Malaria kills at least one million people each year, mostly children - a death every 30 seconds. Almost one half of the world population is at risk from malaria. Antimalarial drugs are the only means for the treatment of about 500 million annual global malaria cases. Because of prevalent drug-resistance it is extremely urgent to identify new drug targets. Many proteins involved in eukaryotic signal transduction and cell cycle progression undergo post-translational lipid modification by a prenyl group. Protein prenyltransferases, which catalyze the post-translational prenyl modification, have been established as a target for anticancer therapy. Research done in our laboratory has demonstrated recently that prenyl modification of proteins could be a novel target for the development of antimalarial drugs.The goal of this study is to understand the molecular mechanism of protein prenylation in Plasmodium. The key to use of prenyltransferase inhibitors for the pharmacological intervention is a thorough understanding of the in vivo prenylation pathways in the malaria parasite. Knowledge of the physiological functions of the cellular protein substrates of malarial prenyltransferases is an important first step in the elucidation of the mechanism of antimalarial action of inhibitors of protein prenylation. The research described in this thesis revealed the evidence for the existence of farnesylated and geranylgeranylated malaria parasite proteins. The study shows that the dynamics of protein prenylation changes with the intraerythrocytic development cycle of the parasite. We detected that prenylated proteins in the 50 kDa range were mostly farnesylated and that the proteins in the 22-25 kDa range were mostly geranylgeranylated. The prenylation of P. falciparum proteins is inhibited by prenyltransferase inhibitors. We have also demonstrated unique features of protein prenylation in P. falciparum compared to the human host such as farnesylation of proteins are sensitive to inhibition by geranylgeranyltransferase inhibitors.. In-silico search of the malarial genome sequence identified potential protein prenyltransferase substrates. One of these substrates is a SNARE protein Ykt6 homologue. The malarial Ykt6 was recombinantly expressed and subjected to an in-vitro prenylation assay. We showed that the recombinant Ykt6 was indeed a substrate for the malarial prenyltransferase.

The isoprenoid biosynthetic pathway (IBP) has been highly implicated in a number of cellular malignancies, including proliferation, invasion, and migration. Epidemiological studies have found clinically relevant inhibitors of the IBP, such as the statin family and nitrogenous bisphosphonates, reduce the risk of prostate cancer advancement. In vitro work has implicated statin's and nitrogenous bisphosphonate's inhibition of GGPP and protein geranylgeranylation as the components responsible for their reduction of prostate cancer progression. However, their depletion of nearly all isoprenoid intermediates as well as their organ specificities make understanding the specific role of protein geranylgeranylation in prostate cancer metastasis impossible. Consequently, we have developed a novel library of seven alkyl bisphosphonate ethers found to potently reduce GGDPS with little to no activity against the related FDPS enzyme. Inhibition of GGDPS in three human prostate cancer cell lines reduced GGPP and protein geranylgeranylation without affecting protein farnesylation, translating into a reduction in cell migration and invasion. Interestingly, the GGDPS inhibitors reduced protein geranylgeranylation at lower concentrations in the highly metastatic PC3 cell line as compared to the less metastatic LNCaP and 22Rv1 cell lines. Additionally, the PC3 cell line was found to have higher levels of endogenous IBP intermediates as compared to the less metastatic cells. Translation in vivo using two murine models of human prostate cancer metastasis found a reduction in soft tissue tumor burden that corresponded to a biochemical reduction in protein geranylgeranylation. In conclusion, selective reduction of GGPP and protein geranylgeranylation was sufficient to reduce the metastatic potential of prostate cancer in vitro and in vivo.

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Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.