Novel bisphosphonates as inhibitors of isoprenoid biosynthesis

Statins and nitrogenous bisphosphonates (NBPs) are clinically used inhibitors of the mevalonate pathway, which is the pathway responsible for cholesterol production as well as other isoprenoid-derived molecules. Through the inhibition of HMGCR and FDPS, respectively, these drugs deplete cells of FPP and can disrupt downstream cellular processes such as protein prenylation. At the major branch point of the mevalonate pathway, SQS utilizes FPP in the first committed step toward cholesterol biosynthesis. In these studies, novel bisphosphonates are identified as potent and specific inhibitors of SQS, including 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1bisphosphonic acid, tetrasodium salt (compound 5; Figure 8). Compound 5 reduced cholesterol biosynthesis, did not effect cell viability, and lead to a substantial intracellular accumulation of FPP in HepG2 cells. At high concentrations, lovastatin and zoledronate impair protein prenylation and are cytotoxic, limiting their use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced depletion of FPP levels and protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation. Co-treatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly ameliorate the reduction of cell 16 viability activity caused by treatment with lovastatin or zoledronate alone. The combination of an SQS inhibitor with HMGCR or FDPS inhibitors provides a rational approach for reducing cholesterol synthesis, while maintaining non-sterol isoprenoid biosynthesis closer to basal levels than treatment with single inhibitors. Introduction Squalene synthase (SQS) is responsible for the first committed step from the isoprenoid biosynthetic pathway toward cholesterol biosynthesis. SQS is a membraneassociated enzyme that localizes to the endoplasmic reticulum (57). SQS catalyzes the reductive dimerization of two units of FPP in a head-to-head orientation in a two step reaction to form squalene (23). In the first step of the reaction, pre-squalene diphosphate is formed, which is then converted to squalene in an NADPH-dependent second step (Figure 5). The structure of SQS is folded as a single domain and is entirely α-helical with two active sites located in a central channel that is lined by two DDXXD motifs containing conserved aspartate residues (58). These residues associate with multiple Mg ions that stabilize binding to the diphosphate portion of FPP, while the hydrophobic portion of FPP extends into a hydrophobic channel (58). Inhibitors containing bulky hydrophobic groups were characterized in a complex with SQS, and the hydrophobic groups were in proximity of the side chains of Phe and Tyr within an inhibitor-binding cavity (58). Statins inhibit HGMCR and impair cholesterol biosynthesis, which causes an upregulation of the LDL receptor in the liver resulting in clearance of cholesterolcontaining LDL particles from the bloodstream. Statins are widely used because elevated LDL levels and total cholesterol are major risk factors for coronary heart disease (31). 17 While the statins are used abundantly and effectively, there are various reasons for developing novel inhibitors of cholesterol biosynthesis. For example, there are sideeffects associated with statin use such as myopathy and hepatotoxicity (59), which are commonly speculated to be due to the depletion of non-sterol components of the mevalonate pathway (60). Furthermore, statin use does not always reduce LDL to desired levels (61), which is particularly important as lower LDL target levels are suggested for some patients (62,63). Inhibition of SQS has drawn much interest as a pharmacological target, and various molecules have been identified as inhibitors (49,50). Lapaquistat (TAK-475, Takeda) progressed to phase III clinical trials, but these trials were discontinued after the U.S. Food and Drug Administration (FDA) recommended suspension of studies with the high dose (100 mg/kg) mono-therapy due to cases of elevated blood levels of liver transaminases, a commonly used measure of hepatotoxicity (50). It is currently unknown whether this was due to inhibition of SQS per se, or if it was specific to the drug. Inhibition of SQS can result in the accumulation of FPP metabolites, such as farnesolderived dicarboxylic acids (64), which could be responsible for the possible hepatotoxicity with the high-dose monotherapy. Farnesol itself can also be pro-apoptotic at high concentrations (65). Other reported results appeared promising with lapaquistat, with cholesterol levels decreased in monotherapy treatment. Moreover, the combination therapy with statins showed additional LDL reduction compared to statins alone (50). Also of interest, T-91485 (the active metabolite of lapaquistat) was capable of preventing statin-induced myotoxicity in a human skeletal muscle cell model (66). Similarly, lapaquistat prevented statin-induced myotoxicity in a guinea pig model (67). In addition 18 to the expected cholesterol depletion, other SQS inhibitors have shown the potential for added benefits due to decreased triglyceride biosynthesis (68), likely resulting from a farnesol-induced mechanism (69). Nitrogenous bisphosphonates (e.g., zoledronate, alendronate) are another class of clinical drugs targeting the mevalonate pathway. These compounds inhibit FDPS and deplete FPP and other downstream isoprenoids (40). Although these compounds have a high affinity for bone, there are also reports of decreased cholesterol levels with patients treated with nitrogenous bisphosphonates (70). The combination of FDPS inhibitors with SQS inhibitors has not been evaluated. While various SQS inhibitors exist, relatively few are based on a bisphosphonate structure (71), and specificity for SQS relative to the prenylation arm of the mevalonate pathway has not been reported. Herein is described the identification of novel bisphosphonates as potent inhibitors of SQS. A structure activity relationship (SAR) was evaluated in the context of potency and specificity for these novel compounds. Emphasis was placed on the evaluation of a lead compound (5) in combination with lovastatin or zoledronate in HepG2 cells. These combinations of inhibitors are hypothesized to decrease cholesterol biosynthesis while preventing the depletion of non-sterol isoprenoid levels, resulting in less adverse cellular effects compared to inhibition of HMGCR or