The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model.

Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed.

Rapid metabolic inactivation of tipredane, a structurally novel topical steroid.

[3H]Tipredane ([3H]TP), [3H]triamcinolone acetonide ([ 3H]TAAC), [3H]hydrocortisone ([3H]HC), and [3H]betamethasone-17 alpha-valerate ([3H]BMV), each at a concentration of 1 microM, were separately incubated with the 10,000 g supernatant fraction of the liver and skin homogenates of humans, rats and mice (BMV was studied only in human liver). Sequential samples were taken for up to 1 h during each incubation. The radioactivity in each sample was extracted with methanol, and the methanolic extracts were analyzed by high performance liquid chromatography. Among the four compounds studied, [3H]TP was most rapidly biotransformed by the liver preparations of the three species. The rates of in vitro biotransformation of TP were 2.5-30 times faster than those of TAAC, HC and BMV. In the human liver preparation, the rates of biotransformation were in the order of: TP greater than TAAC greater than HC = BMV. In the mouse and rat liver preparations, the orders were: TP greater than TAAC greater than HC and TP greater than HC greater than TAAC, respectively. In the skin preparations, little, if any, biotransformation of [3H]TP and [3H]TAAC was observed in any of the species studied; however, [3H]HC underwent a slow, steady biotransformation in the skin preparations of humans and rats but not of mice. [3H]TP was biotransformed by the liver preparations of all three species to numerous metabolites, thirteen of which have been identified. The biotransformation reactions included: (1) sulfoxidation; (2) elimination of either one or both alkylthio groups; and (3) hydroxylation of the steroid nucleus. Some metabolites were synthesized and tested for glucocorticoid receptor binding and anti-inflammatory activities; all were found to be much less potent than TP. The observed separation of local anti-inflammatory activity from systemic side effects of TP is most probably due to its rapid metabolic inactivation; the liver, rather than the skin, is mainly responsible for the metabolic inactivation of TP.

Androstene-17-thioketals. 1st communication: glucocorticoid receptor binding, antiproliferative and antiinflammatory activities of some novel 20-thiasteroids (androstene-17-thioketals).

The unique replacements of the alpha-hydroxyl and beta-ketol groups of corticoids at C17 with selected, simple alkylthio or (2-fluoroalkyl)thio groups resulted in the structurally novel steroids, C17-alkylthioketals of 9 alpha-fluoro-11 beta-hydroxy-androsta-1,4-diene-3,17-dione. The described androstene-17-thioketals (20-thiasteroids) had high affinities for the glucocorticoid receptor protein of rat liver cytosol. Most were more potent than triamcinolone acetonide, a clinically moderately potent corticoid, in antiproliferative and antiinflammatory activities in mice. Specifically, (11 beta, 17 alpha)-17-(ethylthio)-9 alpha-fluoro-11 beta-hydroxy-17-(methylthio) androsta-1,4-dien-3-one (tipredane, SQ 27,239) and (11 beta, 17 alpha)-17-(ethylthio)-9 alpha-fluoro-17-[2-(fluoroethyl)thio] - 11 beta - hydroxy-androsta-1,4-dien-3-one (SQ 28,300), topically applied, were as potent as halcinonide, a clinically highly potent corticoid, in inhibition of croton oil-induced edema in the mouse. It is suggested that both thiasteroids could be moderately to highly potent topical antiinflammatory agents in man.

Androstene-17-thioketals. 2nd communication: pharmacological profiles of tipredane and (11 beta, 17 alpha)-17-(ethylthio)-9 alpha-fluoro-17-[2-(fluoroethyl)thio]-11 beta-hydroxy-androsta-1,4-dien-3-one, structurally novel 20-thiasteroids possessing potent and selective topical antiinflammatory activity.

Two structurally novel alkylthio-substituted steroids, (11 beta, 17 alpha)-17-(ethylthio)-9 alpha-fluoro-11 beta-hydroxy-17-(methylthio)andro-1,4-dien-3-one (tipredane, SQ 27,239) and (11 beta, 17 alpha)-(ethylthio)-9 alpha-fluoro-17-[2-(fluoroethyl) thio]-11 beta-hydroxy-androsta-1,4-dien-3-one (SQ 28,300) were compared to presently available topical corticosteroids for in vitro and in vivo glucocorticoid and antiinflammatory activities. Based upon results of in vitro assays, in vivo antiinflammatory tests in mice, and human vasoconstriction measurements, the thiasteroids most closely resemble moderately potent to highly potent corticoids. These compounds display more modest activity in topical antiinflammatory assays using rats. Both tipredane and SQ 28,300 exhibit favorable separation of local antiinflammatory activity from systemic effects on thymus and hypothalamic-pituitary-adrenal axis function, most probably due to rapid metabolic inactivation. As such, these compounds represent potentially safer therapy for topical treatment of corticoid-responsive skin diseases and bronchopulmonary conditions in humans.

Synthesis and antiinflammatory activity of hexahydrothiopyrano[4,3-c]pyrazoles and related analogues.

A series of novel hexahydrothiopyrano[4,3-c]pyrazoles and related analogues were prepared and tested for antiinflammatory activity by using the mouse active Arthus reaction and the delayed hypersensitivity skin reaction in guinea pigs as primary screens. The compounds of most interest, 18 and 28, were further tested in a model of adjuvant-induced arthritis; in this system, both compounds were active when dosed intraperitoneally but failed to produce significant activity when dosed orally at subtoxic doses.

1-Acyltriazoles as antiinflammatory agents.

Certain 1-acyl-3-phenyl-5-alkyltriazoles were synthesized and evaluated for antiinflammatory activity using the mouse active Arthus (MAA) reaction as the test system. Modification of the acyl group, 4-phenyl substituent, and alkyl group led to the selection of the most active member of this series, 1-acetyl-3-(4-chlorophenyl)-5-methyl-1,2,4-triazole (3c), for further evaluation as a novel nonacidic antiinflammatory agent.