Synthesis of phenazine and phenoxazinone derivatives of geldanamycin as potential polymerase inhibitors

Derivatives of geldanamycin—phenazines and phenoxazinones, some of them analogous to rifazine in the rifamycin series—have been synthesized. Most of the geldanamycin derivatives were highly effective inhibitors of RNA-dependent DNA polymerase from Rauscher leukemia virus. A simple analog containing the aromatic chromophore of the derivatives but without the ansa ring also showed viral inhibition.     

Halogenated epoxides and related compounds as inhibitors of epoxide hydrase

A variety of chlorinated and fluorinated epoxides and related compounds were synthesized and evaluated as inhibitors of epoxide hydrase. The compounds were tested using chicken liver microsomes and a radiometric assay based on [³H]styrene oxide, and using partially purified chicken liver microsomal epoxide hydrase and a continuous photometric assay based on p-nitrostyrene oxide, whose hydration could be monitored at 310 nm. For the 16 compounds studied both assays gave similar patterns of inhibitory activity. As expected from the relative K_m values of the two substrates, all inhibitors were considerably more active against styrene oxide (K_m =1.0 mM) than against p-nitrostyrene oxide (K_m = 4.2 μM), and styrene oxide was a weak alternate-substrate inhibitor against p-nitrostyrene oxide. 1,1,1-Trichloropropene oxide, however, was a potent alternate-substrate inhibitor against p-nitrostyrene oxide. Addition of various substituents to the α-carbon of styrene oxide generated a series of compounds whose inhibitory potency toward p-nitrostyrene oxide increased in the order H ≈ CF₃ < CH₃ < CH₂Cl < CHCl₂ < CCl₃ ≈ 1,1,1-trichloropropene oxide. In contrast, addition of a CH₃ or CCl₃ group to the β-carbon of styrene oxide resulted in only a modest increase in inhibitory potency. 2-Phenyl- and 3-phenyloxetane showed no pronounced inhibitory activity toward either styrene oxide or p-nitrostyrene oxide, but pentafluorophenyl ethylene oxide and 1,1, 1-trichlorobutane-3,4-oxide were moderately active inhibitors, although significantly less potent than 1,1,1-trichloroproene oxide. These results show that electronegativity, steric effects, and hydrophobic effects are each important in governing the interaction of epoxide hydrase substrates with the enzyme, although it is not yet possible to analyze separately the effects of each of these parameters on K_m, V, and the catalytic mechanism.     

Oxidized saccharides as inhibitors of alpha-glucan synthesis by Streptococcus mutans glucosyltransferase

Specific inhibition by periodate-oxidized dextrans of the synthesis of alpha-glucan by S. mutans glucosyltransferase prompted a search for structurally related inhibitors that might be effective as anticaries agents. Clinical dextran derivatives in which from 5 to 50% of the D-glucose units were oxidized acted as potent and specific enzyme-inhibitors, as did 10%-oxidized derivatives of dextran fractions ranging in mol. wt. from 10(4) to 2 X 10(6). Within these limits, differences in oxidation or molecular weight did not significantly affect the high inhibitory potency of the derivatives. In contrast, periodate oxidation of (1 leads to 6)-alpha, (1 leads to 3)-alpha-, and (1 leads to 4)-alpha-linked oligosaccharides containing less than approximately 15 D-glucose units, and of sucrose and structurally related trisaccharides, yielded derivatives that were poor inhibitors. Enzymic hydrolysis of oxidized dextrans caused a loss of their inhibitory power and indicated that, to act as specific inhibitors, oxidized molecules must contain at least 16 to 20 D-glucosyl residues. The similar, minimum size required in order that unoxidized oligosaccharides may act as efficient acceptors in the glucosyltransferase reaction suggests that the inhibitory potencies of oxidized derivatives may reflect their relative abilities to bind at the acceptor site of the enzyme.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Composition of by-products of the essential-oil industry and their potential as feeds for ruminants

Eight plant-residues from the essential-oil industry were assayed for chemical composition. Digestibility of the plant residues was determined by rumen fermentation in vitro and in vivo. Dry-matter digestibility of plant residues from black pepper, vanilla bean, ginger root, cocoa bean and celery seed ranged from 43–65%. Only 15–30% of the residues from nutmeg hulls and cinnamon bark were degraded. These data indicate that anaerobic microbes are capable of degrading significant quantities of the plant residues without further processing of these materials. Long-term feeding studies need to be conducted to determine if ruminants will consume these plant materials, and to investigate the effect of these plant residues on rumen microbial- and host cellular-metabolism.     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Yeast RNA polymerase I: A eukaryotic zinc metalloenzyme

Microwave excitation spectrometry and metal binding inhibition studies show that zinc is a catlytically essential component of the highly purified RNA polymerase I from yeast, the first eukaryotic RNA polymerase I available in quantities sufficient for such studies. It contains 2.4 g-atom of zinc based on a molecular weight of 6.5 × 10⁵ (8). Copper, iron, manganese and magnesium are absent, i.e., below the limits of detection, 10^?13 to 10^?14 g-atoms. A number of derivatives of 1,10-phenanthroline reversibly inhibit the polymerase catalyzed reaction, apparently by forming a ternary polymerase·Zn·OP complex while the nonchelating isomer, 1,7-phenanthroline, is ineffective.     

Inhibition of elastolysis by proteinase inhibitors from chick plasma and aorta

Chick plasma contains inhibitor(s) against trypsin and elastase which also appear to retard the degradation of tropoelastin by arterial tissue Chick aorta extracts also contain similar inhibitors against elastase and trypsin. Both levels of the plasma inhibitor(s) and inhibitor(s) extracted from thoracic aorta increase during early stages of growth and maturation. There is a three- to four-fold increase in the levels of the inhibitor(s) in chick plasma and aorta between one to four weeks after hatching. Of particular interest are the observations that the presence of the inhibitor(s) retards the conversion of soluble elastin (tropoelastin) to smaller elastin peptides. Subsequently, it is speculated that in addition to other vital roles, such proteinase inhibitors may also act in regulating elastogenesis and elastin fiber formation.     

