Mechanism of action of VP1-001 in cryAB(R120G)-associated and age-related cataracts

PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts

Succinylated Octopamine Ascarosides and a New Pathway of Biogenic Amine Metabolism in Caenorhabditis elegans

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLCMS/ MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine- derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in Caenorhabditis elegans

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC–MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles

Investigation Of Small Molecule Signaling In The Model Organism Caenorhabditis Elegans

Caenorhabditis elegans is an important model for the study of aging and disease. The worm has traditionally been used to explore gene function. Notably, one of the first genes shown to extend lifespan was discovered in C. elegans and was found to be conserved in humans. In the past ten years, though, it has been shown that this nematode utilizes a diverse class of small molecules called ascarosides that affect a variety of biological phenomena including development, aging, and mating. Yet many aspects of ascaroside biosynthesis and function remain incomplete. A second class of steroidal small molecules, the dafachronic acids, has also been recently discovered to be endogenous regulators of C. elegans physiology. It is clear that C. elegans is becoming established as a model for chemists. The work presented here highlights recent progress in our understanding of small molecule signaling in C. elegans. My Ph.D. research has contributed novel discoveries in the areas of sex-specific ascaroside biosynthesis, ascaroside modulation of lifespan, and ascaroside based immune system activation. My research has also led to discovery of a small molecule detoxification mechanism in C. elegans and utilized an established 2D NMR-based metabolomics method to investigate a host-pathogen interaction. Finally, this thesis presents a novel statistically powerful 2D NMR metabolomics tool to take small molecule research in C. elegans and higher organisms into the 21st century

Chemical Detoxification of Small Molecules by <i>Caenorhabditis elegans</i>

<i>Caenorhabditis elegans</i> lives in compost and decaying fruit, eats bacteria and is exposed to pathogenic microbes. We show that <i>C. elegans</i> is able to modify diverse microbial small-molecule toxins via both <i>O-</i> and <i>N-</i>glucosylation as well as unusual 3′-<i>O</i>-phosphorylation of the resulting glucosides. The resulting glucosylated derivatives have significantly reduced toxicity to <i>C. elegans</i>, suggesting that these chemical modifications represent a general mechanism for worms to detoxify their environments

Mechanism of Action of VP1-001 in cryAB(R120G)-Associated and Age-Related Cataracts.

PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in <i>Caenorhabditis elegans</i>

In the model organism <i>Caenorhabditis elegans</i>, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC–MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles

ICA reduces virulence of <i>C. rodentium in vitro</i> and <i>in vivo</i>.

<p>(<b>a</b>) Western analysis of Ler expression in <i>C. rodentium</i> treated with various concentrations of indole, ICA or IAA. The non-specific band recognized by the Ler pAb served as a loading control. (<b>b</b>) Quantitation of pedestal formation in 3T3 cells infected with <i>C. rodentium</i> in the presence of indole or ICA at various concentrations. Mean +/− SEM are shown; * corresponds to p<0.01 with respect to untreated control. (<b>c</b>) CFU from colon, liver, and spleen of MyD88^−/− mice infected for 7 days with <i>C. rodentium</i> and administered carrier (DMSO/5%Citric acid/PEG400, (30∶35:35%)) or ICA (100 mg/kg/day) by oral gavage. Mean +/− SEM are shown; * corresponds to p<0.01 with respect to untreated control. 8 mice were used for the control and 9 for the treatment with 100 mg/kg ICA. (<b>d</b>) Images of colons from animals left uninfected and treated with ICA (100 mg/kg/day) for 7 days, or infected with <i>C. rodentium</i> together with carrier or ICA. (<b>e</b>) H&E staining of colon sections from mice left uninfected, or infected with <i>C. rodentium</i> for 7d and administered either ICA (100 mg/kg/day) or carrier. (<b>f</b>) Survival curves of animals treated with carrier or 100 to 300 mg/kg/day ICA. Each curve is from a representative experiment (n = 3 animals per condition), and each experiment was repeated 3 times. Statistical significance was determined by the Kaplan Meyer test.</p

Indole derivatives inhibit Shiga toxin production, biofilm and motility in EHEC and EAEC.

<p>(<b>a</b>) Effects of ICA or the synthetic analog 4-nitroindole-3-carboxaldehyde (N-ICA) on Shiga toxin production by EHEC 8624 or EAEC 3493. Cytotoxic effect on vero cells was used as a measure of Shiga toxin production. (<b>b</b>) Effects of ICA on biofilm in EAEC 3493, EAEC 2071, and EHEC 3023. (<b>c</b>) Effects of Indole, ICA, and IAA on motility in soft agar of EAEC 3493, EAEC 2071, and EHEC O157:H7 EDL933. For b-c, mean +/− SEM are shown; * corresponds to p<0.01 with respect to untreated control.</p

Effects of synthetic indole or extract on killing and conditioning of <i>C. elegans</i>.

<p>(<b>a</b>) Comparison of the effect of synthetic indole or of EPEC agar extracts in which the indole concentration has been estimated based on HPLC measurements of the indole concentration in identically prepared samples. Note that the LD₉₀ for synthetic indole is 3.5 mM, but that the extract kills even at indole concentrations of 1.5 mM, suggesting that additional factors in the extract contribute to killing. Mean +/− SEM are shown; * corresponds to p<0.001 with respect to control at each dilution. (<b>b</b>) Tryptophan and tryptophanase are required for production of additional killing factors. N2 worms were exposed either to EPEC extracts from LB plates lacking tryptophan or to EPECΔ<i>tnaA</i> extracts. Significant killing was evident in either condition only when synthetic indole was added at concentrations greater than 3 mM. Mean +/− SEM are shown; * corresponds to p<0.01 with respect to control at each dilution. (<b>c</b>) Comparison of the effects of conditioning <i>C. elegans</i> with either 3.5 mM indole or with extract derived from EPEC/LBT plates, and challenging with EPEC. Note that extract conditions 2-fold better than indole. Mean +/−95% confidence intervals are shown. Lack of overlapping error bars indicates significance at the 5% level.</p