Kinetics of low-density lipoprotein receptor activity in Hep-G2 cells: derivation and validation of a Briggs-Haldane-based kinetic model for evaluating receptor-mediated endocytotic processes in which receptors recycle.

The process of receptor-mediated endocytosis for receptors that recycle to the cell surface in an active form can be considered as being kinetically analogous to that of a uni-substrate, uni-product enzyme-catalysed reaction. In this study we have derived steady-state initial-velocity rate equations for this process, based on classical Briggs-Haldane and King-Altman kinetic approaches to multi-step reactions, and have evaluated this kinetic paradigm, using as a model system the low-density lipoprotein (LDL)-receptor-mediated endocytosis of the trapped label [14C]sucrose-LDL in uninduced, steady-state Hep-G2 cells. Using the derived rate equations, together with experimentally determined values for Bmax (123 fmol/mg of cell protein), Kd (14.3 nM), the endocytotic rate constant ke (analogous to kcat; 0.163 min-1), Km (80 nM) and maximal internalization velocity (26.4 fmol/min per mg), we have calculated the ratio ke/Km (0.00204 nM-1.min-1), the bimolecular rate constant for LDL and LDL-receptor association (0. 00248 nM-1.min-1), the first-order rate constant for LDL-LDL-receptor complex dissociation (0.0354 min-1), the total cellular content of LDL receptors (154 fmol/mg of cell protein), the intracellular LDL receptor concentration (30.7 fmol/mg of cell protein) and the pseudo-first-order rate constant for LDL receptor recycling (0.0653 min-1). Based on this mathematical model, the kinetic mechanism for the receptor-mediated endocytosis of [14C]sucrose-LDL by steady-state Hep-G2 cells is one of constitutive endocytosis via independent internalization sites that follows steady-state Briggs-Haldane kinetics, such that LDL-LDL-receptor interactions are characterized by a rapid-high-affinity ligand-receptor association, followed by ligand-receptor complex internalization that is rapid relative to complex dissociation, and by receptor recycling that is more rapid than complex internalization and that serves to maintain 80% of cellular LDL receptors on the cell surface in the steady-state. The consistency with which these quantitative observations parallel previous qualitative observations regarding LDL-receptor-mediated endocytosis, together with the high correlation between theoretical internalization velocities (calculated from determined rate constants) and experimental internalization velocities, underscore the validity of considering receptor-mediated endocytotic processes for recycling receptors in catalytic terms.

3-(4-chlorophenyl)-2-(4-diethylaminoethoxyphenyl)-A-pentenonitrile monohydrogen citrate and related analogs. Reversible, competitive, first half-reaction squalene synthetase inhibitors.

Squalene synthetase (SQS) catalyzes the head-to-head condensation of two molecules of farnesyl pyrophosphate (FPP) to form squalene. The reaction is unique when compared with those of other FPP-utilizing enzymes, and proceeds in two distinct steps, both of which involve carbocationic reaction intermediates. In this report, we describe the mechanism of action of, and structure-activity relationships within, a series of substituted diethylaminoethoxystilbenes that mimic these reaction intermediates, through characterization of the biochemical properties of 3-(4-chlorophenyl)-2-(4-diethylaminoethoxyphenyl)-A- pentenonitrile monohydrogen citrate (P-3622) and related analogs. As a representative member of this series, P-3622 inhibited SQS reversibly and competitively with respect to FPP (Ki = 0.7 microM), inhibited the enzymatic first half-reaction to the same extent as the overall reaction, exhibited a 300-fold specificity for SQS inhibition relative to protein farnesyltransferase inhibition, inhibited cholesterol synthesis in rat primary hepatocytes (IC50 = 0.8 microM), in cultured human cells (Hep-G2, CaCo-2, and IM-9; IC50 = 0.2, 1.2, and 1.0 microM), and in chow-fed hamsters (62% at 100 mg/kg) without accumulation of post-squalene sterol precursors, and reduced plasma cholesterol in experimental animals. Structure-activity relationships among 72 related analogs suggest that the phenyl residues and central trans-olefin of the stilbene moiety serve as mimics of the three isoprene units of the donor FPP, that substitutions across the central olefin and para-substitutions on the terminal phenyl residue mimic the branching methyl groups of the donor FPP, and that the diethylaminoethoxy moiety of these molecules mimics the various carbocations that develop in the C1-C3 region of the acceptor FPP during reaction. Members of this series of reversible, competitive, first half-reaction SQS inhibitors that show a high degree of specificity for SQS inhibition relative to inhibition of other FPP-utilizing enzymes and other cholesterol synthesis pathway enzymes may serve as useful tools for probing the unique catalytic mechanisms of this important enzyme.

