N-myristoyltransferases inhibitory activity of ellagitannins from Terminalia bentzoë (L.) L. f. subsp. bentzoë

Abstract

N-myristoylation (Myr) is an eukaryotic N-terminal co- or post-translational protein modification in which the enzyme N-myristoyltransferase (NMT) transfers a fatty acid (C14:0) to the N-terminal glycine residues of several cellular key proteins. Depending on the cellular context, NMT may serve as a molecular target in anticancer or anti-infectious therapy, and drugs that inhibit this enzyme may be useful in the treatment of cancer or infectious diseases. As part of an on-going project to identify natural Homo sapiens N-myristoyltransferase 1 inhibitors (HsNMT1), two ellagitannins, punicalagin (1) and isoterchebulin (2), along with eschweilenol C (3) and ellagic acid (4) were isolated from the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë. Their structures were determined by means of spectroscopic analyses and comparison with literature data. Punicalagin (1) and isoterchebulin (2) showed significant inhibitory activity towards HsNMT1, and also against Plasmodium falciparum NMT (PfNMT) both in vitro and in cellulo, opening alternative paths for new NMT inhibitors development. This is the first report identifying natural products from a botanical source as inhibitors of HsNMT and PfNMT.

Introduction

N-myristoylation consists in the co- and post-translational attachment of myristate, a 14-carbone fatty acid, to the N-terminal glycine residue of various eukaryotic and viral proteins [[1], [2], [3]]. This modification is catalysed by the enzyme myristoyl-CoA: protein N-myristoyltransferase (NMT) and is crucial for biological functions. N-myristoylation contributes to protein stability, protein-protein and protein-membranes interactions and affects number of key proteins involved in signaling pathways, including those of apoptosis [[4], [5], [6]]. NMT activity, which is also essential for growth and cellular proliferation, was shown to be upregulated in breast, lung, ovarian and colorectal cancers, gallbladder carcinoma and brain tumors [7,8]. As a result, NMT inhibition has been suggested as a therapeutic strategy for cancer treatments [9,10]. To date, the most potent HsNMT inhibitors were found to be sulfonamide derivatives with IC₅₀ values in the nanomolar to low micromolar range [7,11].

Given NMT activity requirement for the survival of several human pathogens, this enzyme is also considered as a promising antiviral [[12], [13], [14]], antifungal [15], and anti-parasitic [[16], [17], [18]] drug target. To identify NMT inhibitors, various approaches have been used including rational design or screening followed by medicinal chemistry approaches [11]. Although a common core sequence is recognizable, unicellular eukaryote NMTs including that from parasites display some differences [19] allowing specific recognition and selectivity of the peptide binding pockets. However, only a dozen of inhibitory compounds have been reported on parasite NMTs [11] and none of them, even the most potent ones, display great selectivity and/or therapeutic effects [20]. The available crystal structures of the NMT/inhibitors complexes and comparison of key residues identified for binding and catalysis seem indeed to suggest that it would be difficult to develop specific inhibitors focusing only on those residues and that new NMT inhibitors should target other regions than the classical peptide binding pocket.

In order to identify new natural inhibitors of HsNMT1, a systematic in vitro evaluation using an enzymatic assay developed by Traverso et al. [19] was conducted on 2700 EtOAc extracts prepared from various parts of approximately 1250 tropical plant species. The EtOAc extract of the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë displayed a significant HsNMT1 inhibitory activity and therefore was selected for further chemical investigation. The activity of its MeOH extract was also evaluated and was shown to be interesting too. The bio-guided fractionation of both extracts led to the isolation of punicalagin (1), isoterchebulin (2), eschweilenol C (3) and ellagic acid (4).

Comprising slightly over a hundred of species [21], the genus Terminalia is one of the largest of the Combretaceae family. Most of them occur in tropical regions of Asia, Australia and Africa. Plants of this genus have been extensively used for their medicinal properties by traditional healers. A broad range of therapeutic activities has been reported in the literature and reviewed by Cock [22]. Terminalia species have shown anti-infective, antidiarrheal, analgesic, antioxidant, anti-inflammatory, anticancer and antidiabetic activities as well as wound healing and cardiovascular effects. Most of the constituents isolated from these species are triterpenoids, flavonoids and tannins. Terminalia bentzoë (L.) L. f. subsp. bentzoë is a species endemic to Reunion and Mauritius Islands [23]. Its ethnopharmacological use by natives of Reunion has been exhaustively described by Lavergne [24]. The plant is used for treating respiratory infections, malarial fever, diarrhea, dysentery, and for its sudorific, emmenagogic and depurative properties. The methanolic extract of the bark of T. bentzoë subsp. bentzoë was shown to possess antimalarial activity [25]. In the light of this latter property and of the Reunion traditional use of this species, compounds isolated from T. bentzoë subsp. bentzoë were also evaluated on Plasmodium falciparum N-myristoyltransferase. Assays were completed by an in cellulo evaluation on P. falciparum strains 3D7. Here we report the isolation, structure elucidation and biological evaluation of isolated compounds. Our results show that the identified active compounds present original scaffolds for further optimization of their NMT inhibitory potency against both HsNMT and/or protozoan parasite NMT.