Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1).

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.

Probes for narcotic receptor mediated phenomena. 43. Synthesis of the ortho-a and para-a, and improved synthesis and optical resolution of the ortho-b and para-b oxide-bridged phenylmorphans: compounds with moderate to low opioid-receptor affinity.

N-Phenethyl-substituted ortho-a and para-a oxide-bridged phenylmorphans have been obtained through an improved synthesis and their binding affinity examined at the various opioid receptors. Although the N-phenethyl substituent showed much greater affinity for µ- and κ-opioid receptors than their N-methyl relatives (e.g., K(i)=167 nM and 171 nM at µ- and κ-receptors vs >2800 and 7500 nM for the N-methyl ortho-a oxide-bridged phenylmorphan), the a-isomers were not examined further because of their relatively low affinity. The N-phenethyl substituted ortho-b and para-b oxide-bridged phenylmorphans were also synthesized and their enantiomers were obtained using supercritical fluid chromatography. Of the four enantiomers, only the (+)-ortho-b isomer had moderate affinity for µ- and κ-receptors (K(i)=49 and 42 nM, respectively, and it was found to also have moderate µ- and κ-opioid antagonist activity in the [(35)S]GTP-γ-S assay (K(e)=31 and 26 nM).

Inhibition of yeast lipase (CRL1) and cholesterol esterase (CRL3) by 6-chloro-2-pyrones: comparison with porcine cholesterol esterase.

Previously, it was demonstrated that pancreatic cholesterol esterase is selectively inhibited by 6-chloro-2-pyrones with cyclic aliphatic substituents in the 3-position. Inhibition is reversible and is competitive with substrate. Pancreatic cholesterol esterase is a potential target for treatment of hypercholesterolemia. In the present study, yeast cholesterol esterase from Candida cylindracea (also called C. rugosa CRL3) was compared to porcine pancreatic cholesterol esterase for inhibition by a series of 3-alkyl- or 5-alkyl-6-chloro-2-pyrones. In addition, CRL3 was compared with the related yeast lipase CRL1. Inhibition of CRL3 by substituted 6-chloro-2-pyrones was competitive with binding of the substrate p-nitrophenyl butyrate. Inhibition constants ranged from 0.2 microM to >90 microM. Small changes in the alkyl group had profound effects on binding. The pattern of inhibition of CRL3 is quite distinct from that observed with porcine cholesterol esterase. Molecular modeling studies suggest that the orientation of binding of these inhibitors at the active site of CRL3 can vary but that the pyrone ring consistently occupies a position close to the active site serine. CRL1 is highly homologous to CRL3. Nevertheless, patterns of inhibition of CRL1 by substituted 6-chloro-2-pyrones differ markedly from patterns observed with CRL3. The substituted 6-chloro-2-pyrones are slowly hydrolyzed in the presence of CRL1 and are pseudosubstrates of CRL3, but are simple reversible inhibitors of pancreatic cholesterol esterase

Probes for narcotic receptor mediated phenomena. 46. N-substituted-2,3,4,9,10,10a-hexahydro-1H-1,4a-(epiminoethano)phenanthren-6- and 8-ols - carbocyclic relatives of f-oxide-bridged phenylmorphans.

Oxide-bridged phenylmorphans were conceptualized as topologically distinct, structurally rigid ligands with 3-dimensional shapes that could not be appreciably modified on interaction with opioid receptors. An enantiomer of the N-phenethyl-substituted ortho-f isomer was found to have high affinity for the µ-receptor (K(i) = 7 nM) and was about four times more potent than naloxone as an antagonist. In order to examine the effect of introduction of a small amount of flexibility into these molecules, we have replaced the rigid 5-membered oxide ring with a more flexible 6-membered carbon ring. Synthesis of the new N-phenethyl-substituted tricyclic N-substituted-2,3,4,9,10,10a-hexahydro-1H-1,4a-(epiminoethano)phenanthren-6- and 8-ols resulted in a two carbon-bridged relative of the f-isomers, the dihydrofuran ring was replaced by a cyclohexene ring. The carbocyclic compounds had much higher affinity and greater selectivity for the µ-receptor than the f-oxide-bridged phenylmorphans. They were also much more potent µ-antagonists, with activities comparable to naltrexone in the [(35)S]GTP-γ-S assay.

Enantioselective synthesis of (+)-isolysergol via ring-closing metathesis.

The first enantioselective synthesis of (+)-isolysergol was completed in 12 steps from commercially available materials by a novel approach that features a late stage microwave-mediated, diastereomeric ring-closing metathesis catalyzed by a chiral molybdenum catalyst to simultaneously form the D ring and set the stereocenter at C(8).