Edward S. Inamine

HMG CoA-reductase inhibitors

SummaryLovastatin and simvastatin are the 2 best-known members of the class of hypolipidaemic agents known as HMG CoA reductase inhibitors. Clinical experience with lovastatin includes over 5000 patients, 700 of whom have been treated for 2 years or more, and experience with simvastatin includes over 3500 patients, of whom 350 have been treated for 18 months or more. Lovastatin has been marketed in the United States for over 6 months. Both agents show substantial clinical efficacy, with reductions in total cholesterol of over 30% and in LDL-cholesterol of 40% in clinical studies. Modest increases in HDL-cholesterol levels of about 10% are also reported. Clinical tolerability of both agents has been good, with fewer than 3% of patients withdrawn from treatment because of clinical adverse experiences. Ophthalmological examinations in over 1100 patients treated with one or the other agent have revealed no evidence of significant short term (up to 2 years) cataractogenic potential. One to 2% of patients have elevations of serum transaminases to greater than 3 times the upper limit of normal. These episodes are asymptomatic and reversible when therapy is discontinued. Minor elevations of creatine kinase levels are reported in about 5% of patients. Myopathy, associated in some cases with myoglobinuria, and in 2 cases with transient renal failure, has been rarely reported with lovastatin, especially in patients concomitantly treated with cyclosporin, gemfibrozil or niacin. Lovastatin and simvastatin are both effective and well-tolerated agents for lowering elevated levels of serum cholesterol. As wider use confirms their safety profile, they will gain increasing importance in the therapeutic approach to hypercholesterolaemia and its consequences.

Studies on the biosynthesis of avermectins

To elucidate the pathway of avermectin biosynthesis, the biosynthetic relationships of avermectins A1a, A2a, B1a, B2a, and their respective monosaccharides and aglycones were studied. 14C-labeled avermectin compounds prepared from [1-14C]acetate were fed to Streptomyces avermitilis strain MA5502 and their metabolites were determined. Two furan ring-free aglycones, 6,8a-seco-6,8a-deoxy-5-keto avermectin B1a and B2a, have been isolated from the fermentation broth of a blocked mutant of S. avermitilis. Addition of the compounds and a semisynthetic compound, 5-keto avermectin B2a aglycone, to the fermentation medium of a second blocked mutant established that the two compounds are intermediates in the avermectin biosynthetic pathway immediately preceding avermectin aglycones.

Further studies on the biosynthesis of the avermectins

SummaryThe biosynthesis of avermectins was studied further inStreptomyces avermitilis MA5502 by feeding experiments with labeled precursors.13C-NMR analysis of the compounds biosynthesized from [2-13C]acetate, [1,2-13C2]acetate, [3-13C]propionate and [2,3-13C2]propionate confirmed that the aglycone of avermectins is made from seven intact acetate and five propionate units. Feeding experiments with [1-13C]2-methylbutyrate and [1-13C]isobutyrate have shown that 2-methylbutyrate and isobutyrate are immediate precursors of the starter units of the polyketide chains of avermectin ‘a’ and ‘b’ components, respectively. The3H/14C doublelabeling experiments suggest that the two oleandrose moieties are derived from glucose.

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.

History of yield improvements in the production of asperlicin byAspergillus alliaceus

SummaryThe natural product asperlicin is the first nonpeptide antagonist of cholecystokinin isolated from a microbial source. At discovery, production of asperlicin by the original soil isolate ofAspergillus alliaceus was between 15 and 30 mg/l. Selection of natural variants ofA. alliaceus, use of Plackett & Burman and Simplex experimental designs; formulation of synthetic media; amino acid supplementation of production media; analysis of complex nitrogen sources for their amino acid content; evaluation of promising media in fermentors; substitution of glycerol for glucose as a carbon source and rational mutant selection all contributed to titer increases to >900 mg/l.

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya.

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.

A cofactor for thienamycin biosynthesis produced by a blocked mutant ofStreptomyces cattleya

Thienamycin (THM; Fig. 1), the first naturally occurring [3-1actam antibiotic discovered to possess the novel carbapenem ring system, was isolated from the culture filtrate of Streptomyces cattleya [1]. The biosynthesis of THM was investigated by Williamson et al. [3]. They established that the molecule is made up of cysteine, glutamate, acetate, and two methyl groups of methionine. The methyl groups were found to give rise to the hydroxyethyl side-chain of THM. Our intent was to further explore the biosynthetic route of THM using blocked mutants. During our studies we have isolated non-THM-producing strains by NTG, NMU, EMS and/or UV treatment of a THM-producing strain of S. cattleya. Applying an agar strip method [2], these nonproducers were tested for cosynthetic ability and classified into five groups. The complementation patterns between each group are shown in Table 1. In this communication, we wish to report the isolation of a bioconvertible substance produced by a Class I mutant. The blocked mutants used in these studies were MA 5952 (Class I) and M A 5953 (Class XVI), both of which lacked the ability to elaborate THM. However, MA 5953 growing in the presence of previously prepared MA 5952 filtrate was able to produce THM. Utilizing this response as an analytical tool, we were able to determine the presence of the material produced by MA 5952 that allowed MA

[52] Mannosidostreptomycin hydrolase

Publisher Summary This chapter discusses the assay procedure and properties of mannosidostreptomycin hydrolase. Mannosidostreptomycin hydrolase is an enzyme that transforms mannosidostreptomycin to streptomycin by the hydrolytic removal of the mannose moiety. The two antibiotics are made concurrently during the course of fermentation by strains of Streptomyces griseus. The hydrolase is an inducible enzyme whose formation is subject to catabolite repression so that it is usually synthesized late in fermentation when the repressible carbon source is near depletion. The α-D-mannosidase enzyme is most conveniently assayed by use of the chromogenic substrate p-nitrophenyl-α-D-mannopyranoside. The p-nitrophenol liberated is determined spectrophotometrically at 400 nm. Streptomyces mannosidase hydrolyzes phenyl-α-D-mannopyranoside, mannosidostreptomycin, mannosidodihydrostreptomyein in addition to p-nitrophenyl-α-D-mannopyranoside. The Streptomyces enzyme is bound to the cell until lysis occurs. The cell-bound enzyme can be extracted into water, but its release is inhibited by sodium chloride, phosphate, or Tris.

Guanosine-5′-monophosphate dioxanate: Preparation, characterization, and application

Abstract This paper reports, for the first time, the preparation and characterization of a unique crystalline complex of guanosine-5′-monophosphate, dioxane, and water (GMP-dioxanate) in a 1:1:1 molar ratio. The specificity of this complex is shown by the fact that no other solvent has been found which can replace dioxane. In addition, no other nucleotide or solvent combination has been found which forms a crystalline complex. GMP-Dioxanate is stable to heating and drying and is readily converted to the disodium salt. The compound has been fully characterized by physical, chemical, and biological analyses. Its crystallinity has been confirmed by both microscopic and X-ray analyses. Data on the optical rotation of solutions of GMP-dioxanate as a function of wavelength, concentration, and pH are described and compared with those of GMP and GMP·2Na·2H 2 O. This complex has provided a simplified procedure for the isolation of GMP from acid and salt mixtures, fermentation broths, and ribonucleic acid hydrolyzates.