Andrew A. Kandutsch

Oxysterol binding protein

A binding protein is described for certain oxygenated derivatives of cholesterol which suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis in cultured mammalian cells. This protein is found in the cytosolic fraction of many cell types and is distinct from cytosolic proteins which bind cholesterol. The relative binding affinity of a wide variety of oxysterols correlates with their ability to suppress reductase and it is proposed that the binding protein functions as a receptor for endogenous regulatory oxysterols. The binding protein from cultured mouse fibroblasts (L cells) has been partially purified and characterized. Changes in its molecular form occur when a ligand is bound and further changes in form and binding kinetics occur at acid pH and in the presence of urea. Based on these changes a subunit model for the binding protein is presented.

Inhibitors of sterol synthesis. Hypocholesterolemic action of dietary 5α-cholest-8(14)-en-3β-ol-15-one in rats and mice☆

Abstract 5α-Cholest-8(14)-en-3β-ol-15-one, at a level of 0.1% in a low cholesterol diet has been shown to have a profound hypocholesterolemic effect in rats. In one experiment the mean serum cholesterol level (mg per 100 ml ± S.E.M.) decreased from 71.2 ± 0.9 to 36.9 ± 3.3 after 7 days on the diet. In a second experiment the mean serum cholesterol value decreased from 86.4 ± 1.2 to 33.4 ± 3.9 after 8 days on the ketone-containing diet. The effects of the 15-ketosterol on serum cholesterol levels were significantly (p

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth.

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.

Further studies on the inhibition of sterol biosynthesis in animal cells by 15-oxygenated sterols.

The chemical syntheses of a number of C27 15-oxygenated sterols and their derivatives have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in animal cells in culture. Described herein are chemical syntheses of 3 alpha-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en-15-one-3 alpha-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-3 alpha-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en3,7,15-trione, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha, 14 alpha-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 alpha-cholestan-3 beta, 15 alpha-diol, 5 alpha, 14 alpha-cholestan-15 alpha-ol-3-one, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol, 5 alpha, 14 alpha-cholestan-3,15-dione, and 5 alpha, 14 beta-cholestan-3,5-dione. The effects of 8 of the above compounds and of 5 alpha-cholesta-6,8(14)-dien-3 beta-ol-15-one, 3 beta-he misuccinoyloxy-5 alpha-cholest-8(14)-en-15 one, 3 beta-hexadecanoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3,15-dione, 5 alpha-cholesta-6,8(14)-dien-3,15-dione, 5 alpha-cholest-8-en-3 beta, 15 alpha-diol, 5 alpha-cholest-7-en-3 beta, 15 alpha-diol, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha-cholest-8-en-15 alpha-ol-3-one, and 5 alpha-cholest-7-en-15 alpha-ol-3-one on the synthesis of digitonin-precipitable sterols and on levels of HMG-CoA reductase activity have been investigated and compared with previously published data on 7 other C27 15-oxygenated sterols.

Inhibition of sterol synthesis in animal cells by 15-oxygenated sterols with the unnatural cis-c-d ring junction—5α, 14β-Cholest-7-en-15α-ol-3-one and 5α, 14β-Cholest-7-en-15β-ol-3-one

Abstract 5α,14β-Cholest-7-en-15α-ol-3-one was prepared in 72 per cent yield by selective oxidation of the 3β-hydroxyl function of 5α,14β-cholest-7-en-3β,15α-diol by cholesterol oxidase. 5α,14β-Cholest-7-en-15β-ol-3-one was prepared in a similar manner from 5α,14β-cholest-7-en-3β,15β-diol in 66 per cent yield. The new compounds were found to inhibit sterol synthesis in mouse fibroblasts in culture. The 15α-hydroxy-3-ketosterol and the 15β-hydroxy-3-ketosterol caused a 50 per cent inhibition of the incorporation of [1− 14 C]acetate into digitonin-precipitable sterols at concentrations of 2.0 × 10 −6 M and 2.5 × 10 −7 M, respectively, and suppressed the level of activity of 3-hydroxy-3-methylglutaryl Coenzyme A reductase activity by 50 per cent at concentrations of 3.3 × 10 −7 M and 2.5 × 10 −7 M respectively.

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols☆

Abstract 14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10 −6 M and 8 × 10 −6 M, respectively. Compound II, the Δ 6 -analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10 −7 M and 2 × 10 −6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.

Inhibition of sterol synthesis in L cells by 14α-ethyl-5α-cholest-7-en-15α-ol-3-one at nanomolar concentrations☆

Abstract 14α-Ethyl-5α-cholest-7-en-15α-ol-3-one was prepared in 85% yield by selective oxidation of the 3β-hydroxyl function of 14α-ethyl-5α-cholest-7-en-3β,15α-diol by cholesterol oxidase. 14α-Ethyl-5α-cholest-7-en-15α-ol-3-one caused a 50% inhibition of the incorporation of [1-14C]-acetate into digitonin-precipitable sterols at a concentration of 6 × 10−9M in L cells and a 50% reduction in level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in the same cells at a concentration of 4 × 10−8 M.

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.

Increased synthesis and concentration of dolichyl phosphate in mouse spleens during phenylhydrazine-induced erythropoiesis.

Abstract Erythropoietic spleens from mice treated with phenylhydrazine synthesized dolichol + dolichyl acyl esters at a higher rate than did normal spleens, and this increased synthesis occurred 1–2 days after the peak of cholesterol synthesis. We have further characterized this dolichol synthesis and have found that at 4 days following phenylhydrazine treatment, dolichyl phosphate accounted for 30% of total synthesis, and at this time 60% of tissue dolichol was phosphorylated. In contrast, treatment with erythropoietin caused simultaneous increases in dolichol and cholesterol synthesis, with very low levels of dolichyl phosphate synthesis. The present results show that the synthesis and the mass of dolichyl phosphate increased in the spleens of phenyl-hydrazine- but not erythropoietin- treated mice.

Inhibition of sterol biosynthesis by 9α-fluoro and 9α-hydroxy derivatives of 5α-cholest-8(14)-en-3β-ol-15-one☆

Abstract 5α-Cholest-8(14)-en-3β,9α-diol-15-one has been prepared by chemical synthesis in two steps from 5α-cholest-8(14)-en-3β-ol-15-one. 9α-Fluoro-5α-cholest-8(14)-en-3β-ol-15-one was prepared in 91% yield from the corresponding 9α-hydroxysterol. 9α-Fluoro-5α-cholest-8(14)-en-3,15-dione was prepared, in 84% yield, from the corresponding 3β-hydroxy compound. These compounds have been found to be potent inhibitors of sterol synthesis in animal cells in culture. The concentrations of the steroids required to cause a 50% inhibition of the synthesis of digitonin-precipitable sterols from labeled acetate were comparable to those required to cause a 50% reduction in the levels of HMG-CoA reductase activity in the same cells.