Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major.

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.

Protein farnesyl and N-myristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics.

To accelerate progress in the development of therapeutics for protozoan parasitic diseases, we are studying enzymes active in co- and post-translational protein modification that are already the focus of drug development in other eukaryotic systems. Inhibitors of the protein farnesyltransferases (PFT) are well-established antitumour agents of low cytotoxicity and known pharmokinetic properties, while inhibitors of N-myristoyl transferase show both selectivity and specificity in the treatment of fungal infections. Here, we summarise the current evidence that supports the targeting of these ubiquitous eukaryotic enzymes for drug development against trypanosomatid infections and malaria.

Naturally occurring, tumor-specific, therapeutic proteins.

The emerging approach to cancer treatment known as targeted therapies offers hope in improving the treatment of therapy-resistant cancers. Recent understanding of the molecular pathogenesis of cancer has led to the development of targeted novel drugs such as monoclonal antibodies, small molecule inhibitors, mimetics, antisense and small interference RNA-based strategies, among others. These compounds act on specific targets that are believed to contribute to the development and progression of cancers and resistance of tumors to conventional therapies. Delivered individually or combined with chemo- and/or radiotherapy, such novel drugs have produced significant responses in certain types of cancer. Among the most successful novel compounds are those which target tyrosine kinases (imatinib, trastuzumab, sinutinib, cetuximab). However, these compounds can cause severe side-effects as they inhibit pathways such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor, which are also important for normal functions in non-transformed cells. Recently, a number of proteins have been identified which show a remarkable tumor-specific cytotoxic activity. This toxicity is independent of tumor type or specific genetic changes such as p53, pRB or EGFR aberrations. These tumor-specific killer proteins are either derived from common human and animal viruses such as E1A, E4ORF4 and VP3 (apoptin) or of cellular origin, such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and MDA-7 (melanoma differentiation associated-7). This review aims to present a current overview of a selection of these proteins with preferential toxicity among cancer cells and will provide an insight into the possible mechanism of action, tumor specificity and their potential as novel tumor-specific cancer therapeutics.

Functional analysis of TbARL1, an N-myristoylated Golgi protein essential for viability in bloodstream trypanosomes.

Myristoyl-CoA:protein N-myristoyltransferase (NMT), an essential protein in Trypanosoma brucei and Leishmania major, catalyses the covalent attachment of the fatty acid myristate to the N-terminus of a range of target proteins. In order to define the essential targets contributing to lethality in the absence of NMT activity, we have focused on the ADP-ribosylation factor (Arf) family of GTP-binding proteins, as growth arrest in Saccharomyces cerevisiae mutants with reduced NMT activity correlates with a decrease in N-myristoylated Arf proteins. We have identified nine Arf/Arls in the T. brucei and T. cruzi genomes and ten in L. major. Characterization of the T. brucei ARL1 homologue has revealed that the protein is localized in the Golgi apparatus and is expressed only in the mammalian bloodstream form of the parasite and not in the insect procyclic stage. This is the only reported example to date of a differentially expressed ARL1 homologue in any species. We have used RNA interference to demonstrate that ARL1 is essential for viability in T. brucei bloodstream parasites. Prior to cell death, depletion of ARL1 protein in bloodstream parasites results in abnormal morphology, including disintegration of the Golgi structure, multiple flagella and nuclei, and the presence of large numbers of vesicles. The cells have only a minor apparent defect in endocytosis but exocytosis of variant surface glycoprotein to the parasite surface is significantly delayed. RNA interference of ARL1 in procyclic cells has no effect on parasite growth or morphology. Our results suggest that there may be different pathways regulating Golgi structure and function in the two major life cycle stages of T. brucei.

Myristoyl-CoA:protein N-myristoyltransferase, an essential enzyme and potential drug target in kinetoplastid parasites.

Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.