The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.

Three enzymes of the shikimic acid pathway, isochorismate synthase (IS), anthranilate synthase (AS), and p-aminobenzoate synthase (PABS), exhibit significant sequence homology and may be related mechanistically.Compounds 1, 2, and 3 were designed to mimic, in their all-axial conformations, the putative transition state for these enzymes. The inhibitors were prepared in racemic form starting from Diels-Alder addition of a propiolate ester to a protected 1-oxy-or 1-amino-1,3-butadiene in 14%, 4%, and 9% overall yields, respectively. All three compounds are competitive inhibitors of the three enzymes, binding IS and AS strongly and PABS weakly. For both IS and AS, the affinity of the 6-amino-4-hydroxy isomer 2 is ca. 10-fold that of the 4-amino-6-hydroxy isomer 3, a difference that is largely due to their conformational equilibria; 2 is 25 ± 2% axial and 3 is 6 ± 3% axial, as determined by the temperature dependence of their NMR spectra. The similarity between IS and AS was extended by the finding that IS, like AS, catalyzes formation of 2-amino-2-deoxyisochorismate (ADIC) in the presence of ammonia. These observations are consistent with direct 1,5-substitution mechanisms for both IS and AS; the weak inhibition of PABS by these inhibitors suggests that it operates by a significantly different mechanism.

The macrocyclic arrays of heterocycles and amides present in Lissoclinum peptides can serve as templates for selective metal ion binding and as lead structures for the design of conformationally preorganized peptides and peptidomimetics. The currently available secondary structure information for this class of marine alkaloids, however, is limited to 18- and 24-membered ring isomers. This work provides the first information on the solid state and solution conformation of the 21-membered class of Lissoclinum cyclopeptide alkaloids. The folding of lissoclinamide 7, both in the solid and in the solution state, is dominated by a combination of a type II β-turn formed at the prolyloxazoline moiety and a β-loop segment stabilized by intramolecular five-membered hydrogen bonds at the thiazoline−d-phenylalanine−thiazoline moiety. The resulting rigid backbone geometry controls the preferred stereochemistry at the stereogenic α-carbons, and the natural compound represents the thermodynamically most favorable stereoisomer. In addition to these structural studies, we have compared the relative cytotoxicity of lissoclinamide 7 with analogues with selective oxazoline and thiazoline heterocycle replacements. On the basis of in vitro cell toxicity assays, we can conclude that the substitution of thiazolines in the natural product with oxazoline rings leads to a general decrease in cellular toxicity. However, changes in the stereochemistry of the parent macrocycle also influence cytotoxicity.