Pharmacologic consequences of cholesterol absorption inhibition: alteration in cholesterol metabolism and reduction in plasma cholesterol concentration induced by the synthetic saponin beta-tigogenin cellobioside (CP-88818; tiqueside).

Natural and synthetic saponins inhibit cholesterol absorption and reduce plasma cholesterol levels in experimental animals and are therefore of potential pharmacologic utility in the treatment of hypercholesterolemia. To determine the effects of this class of compounds on cholesterol absorption and metabolism, we evaluated the effects of the synthetic saponin, beta-tigogenin cellobioside (tiqueside; CP-88818), on male golden Syrian hamsters. When administered as either a single oral bolus or as a dietary supplement for up to 2 weeks, tiqueside inhibited cholesterol absorption in a dose-dependent manner in both the presence and absence of dietary cholesterol. Administration of tiqueside to chow-fed hamsters as a 0.2% dietary supplement (150 mg/kg per day) for 4 days resulted in a 68% decrease in intestinal cholesterol absorption with no change in either bile absorption or cholesterol 7 alpha-hydroxylase activity, suggesting that tiqueside inhibits cholesterol absorption without interfering with enterohepatic bile acid recirculation. Under these conditions, hepatic cholesterol levels were also reduced in a dose-dependent manner. Hepatic cholesterol reduction was highly correlated with cholesterol absorption inhibition, and induced compensatory increases in both hepatic HMG-CoA reductase activity and hepatic low density lipoprotein (LDL) receptor levels. Compensatory increases in intestinal HMG-CoA reductase activity were also noted after tiqueside administration, and are consistent with a luminal mechanism for tiqueside action. As a consequence of these changes to cholesterol metabolism, tiqueside administration induced plasma cholesterol reductions that were highly correlated with both hepatic cholesterol reduction and cholesterol absorption inhibition. Tiqueside also produced comparable plasma cholesterol lowering in a variety of other species fed either cholesterol-free diets (hamster, rat, mouse, dog) or cholesterol-containing diets (hamster, rat, rabbit, mouse, cynomolgus monkey, rhesus monkey, SEA quail) indicating the ubiquity of tiqueside action. For all species evaluated except the dog, the reduction in plasma cholesterol was due primarily to a reduction in circulating non-HDL cholesterol levels with little or no change in HDL cholesterol levels. Taken together, these results indicate that inhibition of cholesterol absorption by tiqueside produces profound effects on cholesterol metabolism without affecting bile acid metabolism, and that these changes lead to reductions primarily in plasma non-HDL cholesterol concentrations. The synthetic saponin, tiqueside, may thus represent a prototypical form of therapy for the treatment of hypercholesterolemia.

Efficacy, tissue distribution and biliary excretion of methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate (CP-83101), a hepatoselective inhibitor of HMG-CoA reductase activity in the rat.

Methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate, CP-83101, was identified as a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, inhibiting enzyme activity in vitro with an IC50 of 8.5 +/- 0.7 microM and a Ki with respect to HMG-CoA of 2.6 microM. CP-83101 also inhibited rat hepatic sterol biosynthesis by 39 +/- 7% at a dose of 100 mg/kg. [3H]CP-83101, administered orally to rats, exhibited peak plasma levels at approximately 1 hr that declined thereafter with an apparent half-time of 2-3 hr. Peak tissue levels also occurred 1 hr following oral administration of [3H]CP-83101. The decline in radioactivity in the liver, however, was considerably slower than that noted in blood, whereas the half-life in non-hepatic tissues was approximately 1 hr. Liver/blood ratios of 14, and liver/lens ratios of greater than 3000, following oral administration of [3H]CP-83101, were similar to those previously reported for other HMG-CoA reductase inhibitors, suggesting a high degree of tissue selectivity. In addition, liver/adrenal and liver/ovary ratios were approximately 1000 at all time points examined between 30 min and 24 hr following oral [3H]CP-83101 administration, indicating a high specificity for hepatic versus other steroidogenic tissues. Evaluation of intravenous versus oral administration of the water-soluble, free acid, sodium salt of [3H]CP-83101 in bile duct canulated rats indicated that approximately 20% of orally administered CP-83101 is absorbed from the gastrointestinal tract, and that absorbed CP-83101 is cleared rapidly from the plasma via the liver and from the liver via the bile. In addition, several lines of evidence suggest that CP-83101 may undergo enterohepatic recirculation. Agents of this synthetic series may thus possess advantages over other HMG-CoA reductase inhibitors with respect to tissue kinetics and specificity.