wide and much effort is put in developing methods to either prevent infection or to eradicate the virus after infection has occurred. Here, we describe the potential use of electrospun cellulose acetate phthalate (CAP) fibers as a tool to prevent HIV transmission. During the electrospinning process, anti-viral drugs can easily be incorporated in CAP fibers. Interestingly, as a result of the pH-dependent solubility of CAP, the fibers are stable in vaginal fluid (the healthy vaginal flora has a pH of below 4.5), whereas the addition of small amounts of human semen (pH between 7.4 and 8.4) immediately dissolves the fibers which results in the release of the encapsulated drugs. The pH-dependent release properties have been carefully studied and we show that the released anti-viral drugs, together with the CAP which has been reported to have intrinsic antimicrobial activity, efficiently neutralize HIV in vitro.

Galactose-modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α‐galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1‐biasing glycolipid, α‐C‐GalCer, whose CD1d binding follows a conventional key‐lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose‐modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia

Burkholderia cepacia complex strains are opportunistic pathogens causing life-threatening infections in cystic fibrosis patients. B. cepacia complex strains are resistant to many antimicrobial agents and commonly produce biofilms in vitro and in vivo. This contributes to their virulence and makes Burkholderia infections difficult to treat. Recently, the quorum sensing (QS) system of Burkholderia spp. has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. However, detailed information about the anti-biofilm effect of these compounds is still lacking. In the present study, we evaluated the anti-biofilm effect of several known QS inhibitors. The effect on Burkholderia spp. biofilm formation was examined using crystal violet, resazurin and SYTO9 staining, confocal laser scanning microscopy as well as plating. When used at subinhibitory concentrations, several compounds interfered with biofilm formation by Burkholderia spp. Our results suggest that the QS inhibitors affect later stages of biofilm formation and detachment.

Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg2+ ion. The crystallographic structure of the complex of one of the compounds, 5-CH2OH-dUMP, with TMPK has been determined at 2.0 Å. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser99. Looking for a role for Ser99, we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu6, Ser99, Arg95, and Asp9) that probably serves to protonate the transferred PO3 group. The crystallographic structure of the commercially available bisubstrate analogueP 1-(adenosine-5′)-P 5-(thymidine-5′)-pentaphosphate bound to TMPK is also reported at 2.45 Å and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.

Structure-activity relationship of short-chain sphingoid bases as inhibitors of sphingosine kinase.

Short-chain sphinganine analogues 8, 9, 18, and 19, as well as 3-fluoro-sphingosine analogues 25 and 26 were synthesized. Their potential as sphingosine kinase inhibitors was investigated, in combination with previously synthesized sphingosine and fluorinated sphinganine analogues.

Structure-Activity Relationship of Cinnamaldehyde Analogs as Inhibitors of AI-2 Based Quorum Sensing and Their Effect on Virulence of Vibrio spp

Background Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs. Methodology/Principal Findings By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,β unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus. Conclusions/Significance Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells.

Activation of invariant NK T (iNKT) cells with the glycolipid α-galactosylceramide promotes CD8+ cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8+ T cells occur on the same APCs. We demonstrate that injection of α-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8+ T cell response and abolishes the efficacy of α-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8+ T cells.