Novel bisphosphonates as inhibitors of isoprenoid biosynthesis

Products of the isoprenoid biosynthetic pathway are involved in diverse biological functions. For example, the isoprenoid diphosphate, farnesyl diphosphate (FPP), is used for synthesis of squalene, a precursor of cholesterol. In addition, FPP and geranylgeranyl diphosphate (GGPP) are used for protein prenylation, which is a post-translational modification of certain proteins required for their proper membrane localization and function. Enzymes within the isoprenoid biosynthetic pathway have been inhibited successfully by drugs that are now used clinically, including statins and nitrogenous bisphosphonates (NBPs). Statins and NBPs are inhibitors of isoprenoid biosynthetic enzymes, due to their structural resemblance to substrates within the pathway. The bisphosphonate core resembles the diphosphate portion of isoprenoid diphosphate intermediates within the isoprenoid biosynthetic pathway. It is hypothesized that distinct isoprenoid biosynthetic enzymes can be inhibited by bisphosphonates in a manner dependent upon the overall bisphosphonate structure.

Along with our collaborators, we have developed novel bisphosphonate inhibitors of multiple isoprenoid biosynthetic enzymes. Potent in vitro inhibitors of squalene synthase (SQS) were identified and evaluated in HepG2 liver cells. A lead inhibitor of squalene synthase was combined with a statin and a nitrogenous bisphosphonate, and focus was placed on these combinations as potential novel mechanisms to reduce cholesterol synthesis while minimizing impairment of non-sterol synthesis. Specifically, it was found that the lead SQS inhibitor prevents lovastatin-mediated impairment of protein farnesylation but not geranylgeranylation. Also, the lead SQS inhibitor prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation.

Novel bisphosphonates were also identified as inhibitors of geranylgeranyl diphosphate synthase (GGDPS) and protein prenylation in K562 leukemia cells. A novel cellular consequence of GGPP depletion was also established. In PC3 cells, zoledronate and digeranyl bisphosphonate (DGBP; a lead inhibitor of GGDPS) were determined to induce autophagy as measured by accumulation of the autophagic marker LC3-II. GGPP depletion was implicated as the cause of autophagic induction in this system. Specifically, results suggest that impairment of proteins geranylgeranylated by geranylgeranyl transferase II is responsible for the induction of autophagy.

Mycobacterium isoprenoid biosynthetic enzymes were also evaluated as inhibitory targets for bisphosphonates. Novel inhibitors of Mycobacteria tuberculosis omega-E,Z-FPP synthase and decaprenyl diphosphate synthase were identified. A lead inhibitor of decaprenyl diphosphate synthase was also evaluated in Mycobacterium smegmatis, which was utilized as a surrogate model. The lead inhibitor was found to have no effect on M. smegmatis growth; however it enhanced growth inhibition mediated by ethambutol. This effect was prevented by addition of exogenous decaprenyl diphosphate, suggesting that the growth inhibition was due to decaprenyl diphosphate depletion. Decaprenyl diphosphate was also found to prevent the growth inhibitory effect of SQ109, a novel anti-mycobacterial drug in clinical development with an unknown mechanism of action.