James D. Bergstrom

Deficiency in Inducible Nitric Oxide Synthase Results in Reduced Atherosclerosis in Apolipoprotein E-Deficient Mice

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

Phytosphingosine 1-phosphate: a high affinity ligand for the S1P(4)/Edg-6 receptor.

It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P(4) receptor has generated interest due to its lymphoid tissue distribution. While the S1P(4) receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4D-hydroxysphinganine 1-phosphate, more commonly known as phytosphingosine 1-phosphate (PhS1P), binds to S1P(4) with higher affinity. Using radiolabeled S1P (S133P), the affinity of PhS1P for the S1P(4) receptor is 1.6nM, while that of S1P is nearly 50-fold lower (119+/-20nM). Radiolabeled PhS1P proved to be superior to S133P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS133P, for in vitro studies of the S1P(4) receptor pharmacology.

Hepatic responses to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase: a comparison of atorvastatin and simvastatin

We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC50s for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3 +/- 9.1 fold and 0.05% Atorva induced activity 26.9 +/- 4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM-10 microM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 microM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.

The isolation and structure elucidation of zaragozic acid C, a novel potent squalene synthase inhibitor.

Abstract The novel zaragozic acid C ( 1 ) has been isolated as a potent inhibitor of squalene synthase. It was found to be a competitive inhibitor of rat liver squalene synthase with an apparent K i of 45 ± 15 pM, and a broad spectrum antifungal agent against both yeast and filamentous fungi.

Identification of Leu276 of the S1P1 Receptor and Phe263 of the S1P3 Receptor in Interaction with Receptor Specific Agonists by Molecular Modeling, Site-Directed Mutagenesis, and Affinity Studies

Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P1 against S1P3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P1 and S1P3 receptors. Computational models of human S1P1 and S1P3 receptors bound with two nonselective agonists or two S1P1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P1 Leu276 and S1P3 Phe263 contribute to the S1P1/S1P3 selectivity of the two S1P1-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P1-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P1 and S1P3 receptors, which should aid development of more selective S1P1 receptor agonists with immunosuppressive properties and improved safety profiles.

Distribution of zaragozic acids (squalestatins) among filamentous ascomycetes

The search for squalene synthase inhibitors of microbial origin has resulted in the discovery of a new class of fungal metabolites, the zaragozic acids (squalestatins). During our survey of representatives of most major groups of fungi and filamentous bacteria, the zaragozic acids were not found in prokaryotes Zygomycotina, or Basidiomycotina. All the fungal producers encountered to date are Ascomycotina, their related anamorphic states or sterile organisms with ascomycete affinities. Members of at least II different taxa of fungi are capable of making zaragozic acids. Zaragozic acid A (squalestatin 1) appears to be the most prevalent among the different fungal taxa. In several cases we have observed production in multiple strains of the same species; for example, nearly all strains of Sporormiella intermedia, that we have examined, produce zaragozic acid B. The discovery of the zaragozic acids illustrates how knowledge of fungal biology and biochemistry can enhance the search for new chemical entities. Simultaneous screening of fungi from diverse phylogenetic and ecological origins was emphasized to discover new zaragozic acids rather than simply relying on organisms from a single kind of substratum from geographically disparate sources.

Isolation, structure determination and squalene synthase activity of L-731,120 and L-731,128, alkyl citrate analogs of zaragozic acids A and B

Abstract Two new alkyl citrates, L-731,120 and L-731,128, with alkyl chains corresponding to those of zaragozic acids A and B, were isolated as minor components of large scale fungal fermentations producing zaragozic acid A and B. They are submicromolar inhibitors of squalene synthase in vitro .

Diesters of zaragozic acid A: Synthesis and biological activity

Abstract Diesters of zaragozic acid A were synthesized and tested in rat liver squalene synthase and oral mouse cholesterol biosynthesis assays. Of these, 3-isopentyl-4-pivaloyloxymethyl and 3-isopentyl-4-acetoxymethyl esters gave ED50s of 9 mg/kg and 6 mg/kg, respectively, in the oral mouse assay.

Design and synthesis of squalene synthase inhibitors - acylic mimics of the zaragazic acids

Abstract A simple and versatile synthetic scheme employing d -malic acid as the principal building block permits the ready preparation of acyclic mimics of the squalene synthase inhibitor zaragozic acid A (ZA-A). Several compounds which were synthesized according to this approach have rat liver squalene synthase (RLSS) enzyme activities ranging from 200 to 470 nM, as compared to 0.3 nM for ZA-A. These analogs represent the first examples in this series which possess biological activity.

3,4-Diesters of zaragozic acid A

A series of 3,4-diesters of zaragozic acid A was synthesized and tested in rat liver squalene synthase and oral mouse cholesterol biosynthesis assays. Several diesters gave ED50s <4.0 mg/kg in the oral mouse assay.