A novel series of highly potent benzimidazole-based microsomal triglyceride transfer protein inhibitors.

A series of benzimidazole-based analogues of the potent MTP inhibitor BMS-201038 were discovered. Incorporation of an unsubstituted benzimidazole moiety in place of a piperidine group afforded potent inhibitors of MTP in vitro which were weakly active in vivo. Appropriate substitution on the benzimidazole ring, especially with small alkyl groups, led to dramatic increases in potency, both in a cellular assay of apoB secretion and especially in animal models of cholesterol lowering. The most potent in this series, 3g (BMS-212122), was significantly more potent than BMS-201038 in reducing plasma lipids (cholesterol, VLDL/LDL, TG) in both hamsters and cynomolgus monkeys.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 microM) with 5 min of ultraviolet irradiation, or 3.0 microM BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose- and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

1,1-Bisphosphonate squalene synthase inhibitors: interplay between the isoprenoid subunit and the diphosphate surrogate.

Inhibitors of squalene synthase have the potential to be superior cholesterol-lowering agents. We previously disclosed that lipophilic 1,1-bisphosphonates I are potent squalene synthase inhibitors and orally active cholesterol-lowering agents in animal models (Ciosek, C. P., Jr.; et al. J. Biol. Chem. 1993, 268, 24832-24837). In this paper, we describe modifications to the bisphosphonate moiety, in an attempt to reduce the number of acidic functions contained in these inhibitors. Replacing one of the acidic groups with a methyl (II, R2 = CH3) results in potent inhibitors when paired with a close mimic of the naturally occurring farnesyl moiety (R1 = farnesylethyl) but not when paired with the shorter isoprene surrogates (R1 = geranylethyl or 4-biphenylpropyl). In contrast, all three corresponding bisphosphonates I are potent squalene synthase inhibitors. Inhibitory potency is recovered with the shorter isoprene surrogates when R2 is CH2OH or CH2OCH3. It is proposed that these R2 groups serve as hydrogen bond acceptors with the active site of the enzyme. The properties of these compounds as cholesterol biosynthesis inhibitors in rats are described, and synthetic routes to these and related compounds are detailed.

Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase.

The rational design, synthesis, and biological activity of farnesyl diphosphate (FPP)-based inhibitors of the enzyme Ras farnesyl protein transferase (FPT) is described. Compound 3, wherein a beta-carboxylic phosphonic acid type pyrophosphate (PP) surrogate is connected to the hydrophobic farnesyl group by an amide linker, was found to be a potent (I50(FPT) = 75 nM) and selective inhibitor of FPT, as evidenced by its inferior activity against squalene synthetase (I50(SS) = 516 microM) and mevalonate kinase (I50(MK) = > 200 microM). A systematic structure-activity relationship study involving modifications of the farnesyl group, the amide linker, and the PP surrogate of 3 was undertaken. Both the carboxylic and phosphonic acid groups of the beta-carboxylic phosphonic acid PP surrogate are essential for activity, since deletion of either group results in 50-2600-fold loss in activity (6-9, I50 = 4.6-220 microM). The farnesyl group also displays very stringent requirements and does not tolerate one carbon homologation (12, I50 = 17.7 microM), substitution by a dodecyl fragment (14, I50 = 9 microM), or introduction of an extra methyl group at the allylic position (18, I50 = 55 microM). Modifications around the amide linker group of 3 were more forgiving, as evidenced by the activity of N-methyl analog (21, I50 = 0.53 microM), the one carbon atom shorter farnesoic acid-derived retroamide analog (32, I50 = 250 nM), and the exact retroamide analog (49, I50 = 50 nM). FPP analogs such as 3, 32, and 49 are novel, potent, selective, small-sized, nonpeptidic inhibitors of FPT that may find utility as antitumor agents.

Microsomal triglyceride transfer protein. Specificity of lipid binding and transport.

Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of very low density lipoproteins by the liver and chylomicrons by the intestine. To further elucidate the nature of the lipid molecule binding and transport site on MTP, we have studied the relative rates at which MTP transports different lipid species. Assay conditions were chosen in which there were minimal changes in the physical properties of the substrate membranes so that transfer rates would reflect MTP-lipid interactions at a membrane surface. Lipid transport rates decreased in order of triglyceride > cholesteryl ester > diglyceride > cholesterol > phosphatidylcholine. Changes in the hydrophobic nature of a lipid molecule by the addition of a fatty acid, modulated the ability of MTP to transport it. Addition of one acyl chain from diglyceride to triglyceride, lysophosphatidylcholine to phosphatidylcholine, or cholesterol to cholesteryl ester increased the rate of MTP-mediated transport 10-fold. In contrast, the lipid transport rate was insensitive to the changes in the structure or charge of the polar head group on phospholipid substrates. Zwitterionic, net negative, or net positive charged phospholipid molecules were all transported at a comparable rate. The ability of MTP to transport lipids is strongly correlated to the binding of these lipids to MTP. Thus, MTP has a specific preference for binding and transporting nonpolar lipid compared with phospholipids, and within a class of lipid molecules, a decrease in polarity increases its tendency to be transported.