Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid

Genetic studies on Cherax destructor Clark in relation to aquaculture

Investigated three strains of the common freshwater crayfish (yabby), to determine the potential for genetic improvement of this species for aquaculture. Crossbreeding generated viable progeny and differences were found between strains in reproductive performance, sex ratios, morphology and overall genetic divergence measured by DNA sequencing

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation

Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target.

BackgroundReliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals).Methodology/principal findingsA phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target.Conclusions/significanceThe orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets

Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target

<div><p>Background</p><p>Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals).</p><p>Methodology/Principal findings</p><p>A phenotypic screen involving <i>Schistosoma mansoni</i> and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated <i>Caenorhabditis elegans</i> lines that express a cDNA for <i>smpde4a</i> on a <i>pde4(ce268)</i> mutant (hypermotile) background: the <i>smpde4a</i> transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the <i>C</i>. <i>elegans</i> lines that express the <i>smpde4a</i> transgene, thereby confirming SmPDE4A as the relevant target.</p><p>Conclusions/Significance</p><p>The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic <i>C</i>. <i>elegans</i> is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.</p></div

Phenotypic screening of a benzoxaborole collection with <i>S</i>. <i>mansoni</i> somules suggests a PDE4 as a molecular target of interest.

<p>The screen involving 1,085 benzoxaboroles was performed at 5 μM for 6 days with observations taken every day using a constrained nomenclature, as noted in the text. Three main phenotype response groups could be adjudicated by microscopical observation: (i) 104 compounds eliciting an early and sustained hypermotile phenotype, of which, 30% was also associated with a parasite degeneration; (ii) 94 compounds that yielded a range of phenotypic responses (<i>e</i>.<i>g</i>., rounding, darkening), including hypermotility, which were either transient (noted at 24 h only) or appeared later (on or after day 3 of the incubation), and (iii) 887 compounds that produced no phenotype. Of the 1,085 benzoxaboroles screened, 174 also had IC₅₀ data for inhibition of huPDE4B2 that were distributed as 77, 82 and 15 compounds across the sustained hypermotile, no phenotype and transient hypermotile groups, respectively. Sixty-five of 77 compounds in the sustained hypermotile group inhibited huPDE4B2 with IC₅₀ values of < 1 μM. In contrast, for the no phenotype group, only 16 of 82 compounds had IC₅₀ values of < 1 μM. The association between the sustained hypermotile phenotype and sub-micromolar inhibition of huPDE4 was highly significant with a Fishers exact <i>p</i>-value of <0.0001. The 5-(3-cyanopyridyl-6-oxy) benzoxaborole scaffold, which is known to preferentially inhibit huPDE4 over other PDEs [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005680#pntd.0005680.ref073" target="_blank">73</a>], was well represented in Group 1.</p

A <i>smpde4A</i> transgene restores wild type motility rates to <i>pde4</i>-deficient <i>C</i>. <i>elegans</i>.

<p>Relative to wild type (WT) <i>C</i>. <i>elegans</i>, a loss of function allele of <i>pde4</i>, namely <i>ce268</i> [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005680#pntd.0005680.ref103" target="_blank">103</a>], causes hypermotility. This hypermotility is reverted back to WT rates upon transgenic expression of a full-length cDNA for <i>smpde4a</i>. Results for two independently generated lines, <i>smpde4a(a)</i> and <i>smpde4a(b)</i> are shown. The same transgenes do not alter the motility of otherwise WT animals. Error bars indicate the standard deviations around the mean motility in a representative experiment with at least 10 worms for each strain. The asterisks indicate significance by Student’s <i>t</i>-test (*p<0.005; **p<0.0005) relative to the hypermotility recorded for the <i>pde4(ce268)</i> mutant.</p

2D interaction diagram of rolipram and roflumilast with huPDE4B and SmPDE4A.

<p>Molecular models of each enzyme in complex with rolipram and roflumilast were built using ICM-pro and huPDE4B1 as a template (PDB ID: 4X0F) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005680#pntd.0005680.ref079" target="_blank">79</a>]. The amino acid residues in the huPDE4B1 and SmPDE4 binding sites that interact directly with the ligands are shown as ovals. Those that distinguish the schistosome ortholog are shown in magenta and the consequent changes in binding free energies are indicated underneath. The residue numbers are consistent with the alignment presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005680#pntd.0005680.g002" target="_blank">Fig 2</a>.</p

Purification and activity analysis of the recombinant catalytic domain of SmPDE4A.

<p>(<b>A</b>) The three-step purification scheme described in the text resulted in purified His₆-tagged SmPDE4A with the expected molecular mass of 44.2 kDa. Each lane (1–4) contains an increasing amount of protein (3, 6, 21 and 59 μg) demonstrating the absence of major contaminants. Molecular mass markers (in kDa) are indicated on the left. (<b>B</b>) Determination of K_m and V_max. Enzyme reaction rates were measured over increasing concentrations of the cAMP substrate up to 10 μM. All reactions ran for six minutes and contained 23.5 units/ml SmPDE4A. K_m and V_max values were determined by nonlinear regression analysis of the data (Prism GraphPad version. 6.03) using a Michaelis-Menten enzyme kinetics model. All data points were determined in triplicate.</p