Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF's tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein's flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC50 values lower than 10 microM (three hits with IC50 lower than 1 microM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC50=300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF's tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs

Small Molecule Based Approaches to Inhibit Macrophage Migration Inhibitory Factor (MIF) Activities and Elucidate its Role in Health and Disease

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease

Method and compositions for inhibition of macrophage migration inhibitory factor (mif)

The present invention relates to methods for inhibition of tautomerase activity of macrophage migration inhibitory factor (MIF). The invention further relates to substances for irreversibly covalently binding to MIF for inhibition of tautomerase activity

Effect of PA2226 on induction of TTSS in PAO1.

<p>PAO1 cells harbouring vector pHO9.1 (PA2226 and PA2225), pHO12.1 (PA2225) and pHO13.1 (PA2226) were grown in LB-medium for 4 h, supplemented or not with 5 mM EGTA, and 20 mM MgCl₂. Proteins in the supernatants were precipitated with TCA. Samples were run on 10% SDS-PAGE and stained with Coomassie. Secreted proteins of the TTSS are indicated on the left and marked by arrows. Expression of PA2226 and PA2225 was sufficient to prevent induction of the TTSS.</p

Growth curves and 3-oxo-C12-HSL autoinducer concentrations in culture supernatants.

<p><i>P. aeruginosa</i> strain PAO1 carrying the empty vector pIApX2, a <i>lasI</i> mutant of PAO1 (PT466), the QS-deficient deletion mutant (PT1617) and PAO1 harbouring plasmid pOH13.1, expressing PA2226 showed comparable growth curves in LB medium at 37°C (A). Culture supernatant samples were taken at indicated time points and 3-oxo-C12-HSL concentrations were determined using the <i>E. coli</i> reporter system based on a <i>lasR</i>-<i>lasI::luxCDABE</i> fusion harboured on plasmid pSB1075 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087814#pone.0087814-Winson1" target="_blank">[33]</a> (B). Results for 3-oxo-C12-HSL measurements are shown as average values and standard deviations from two independent experiments performed in triplicate.</p

Sequence alignment of PA2226 from PAO1 and five ORFs from completely sequenced <i>P. aeruginosa</i> strains.

<p>PA2226 has 46% amino acid identity with an ORF (RL113) from PA14, located on pathogenicity island PAG1. This ORF is not preceded by a <i>vqsM</i> homologue in PA14. Identical residues are boxed in black.</p

DNA region of PAO1 surrounding the 3,552

<p><i>PslA</i> is the first gene of the <i>psl</i> polysaccharide synthesis operon. Arrows indicate putative operon structures. The numbers above the gene symbols indicate the GC content (%GC). The average GC content of the <i>P. aeruginosa</i> genome is 66%.</p

QsrO a Novel Regulator of Quorum-Sensing and Virulence in <i>Pseudomonas aeruginosa</i>

<div><p>In <i>Pseudomonas aeruginosa</i>, the production of many secreted virulence factors is controlled by a quorum-sensing (QS) circuit, constituted of transcriptional activators (LasR, RhlR, PqsR) and their cognate signaling molecules (3-oxo-C12-HSL, C4-HSL, PQS). QS is a cooperative behavior that is beneficial to a population but can be exploited by “QS-cheaters”, individuals which do not respond to the QS-signal, but can use public goods produced by QS-cooperators. In order to identify QS-deficient clones we designed a genetic screening based on a <i>lasB</i>-<i>lacZ</i> fusion. We isolated one clone (PT1617) deficient in QS-dependent gene expression and virulence factor production despite wild type <i>lasR</i>, <i>rhlR</i> and <i>pqsR</i> alleles. Whole genome sequencing of PT1617 revealed a 3,552 bp deletion encompassing ORFs PA2228-PA2229-PA2230 and the <i>pslA</i> gene. However, complementation of PT1617 by plasmid-encoded copies of these ORFs, did not restore QS. Unexpectedly, gene expression levels of ORFs PA2228, PA2227 (<i>vqsM</i>) and PA2222, located adjacent to the deletion, were 10 to 100 fold higher in mutant PT1617 than in PAO1. When expressed from a constitutive promoter on a plasmid, PA2226, alone was found to be sufficient to confer a QS-negative phenotype on PAO1 as well as on PA14. Co-expression of PA2226 and PA2225 in PAO1 further prevented induction of the type III secretion system. In summary, we have identified a novel genetic locus including ORF2226 termed <i>qsrO</i> (QS-repressing ORF), capable of down-regulating all three known QS-systems in <i>P. aeruginosa</i>.</p></div