Although much attention has been directed to the antiviral properties of the HMG-CoA reductase inhibitors, inhibition of the enzyme that immediately precedes it in the mevalonate pathway, HMG-CoA synthase, also inhibits HCV replication [5
]. Other investigators have demonstrated that F-244 (L-659,699), a small molecule inhibitor of HMG-CoA synthase [21
], inhibits HCV replication [5
]. Herein, we report that ceestatin, a novel inhibitor of HCV replication identified in a compound screen [11
], also inhibits HMG-CoA synthase. Furthermore, by restoring intermediates in the mevalonic acid pathway found downstream of HMG-CoA synthase, we were able to eliminate ceestatin’s anti-HCV effects and rescue HCV replication. Ceestatin, therefore, probably exerts its antiviral effects via HMG-CoA synthase inhibition.
One potential drawback of using small molecules as probes to examine biologic phenomena is that off-target effects may account for a portion of their activity. Although it is possible that ceestatin may act on other unknown targets, both our rescue and siRNA experiments strongly suggest that HMG-CoA synthase inhibition alone is sufficient to inhibit HCV replication. In fact, HMG-CoA synthase inhibition via siRNA knockdown inhibits HCV replication as much as the knockdown produced by siRNA against HMG-CoA reductase, indicating that both act as equivalent antiviral host targets.
The mechanism by which inhibition of HMG-CoA synthase impairs HCV replication remains unknown. One possible hypothesis is based on observations noted in studies of the anti-HCV properties of the statins. HMG-CoA reductase inhibitors shut down cholesterol biosynthesis by preventing the formation of mevalonate from 3-hydroxy-3-methyl-glutaryl CoA. In addition to lowering intracellular levels of sterols, statins also reduce levels of isoprenoids, which are derived from mevalonate. Isoprenoids such as geranylgeranyl pyrophosphate serve as lipid attachments for a variety of intracellular signaling molecules. It has been reported that inhibition of geranylgeranylation, rather than the synthesis of cholesterol itself, is responsible for the inhibition of HCV RNA replication [3
]. In fact, the anti-HCV effect of the statins was reversed by addition of geranylgeraniol, but not by farnesol or cholesterol [3
]. Because the HCV genome does not encode a geranylgeranylated protein, it is hypothesized that a host geranylgeranylated protein, such as FBL2 [22
], must play an important role in HCV replication and that inhibition of the geranylgeranylation of this protein represents a potential strategy for blocking HCV replication. Thus, for statins to exert their anti-HCV effects, they must deplete mevalonate sufficiently to lower the cellular pools of geranylgeranyl pyrophosphates [3
]. Because HMG-CoA synthase immediately precedes HMG-CoA reductase in the mevalonate pathway, one can logically infer that the impairment of HCV replication resulting from its inhibition is mediated by the same mechanisms by which the statins exert their anti-HCV effects. Indeed, the anti-HCV effects of ceestatin were reversed by the addition of geranylgeraniol, but not by the addition of either farnesol or cholesterol, as has been observed previously with the statins (). These findings further underscore the importance of maintaining cellular pools of geranylgeraniol for HCV replication.
No general algorithm currently exists to identify the targets of novel uncharacterized anti-HCV compounds. We employed a systematic process to uncover the mechanism of action of ceestatin. Our paradigm, a stepwise logical method, begins with a broad assessment of activity against both specific viral enzymatic targets (through in vitro enzymatic assays) and other possible nonenzymatic viral targets (through sequence evolution analysis). Should the compound in question not act on a viral target, affinity chromatography would then be used to identify potential host targets, followed by the appropriate confirmatory biochemical assays for target validation. Following this paradigm, we first eliminated potential viral targets before embarking on the more technically challenging, but ultimately successful, search for the host target, HMG-CoA synthase.
In summary, we have found that ceestatin exerts its anti-HCV effects through inhibition of the host cofactor HMG-CoA synthase. Ceestatin may therefore prove to be useful not only as an antiviral agent, but also as a cholesterol-lowering agent. Furthermore, it also can be used as a small molecule probe to further define the relationship between HMG-CoA synthase, the mevalonate pathway, and HCV replication. Indeed, we have provided further evidence supporting the hypothesis that inhibition of the mevalonic acid pathway leads to depletion of cellular pools of geranylgeraniol necessary for HCV replication. Finally, the logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to reveal the targets on which novel uncharacterized anti-HCV compounds act. Our discovery and characterization of the novel anti-HCV compound ceestatin strongly links basic research in chemical biology to an ongoing problem in the practice of medicine, thereby representing a paradigm of the power of translational medicine.