Non-linear relationship between fluorescence and membrane potential

The fluorescence intensity of the cynanine dye DiO-C₆-(3)_in 0.32% suspension of Ehrlich ascites tumor cells was determined under various potassium concentrations. In addition, the fluorescence levels of cell-free buffer solutions and those of supernatants were measured. For every potassium concentration the partition of the dye between cells and medium was calculated and its relation to the potassium gradient was given. A model was developed which assumes the fluorescence of a cell suspension to be the sum of the fluorescence signal of dye in the extracellular medium and that of cell-associated dye. A calibration curve of fluorescence vs. membrane potential was constructed. Neither the fluorescence of the cell suspension nor that of the supernatants was a linear function of the membrane potential. The limitations of membrane potential determination by fluorimetric methods are discussed.     

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.     

Synthesis of adiposin-1 and related compounds

The synthesis is described of adiposin-1 (2a), isolated from an α-d-glucosidase inhibitor complex, adiposin, produced by Streptomyces caluvs TM-521. The synthesis involved the coupling of 1,6-anhydro-4-O-(3,4-anhydro-α-d-galactopyranosyl)-β-d-glucopyranose (13) with the di-O-isopropylidene derivative (7) of dl-(1,4,$\text{6}\text{5}$)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexenylamine. All possible diastereoisomers of the secondary amine were isolated by chromatography on silica gel. Their structures were tentatively assigned on the basis of ¹H-n.m.r. spectroscopy and optical rotation. Likewise, both the core-structure (4) of adiposin and the saturated analog (22) of 2a were synthesized.     

Inhibition of Escherichia coli DNA polymerase I by rose bengal

The fluorescein dye, rose bengal, inhibits Escherichia coli DNA polymerase I reversibly in the dark and irreversibly in the light. The reversible inhibition, which occurs in the micromolar concentration range, is competitive with respect to the poly(dA-T) template/ primer and noncompetitive with respect to the complementary deoxynucleoside triphosphates. The Hill coefficient for the inhibition by rose bengal is 3.0. Equilibrium dialysis experiments using ¹³¹I-labeled rose bengal have demonstrated direct binding of the inhibitor to the enzyme. No dye binds to poly(dA-T) at concentrations where the inhibition is observed. There are 22 ± 3 rose bengal binding sites per polymerase which can be subdivided into a class of high affinity sites and one of low affinity sites. The high affinity sites (3 μm) bind rose bengal with a Hill coefficient of 1.7 and are responsible for the observed inhibition. The low affinity sites (7μm) are more numerous (about 16) and bind rose bengal in a noncooperative manner. The displacement of rose bengal from the enzyme by poly(dA-T) at equilibrium confirms the competition between poly(dA-T) and rose bengal inferred from the kinetic data for the polymerization reaction. The inhibition of the 3′,5′ exonuclease activity and the template-directed dATP ? P-P exchange reaction by rose bengal is fully consistent with the interaction of rose bengal at the polynucleotide binding site. The enzyme induces an extrinsic Cotton effect in the visible absorption of rose bengal. The abolition of this Cotton effect by poly(dA-T) further supports the proposed site of binding of the dye.     

Subcellular distribution of the mannan-binding protein and its endogenous inhibitors in rat liver

The subcellular distribution of the mannan-binding protein from rat liver, a lectin specific for mannose and N-acetylglucosamine, was studied. Approximately 75% of the binding activity of the homogenate was recovered in microsomes, approximately 76% of which was accounted for by rough microsomes. Rough microsomes had the highest specific activity of binding, followed by the Golgi apparatus and smooth microsomes, whereas plasma membranes, lysosomes, mitochondria, and the soluble fraction had little or no binding activity. A topographical survey indicated that the binding protein was localized exclusively on the cisternal surface of microsomal vesicles. Thus, the binding protein of microsomal vesicles was protected from protease digestion and was released from the vesicles by mild detergent treatment. Competitive inhibitors, which presumably represent endogenous ligands of the binding protein, were found among subcellular fractions. More than 50% of the inhibitory activity of the homogenate was recovered in rough microsomes, while the highest specific activity of inhibition was found in lysosomes. The K_i values estimated for rough microsomes and lysosomes were 25.9 and 8.67 μg/ml, respectively. The distribution profiles of inhibitors were correlated roughly with those of the binding protein, resulting in masking of the binding activity in organelles up to the level of 86%. On the basis of the known localization and topology of the binding protein and endogenous inhibitors (ligands), possible physiological functions of the binding protein relevant to the transport of biosynthetic intermediates of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus and from the Golgi apparatus to lysosomes were discussed.     

Synthesis and properties of the cyclopentapeptide desthiomalformin

Desthiomalformin, cyclo-d-alanyl-d-alanyl-l-valyl-d-leucyl-l-isoleucyl, was synthesized by conventional methods. Cyclization of the open chain intermediate through the azide produced the cyclopentapeptide in short reaction time and in high yield and was not accompanied by cyclodimerization. The extreme readiness of the pentapeptide azide to form a cyclic structure can be attributed to the presence of alternating d and l residues in the sequence, a feature that should result in reverse turns stabilized by intramolecular hydrogen bonds. Thus the open chain intermediate could have a preferred cyclic conformation.Desthiomalformin has high thermal stability; it can be sublimed in vacuo without decomposition. It lacks, however, the biological activity of its parent compound, malformin.