Mode of interaction of .beta.-hydroxy-.beta.-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

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Abstract
The Na salts of compactin (1) and trans-6-[2-(2,4-dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-2H-pyran-2-one (3) are inhibitors of yeast .beta.-hydroxy-.beta.-methyglutaryl coenzyme A (HMG-CoA) reductase [a rate-limiting enzyme in cholesterol biosynthesis]. The dissociation constants are 0.24 .times. 10-9 and 0.28 .times. 10-9 M, respectively. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. The basis for the strong interaction between HMG-CoA reductase and these inhibitors was investigated. HMG-CoA and coenzyme A (CoASH), but not reduced NADPH, prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to enzyme. The inhibitory activity of molecules that resemble structural components of compactin was investigated. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 .+-. 0.9) .times. 10-2 M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be .gtoreq. 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The Na salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 .mu.M. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. These inhibitors occupy 2 sites on the enzyme: 1 site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site. The high affinity of these inhibitors for HMG-CoA results from the simultaneous interaction of these inhibitors with 2 separate binding sites on the enzyme. The binding advantage gained from connecting the lactone and the decalin portions of compactin may be .gtoreq. 5 .times. 104 M. When HMG-CoA (150 .mu.M) and compactin (0.07-0.70 .mu.M) are added to the enzyme, the enzyme is rapidly converted to enzyme-compactin and E.cntdot.HMG-CoA. The amount of E .cntdot. EMG-CoA is converted to enzyme-compactin until equiibrium is reached. Formation of E-compactin is, thus, a biphasic process. The overshoot in the formation of E.cntdot.EMG-CoA is a consequence of the concentration of HMG-CoA and compactin and the slow dissociation rate of E.cntdot.EMG-CoA. The on- and off-rate constants of HMG-CoA are 1.9 .times. 105 M-1 s-1 and 0.11 s-1, respectively, and the on- and off-rate constants of compactin are 2.7 .times. 107 M-1 s-1 and 6.5 .times. 10-3 s-1, respectively. The on-rate constant for compactin is nearly diffusion limited and is 2 orders of magnitude faster than on-rate constant for HMG-CoA.