Sunday, April 5, 2015

Dafachronic Acid, A Bile Acid-Like Steroid That Regulates Nematode Development and Lifespan

            Caenorhabditis elegans (C. elegans) is a free-living (non-parasitic), transparent nematode that resides in temperate soil environments. C. elegans was the first multicellular organism to have its genome sequenced and, given that approximately 35% of its genes have human homologs, the roundworm is used extensively in biotechnology applications as a model organism. C. elegans is a multicellular eukaryotic organism, yet it is simple enough to be studied in great detail. For example, the transparency of C. elegans facilitates the study of cellular differentiation and other developmental processes in the intact organism. C. elegans is also one of the simplest organisms with a central nervous system.
            The life cycle of the nematode C. elegans (shown schematically above) is also relatively uncomplicated. The organism progresses from egg, through four intermediary larval stages, to reproductive adult. However, in response to adverse environmental conditions such as crowding or lack of nutrition, larvae undergo developmental arrest at the second molt. In other words, C. elegans is encountered with a binary decision at the L2 larval stage of development: either progess to the normal third larval stage or enter into an alternate dormant stage, referred to as dauer. Dauer is a larval stage of arrested development in a protected dormant diapause. In this stage, C. elegans exhibits entirely different physiology, morphology and behavior. During dauer, the worm is sealed in a thickened body wall cuticle that serves as a protective capsule, enabling survival during harsh environmental challenges. Eventually, when favorable environmental conditions resume, a pathway is activated to complete the second larval molt into L4. Curiously, the dauer stage is non-aging. Dauers can persist for months before recovery and development into an adult that lives a life span of only a few weeks.
            It turns out that small molecules exert control over the binary decision made at the branching point of C. elegans’ developmental life cycle pathway. Dauer pheromones called ascarosides promote dauer formation. The ascarosides are lipidated derivatives of the dideoxy-sugar, ascarylose. The molecular mechanism of action of the ascarosides has not been fully elucidated but is thought to involve GPCRs. Dauer recovery is controlled by an endocrine signal called dafachronic acid (specifically, (25S)-D7-dafachronic acid). Dafachronic acid (DA) is produced in response to favorable environmental cues and activates the nuclear receptor DAF-12 to promote dauer recovery and molt to L4. Because C. elegans is auxotrophic for cholesterol, DA must be biosynthesized from dietary sterols. Consequently, the presence of environmental nutrients such as cholesterol signals a fed state that is conducive to reproduction. The biosynthesis of DA starting from cholesterol (shown above; adapted from PLoS Biol. 2012, 10, 1.) involves an enzyme with 3b-dehydrogenase activity called DHS-16 and a cytochrome P450 called DAF-9. The latter oxidizes the diastereotopic C27 position of the cholestane side chain to the carboxylic acid oxidation state. The stereoselectivity of this biotransformation governs the stereochemical configuration of the DA C25 position. Dauer pheromone (ascarosides) has been shown to inhibit DA biosynthesis.
            The binding mode of DA to the DAF-12 nuclear receptor has been deduced from X-ray crystal structures of the ligand-binding domain of the receptor complexed with DA. For example, in the top panels shown above (reproduced from PNAS 2009), the X-ray structure Strongyloides stercoralis DAF-12 is depicted. The binding mode of DA to this receptor is similar to the mode in which bile acids bind to the mammalian farnesoid X receptor. In this binding mode, the oxygen atoms at either end of the DA structure are molecular determinants for ligand recognition. The C3-ketone and C27-carboxylate accept hydrogen bonds donated by polar side chains contained within the binding domain of the receptor. As depicted above in the lower panel, the analogous binding mode has been characterized in the hookworm Ancylostoma ceylanicum. In general, orthologs of the DAF-12 receptor found in parasitic nematodes share ~50% sequence homology with C. elegans DAF-12 in the ligand-binding domain. This leads to differential relative affinities for DA across different species. In fact, the precise physiologic ligand for different parasite species may vary in terms of molecular structure and pharmacophore.
            E. J. Corey first pointed out the parallel nature of the role of DA in the regulation of C. elegans development to that of glycinoeclepin A (chemical structure shown above) on an alternate nematode. Glycinoeclepin A is the hatch-stimulating substance for the soybean cyst nematode Heterodera glycines, a predator of the soybean plant and various other beans. Glycinoeclepin A, a metabolite derived from the plant triterpene cycloartenol, is produced and released from the roots of the soybean plant. Its release stimulates the hatching of previously dormant eggs of H. glycines. The analogous hatching stimulus for the potato cyst nematode is the structurally complex nortriterpenoid, solanoeclepin A. Potential agrochemical applications for molecules capable of inducing nematodes to hatch prematurely and then die shortly thereafter have been discussed at this site previously. The three intriguing molecules depicted above are quite unique in that they all link nematode development and lifespan to discrete environmental signals. In the case of DA, the regulatory mechanism involves interaction with a nuclear receptor and regulation of gene expression. Additional oxidized sterols and triterpenoids with similar endocrine signaling properties in free-living and parasitic nematodes will surely be uncovered in the not-too-distant future. Discoveries of this nature may unlock new therapeutic targets in parasitic nematodes that will deliver the next generation of anthelmintic drugs against endoparasites.
            In a forthcoming post, we will provide an overview of the current state-of-the-art with regard to synthetic approaches that are well suited to provide access to large quantities of (25S)-D7-dafachronic acid, beginning from readily available sterol precursors.

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