In 1901 a physician, Archibald Garrod, observed a patient with black urine. He used this simple observation to demonstrate that a single mutant gene can produce a discrete block in a biochemical pathway, which he called an “inborn error of metabolism”. Garrod’s brilliant insight anticipated by 40 years the one gene-one enzyme concept of Beadle and Tatum. In similar fashion the chemist Linus Pauling and the biochemist Vernon Ingram, through study of patients with sickle cell anemia, showed that mutant genes alter the amino acid sequences of proteins. Clearly, many fundamental advances in biology were spawned by perceptive studies of human genetic diseases (1). We began our work in 1972 in an attempt to understand a human genetic disease, familial hypercholesterolemia or FH. In these patients the concentration of cholesterol in blood is elevated many fold above normal and heart attacks occur early in life. We postulated that this dominantly inherited disease results from a failure of end-product repression of cholesterol synthesis. The possibility fascinated us because genetic defects in feedback regulation had not been observed previously in humans or animals, and we hoped that study of this disease might throw light on fundamental regulatory mechanisms. Our approach was to apply the techniques of cell culture to unravel the postulated regulatory defect in FH. These studies led to the discovery of a cell surface receptor for a plasma cholesterol transport protein called low density lipoprotein (LDL) and to the elucidation of the mechanism by which this receptor mediates feedback control of cholesterol synthesis (2,3). FH was shown to be caused by inherited defects in the gene encoding the LDL receptor, which disrupt the normal control of cholesterol metabolism. Study of the LDL receptor in turn led to the understanding of receptor-mediated endocytosis, a genera! process by which cells communicate with each other through internalization of regulatory and nutritional molecules (4). Receptor-mediated endocytosis differs from previously described biochemical pathways because it depends upon the continuous and highly controlled movement of membraneembedded proteins from one cell organelle to another in a process termed

https://science.sciencemag.org/content/sci/232/4746/34.full.pdf

A receptor-mediated pathway for cholesterol homeostasis.

▪ Abstract Statins are potent inhibitors of cholesterol biosynthesis. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. However, the over...

/pdf/pleiotropic-effects-of-statins-1b9b1g8457.pdf

Pleiotropic effects of statins

Mevinolin, a fungal metabolite, was isolated from cultures of Aspergillus terreus. The structure and absolute configuration of mevinolini and its open acid form, mevinolinic acid, were determined by a combination of physical techniques. Mevinolin was shown to be 1,2,6,7,8,8a-hexahydro-beta, delta-dihydroxy-2,6-dimethyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-hepatanoic acid delta-lactone. Mevinolin in the hydroxy-acid form, mevinolinic acid, is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]; its Ki of 0.6 nM can be compared to 1.4 nM for the hydroxy acid form of the previously described related inhibitor, ML-236B (compactin, 6-demethylmevinolin). In the rat, orally administered sodium mevinolinate was an active inhibitor of cholesterol synthesis in an acute assay (50% inhibitory dose = 46 microgram/kg). Furthermore, it was shown that mevinolin was an orally active cholesterol-lowering agent in the dog. Treatment of dogs for 3 weeks with mevinolin at 8 mg/kg per day resulted in a 29.3 +/- 2.5% lowering of plasma cholesterol.

https://www.pnas.org/content/pnas/77/7/3957.full.pdf

Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portion of human HMGR complexed with six different statins. The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. Near the carboxyl terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. If these residues were not flexible, they would sterically hinder statin binding.

https://science.sciencemag.org/content/sci/292/5519/1160.full.pdf

Structural mechanism for statin inhibition of HMG-CoA reductase.

