51 research outputs found

    Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function

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    Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms

    Ubistatins Inhibit Proteasome-Dependent Degradation by Binding the Ubiquitin Chain

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    To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys^(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system

    New Methodology toward α,β-Unsaturated Carboxylic Acids from Saturated Acids

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    A carefully designed three-step unsaturation of carboxylic acids is described. Briefly, carboxylic acids were converted to the trifluoromethyl ketone. Subsequent treatment with selenium dioxide followed by hydrolysis afforded α,β-unsaturated carboxylic acids. The mechanism of the reported transformation was investigated, which led us to propose a novel explanation featuring selenium dioxide assisted enolizaion of a trifluoromethyl ketone followed by β-deprotonation

    Bile Acid Recognition by Mouse Ileal Bile Acid Binding Protein

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    Ileal bile acid binding protein (I-BABP, gene name FABP6) is a component of the bile acid recycling system, expressed in the ileal enterocyte. The physiological role of I-BABP has been hypothesized to be either an intracellular buffering agent to protect against excess intracellular bile acids or separately as a modulator of bile acid controlled transcription. We investigated mouse I-BABP (mI-BABP) to understand the function of this protein family. Here, we studied energetics and site selectivity of binding with physiological bile acids using a combination of isothermal calorimetric analysis and NMR spectroscopy. We found that the most abundant bile acid in the mouse (β-muricholic acid) binds with weak affinity individually and in combination with other bile acids. Further analysis showed that mI-BABP like human I-BABP (hI-BABP) specifically recognizes the conjugated form of cholic acid:chenodeoxycholic acid (CA:CDCA) in a site-selective manner, displaying the highest affinity of any bile acid combination tested. These results indicate that I-BABP specifically recognizes the ligand combination of CDCA and CA, even in a species such as the mouse where CDCA only represents a trace component of the physiological pool. Specific and conserved recognition of the CDCA and CA ligand combination suggests that I-BABP may play a critical role in the regulation of bile acid signaling in addition to its proposed role as a buffering agent

    Approach for Expanding Triterpenoid Complexity via Divergent Norrish-Yang Photocyclization

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    Triterpenoids comprise a very diverse family of polycyclic molecules that is well-known to possess a myriad of medicinal properties. Therefore, triterpenoids constitute an attractive target for medicinal chemistry and diversity-oriented synthesis. Photochemical transformations provide a promising tool for the rapid, green, and inexpensive generation of skeletal diversity in the construction of natural product-like libraries. With this in mind, we have developed a diversity-oriented strategy, whereby the parent triterpenoids bryonolic acid and lanosterol are converted to the pseudosymmetrical polyketones by sequential allylic oxidation and oxidative cleavage of the bridging double bond at the B/C ring fusion. The resultant polyketones were hypothesized to undergo divergent Norrish-Yang cyclization to produce unique 6/4/8-fused triterpenoid analogues. The subtle differences between parent triterpenoids led to dramatically different spatial arrangements of reactive functionalities. This finding was rationalized through conformational analysis to explain unanticipated photoinduced pinacolization, as well as the regio- and stereochemical outcome of the desired Norrish-Yang cyclization

    Synthesis of [3,4- 13

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