The purpose of the present study was to determine the lead structure in cardiac glycosides at the receptor level, i.e. the minimal structural requirement for specific and powerful receptor recognition. Accordingly 73 digitalis-like acting steroids were characterized as to the concentration effecting half-maximum inhibition of Na,K-ATPase from human cardiac muscle under standardized turnover conditions. Since the Ki value equaled the apparent KD value, K'D was expressed in terms of the apparent standard Gibbs energy change delta G degrees' of steroid interaction with Na,K-ATPase. This allowed the use of the extrathermodynamic approach as a rational way of correlating in a quantitative manner, the potency and structure of the various steroidal compounds. The results of the present analysis taken in conjunction with relevant findings reported in the literature, favour the following conclusions. Cassaine, canrenone, prednisolone- and progesterone-3,20-bisguanylhydrazone, and chlormadinol acetate are compounds that are not congeneric with digitalis. The butenolide ring of cardenolides or the analogous side-chains at C17 beta of 5 beta, 14 beta-androstane-3 beta, 14-diol are not pharmacophoric substructures, but merely amplifiers of the interaction energy of the steroid lead. All modifications of the structure, geometry and spatial relationship between the steroid nucleus and butenolide side chain of digitoxigenin all at once weaken the close fit interaction with the steroid and butenolide binding subsites of the enzyme in such way that the cardenolide derivatives interact with the receptor binding site area in whatever orientation that will minimize the Gibbs energy of the steroid-receptor-solvent system. The "butenolide carbonyl oxygen distance model" (Ahmed et al. 1983) for the interpretation of the differences in potency of the cardenolide derivatives describes the change in interaction energy through structural modification as a function of the entire molecule. 5 beta, 14 beta-androstane-3 beta, 14-diol, the steroid nucleus of cardiac glycosides of the digitalis type, is the minimum structure for specific receptor recognition and the key structure for inducing protein conformational change and thus Na,K-ATPase inhibition. It is also the structural requirement for maximum contributions of the butenolide substituent at C17 beta and the sugar substituent at C3 beta-OH to the overall interaction energy, i.e. this steroid nucleus is the lead structure.(ABSTRACT TRUNCATED AT 400 WORDS)
A Free-Wilson analysis of 95 digitalis-like steroids inhibiting the Na/K-ATPase is presented. The chosen parent structure -5P,14P-androstane-3P,14-diolwas varied at 12 individual substitution sites. The high significance of the multiple regression of the data obtained with the great majority of the derivatives tested revealed the general applicability of the additivity model, allowing the prediction of the potency of hypothetical compounds from the derived 57 de noljo substituent constants. Derivatives carrying voluminous substituents at C3 or C17, in which the cis/trans/ cis-junction of the steroid rings were altered, disclosed incompatibilities with the model.
Recently, we have proposed that (Na, K)-ATPase functions according t o the mechanism of the "flip-flop" enzymes.' In this paper, we will not repeat the arguments favoring our flip-flop model of coupled Na+/K+ transport, but rather will present briefly the results of various experimental and theoretical examinations of some predictions of the model. A fuller account will be given elsewhere.2
Some Basic Features and Predictions o f the Flip-Flop ModelAs detailed in an earlier paper,' (Na, K)-ATPase shows half-of-the-sites reactivity and thus behaves like a functional dimer. The unliganded dimeric enzyme may then be described by where F represents the free substrate reactive moieties and f the free cation carrier moieties, and the colon indicates a decoupled state of the two half units.
5'z % 0 a n w z
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