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

https://asbmr.onlinelibrary.wiley.com/doi/pdf/10.1359/jbmr.2005.20.7.1265

Nitrogen-Containing Bisphosphonates Inhibit the Mevalonate Pathway and Prevent Post-Translational Prenylation of GTP-Binding Proteins, Including Ras

https://www.jstage.jst.go.jp/article/antibiotics1968/29/12/29_12_1346/_pdf

Ml-236a, ml-236b, and ml-236c, new inhibitors of cholesterogenesis produced by penicillium citrinum

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2876%2980996-9

Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity

Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ml-236a and ml-236b fungal metabolites, having hypocholesterolemic activity

A compound active against sterol biosynthesis was isolated from the culture filtrate of the fungus Prthium ultimum IAM 6073 and identified as citrinin which had been isolated as an antibiotic.') The sequence of reactions by which cholesterol is formed from acetyl-CoA is one of the most complex biosynthetic pathways in eukaryotic cells. In every vertebrate species so far studied, potent activity in cholesterol synthesis can be detected in the liver which seems to be the sole organ supplying plasma cholesterol. Because of the importance of the plasma cholesterol level in atherosclerosis, several approaches have been made to control it by decreasing cholesterol synthesis in the liver.2~4) The inhibition of endogenous cholesterol synthesis could lead to a lowering of its level in the plasma. This communication describes inhibition by citrinin of cholesterol and ergosterol synthesis and its hypocholesterolemic activity in rats. The enzymatic synthesis of cholesterol was assayed by measuring the radioactive nonsaponifiable products derived from 14C-acetate in a rat liver enzyme system by the method of KNAUSS et al.5) Culture filtrates of microorganisms including fungi, bacteria, and actinomycetes were tested for inhibitory activity in this assay system, and Pvthium ultimum was selected as one of the producers of an inhibitor. The fungus was cultured aerobically in a medium containing 2.0% glucose, 2% peptone and 0.3 % corn steep liquor in a 30-liter fermentor for 4 days. The culture filtrate was adjusted to pH 2, and the active compound was extracted with benzene. The extract was concentrated in vacuo, resulting in the formation of yellowish crystals of the active compound. The compound was recrystallized from hot chloroform. Calcd.: C 62.39, H 5.64, O 31.97 Found: C 62.39, H 5.58, O 32.03 The chemical structure of 4,6-dihydro-8-hydroxy-3,4, 5-trimethyl 6 oxo3 H-2-benzopyran-7 carboxylic acid was suggested by n.m.r. spectrum. The identity with citrinin was proven by comparison with authentic samples (kindly supplied by Dr. S. UDAGAWA, National Institute of Hygienic Science, Tokyo). As shown in Fig. 1, citrinin strongly inhibited cholesterol synthesis from 14C-acetate. The concentration at which cholesterol synthesis was inhibited by 50 % was about 8.5 mcg/ml (3.4 x 10-5M). Under the same conditions, 14C-acetate incorporation into fatty acid fraction was also reduced by citrinin but to a lesser extent. The concentration of the inhibitor required for 50 inhibition of fatty acid synthesis was approximately 50 mcg/ml. Citrinin was also inhibitory in the sterol synthesizing system of the yeast Saccharomyces cerevisiae in which 14C-acetate incorporation into nonsaponifiable lipids was determined as described by KAWAGUCHI (Fig. 1).e) At a concentration of 100 mcg/ml, the inhibitor reduced the sterol synthesis by approximately 50%. For studies of hypocholesterolemic effect of

https://www.jstage.jst.go.jp/article/antibiotics1968/29/8/29_8_841/_pdf

Citrinin, an inhibitor of cholesterol synthesis.

Derivatives of ML-236B carboxylic acid, its salts and esters and corresponding lactone have a hydroxy group at the 3"-position and optionally also at the 6'-position. These derivatives may be prepared by the enzymatic hydroxylation of a corresponding ML-236B compound employing an enzyme produced by a microorganism of the genus Streptomyces or Amycolata, especially Streptomyces sp. SANK 62285, Streptomyces sp. SANK 62385, Streptomyces sp. SANK 62485, Streptomyces sp. SANK 62585, Amycolata autotrophica SANK 62781, Amycolata autotrophica subsp. canberrica SANK 62882 or Amycolata autotrophica subsp. amethystina SANK 62981. These derivatives have the ability to inhibit cholesterol biosynthesis.

Masao Kuroda

Papers

Ml-236a, ml-236b, and ml-236c, new inhibitors of cholesterogenesis produced by penicillium citrinum

Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity

Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ml-236a and ml-236b fungal metabolites, having hypocholesterolemic activity

Citrinin, an inhibitor of cholesterol synthesis.

Hydroxy-ml-236b derivatives, their preparation and use