Apomine, a Novel Hypocholesterolemic Agent, Accelerates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase and Stimulates Low Density Lipoprotein Receptor Activity

Abstract

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterolregulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.In mammalian cells, cholesterol homeostasis is maintained by balancing cholesterol uptake and production. Cholesterol uptake is regulated through modulating the levels of the cell surface low density lipoprotein (LDL) 1 receptor (LDLR), and cholesterol synthesis is regulated primarily by changes in levels and activity of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (1, 2). HMGR is an endoplasmic reticulum (ER) enzyme that catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA into mevalonate (MVA), the first committed step in the MVA pathway that leads to the synthesis of cholesterol as well as of essential non-sterol isoprenoid compounds (1, 2). When cells are starved for cholesterol and/or MVA, levels of HMGR and LDLR are elevated, thus increasing the rate of endogenous sterol synthesis and of LDL uptake (3, 4). Conversely, in cholesterol-replete cells, the levels of HMGR and LDLR decline, thereby lowering sterol production and LDL internalization.HMGR and LDLR are regulated at the transcriptional level by sterol-regulatory elements (SREs) in the promoter of their genes (1, 5). Specific transcription factors, designated SREbinding proteins (SREBPs), bind to these elements and activate transcription (5, 6). The SREBP family comprises three members that control different sets of genes. SREBP-2 regulates the transcription of genes mainly involved in sterol synthesis, whereas SREBP-1a and -1c regulate principally fatty acid biosynthetic genes (7). The nuclear, transcriptionally active SREBPs are derived from the NH 2 -terminal domain of large ER membrane-bound prec...

Apomine™, an inhibitor of HMG‐CoA‐reductase, promotes apoptosis of myeloma cells in vitro and is associated with a modulation of myeloma in vivo

“…Its actions differ from statins since it mediates its effects by increasing the rate of degradation of HMG-CoA reductase, rather than by inhibiting the enzyme directly. 26 The present study has demonstrated that Apomine is able to induce a direct anti-myeloma effect by inducing myeloma cell apoptosis in a concentration-dependent manner. This supports recent reports that showed that Apomine could also induce apoptosis in several tumor cell lines including HL60 leukemia cells and, MCF-7 and MDA-MB-231 breast cancer cells.…”

“…25,26 Apomine has also been identified as an activator of the farnesoid X receptor suggesting it may have alternative effects, independent of the mevalonate pathway, as observed in breast cancer cells. 25,27 Apomine does differ in its mechanism of action from the other inhibitors of HMG-CoA reductase, the statins, in that it is not a competitive inhibitor.…”

Sterol-induced dislocation of 3-Hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

“…In cultured cells, the 1,1-bisphosphonate esters Apomine and SR-12813 mimic 25-HC in stimulating Insig-mediated ubiquitination and subsequent degradation of reductase in intact cells (Roitelman et al, 2004;Sever et al, 2004). Having optimized the assay for sterol-induced dislocation of reductase in permeabilized cells using either ubiquitin aldehyde or USP2-cd (Figure 4 The results of Figure 4A are consistent with a scenario in which removal of polyubiquitin chains from dislocated reductase enhanced detection of nonubiquitinated, full-length reductase in the supernatant of permeabilized cells.…”

Population pharmacokinetics of APOMINE™: A meta‐analysis in cancer patients and healthy males

“…One of its unique mechanisms of action is to decrease cholesterol synthesis by accelerating HMG-CoA reductase degradation [1]. Another action is the activation of the farnesoid nuclear receptor (FXR), which has been implicated in inducing apoptosis [2].…”