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

Abstract

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 myristoylation in health and disease

“…NMT enzymes have been characterised in L. major [32] (which causes leishmaniasis), T. brucei (responsible for African sleeping sickness) [32] and P. falciparum (the parasite causing most cases of malaria in humans) [25]. Several studies have sought to identify selective inhibitors of the NMTs in these organisms [26,113,114]. Another potential target for NMT inhibitors is HIV-1 infection.…”

“…It appears to be ubiquitous in eukaryotes and has been isolated and characterised in yeast and fungi (Candida albicans, Saccharomyces cerevisiae [22], Cryptococcus neoformans [23] and Aspergillus nidulans [24]), parasitic protozoa (plasmodium species [25], Leishmania major [26] and, Trypanosoma brucei [26]), insects (Drosophila melanogaster [27]), plants (Arabidopsis thaliana [28]) and mammals (including mouse, rat, cow and human). NMT has been shown to be essential for the survival of S. cerevisiae [29], C. albicans [30], C. neoformans [31] and the bloodstream forms of the parasites L. major and T. brucei [32].…”

Crystal Structures of Saccharomyces cerevisiae N-Myristoyltransferase with Bound Myristoyl-CoA and Inhibitors Reveal the Functional Roles of the N-terminal Region

“…These properties make NMT an attractive therapeutic target for antifungal agents (19 -21) designed to occupy the peptide substrate-binding site. In addition, because of its essential role in cell viability, NMT is a potential target for antiviral, antiparasitic, and even antineoplastic chemotherapy (18,(22)(23)(24). Thus, understanding the structural basis of the recognition and binding of the substrates with the enzyme should be useful for the development of new therapeutic agents.…”

“…Based on this preference, a large number of peptide-based and peptide-like inhibitors that are effective against C. albicans and C. neoformans (19 -21, 31) and that have antiparasitic activity (24) have been developed. Because of the limitations of peptidebased therapeutics, however, alternative small non-peptidic inhibitors have been screened, and some have been identified with very high activity and selectivity against NMTs of pathogenic fungal species (26,32,33).…”

N-myristoyltransferase in the leukocytic development processes

“…The N-terminal glycine myristoylation of various key proteins is necessary for normal cell functioning, and thus NMT is essential for survival and growth in a number of organisms (Duronio et al 1989;Lodge et al 1994;Weinberg et al 1995;Boutin 1997;Wright et al 2010). In organisms in which a single NMT isoform exists, the targeting of the endogenous NMT functions has been the candidate of choice for the treatment of many human pathogenic states with a focus on developing species-specific NMT inhibitors as anti-fungal, antiparasitic, and antiviral agents (Duronio et al 1991;Sikorski et al 1997;Lodge et al 1998;Georgopapadakou 2002;Price et al 2003;Gelb et al 2003;French et al 2004;Panethymitaki et al 2006;Bowyer et al 2007;Brannigan et al 2010;Frearson et al 2010). In the higher eukaryotes, the two isoforms NMT1 and NMT2 have overlapping but distinct substrate specificities (Giang and Cravatt 1998;Ducker et al 2005).…”