Research paperInhibition of trypanosome alternative oxidase without its N-terminal mitochondrial targeting signal (ΔMTS-TAO) by cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde derivatives active against T. brucei and T. congolense
Graphical abstract
Introduction
Alternative oxidases (AOXs), which are found across a broad range of organisms, including plants, nematodes, algae, yeast and certain disease–causing microorganisms including Trypanosoma spp., are mitochondrial, cyanide–insensitive, membrane-bound proteins that catalyse the oxidation of ubiquinol and the four-electron reduction of oxygen to water [1]. In T. brucei, a parasite that causes African trypanosomiasis in humans (sleeping sickness) [2] and in livestock (nagana) [3] throughout sub-Saharan Africa, the trypanosome alternative oxidase (TAO) is essential for the respiration of bloodstream form (BSF) parasites. In effect, in BSF trypanosomes, TAO is the sole terminal oxidase enzyme to re-oxidize the NADH that accumulates during glycolysis, and, as TAO has no counterpart in mammalian cells and is conserved among T. brucei subspecies [4], it has been validated as a promising target for the chemotherapy of African trypanosomiasis [[5], [6], [7]].
TAO is a cyanide-resistant and cytochrome-independent ubiquinol oxidase, formerly known as glycerol-3-phosphate oxidase, which is sensitive to the specific inhibitors salicylhydroxamic acid (SHAM) and ascofuranone (AF) [[8], [9], [10], [11]]. Recently, we showed that dihydroxybenzoates and salicylhydroxamates could be efficiently targeted to the T. brucei mitochondrial matrix, by coupling them to a lipophilic cation [12]. A preliminary assessment of their antitrypanosomal activity found that some of these compounds appeared to inhibit TAO, which inspired the current strategy of synthesizing a small library of analogues optimized for (a) mitochondrial import and (b) TAO inhibition. As such, a series of 4-Hydroxybenzoate and 4-Alkoxybenzaldehyde derivatives was attached to triphenylphosphonium (TPP) and to quinolinium lipophilic cations, through linkers of variable length that would allow optimal engagement with the TAO binding pocket.
Another issue we addressed for the first time is that previous efforts to screen for, and optimize TAO inhibitors have used a non-physiological version of recombinant TAO that retains the N-terminal Mitochondrial Targeting Sequence (MTS) [13], despite its relatively poor stability and solubility, and its low yield [14]. The AOX gene of T. brucei contains 990 nucleotides, encoding the 330 amino acids full length protein, which includes the MTS. This sequence was predicted to be 25 amino acids long, using the computer program MITOPROT (http://www.expasy.org/tools/). As the MTS is cleaved off after transportation of the protein into the mitochondrion, physiologically functional and relevant form of TAO is lacking the MTS sequence [15].
Therefore, in the present study, we report for the first time the production of recombinant TAO enzyme in its more active, physiological state without the N-terminal MTS sequence (ΔMTS-TAO). We also report the novel use of a SUMO expression system to optimize the production of this protein. The ΔMTS-TAO enzyme was used to study the activity of new TAO inhibitors based on the 4-hydroxybenzoate and 4-alkoxybenzaldehyde scaffolds (Fig. 1). These compounds were designed as analogues of lead compound 1, a low micromolar TAO inhibitor with potent activity against African trypanosomes [12]. The results of rTAO inhibition analysis, trypanocidal activity against wild type and several multi-drug-resistant strains of trypanosomes, and metabolic stability in mouse serum allowed the production of structure-activity relationships (SAR) with these potent TAO inhibitors and the identification of strong candidates for in vivo studies and preclinical development. This is the first time that not only the problem of local drug concentration is addressed as part of the inhibitor-design for a mitochondrial target in a protozoan parasite, but that it has been demonstrated that inhibitors coupled to such targeting moieties still inhibit the intended target without loss of affinity, and with greatly improved anti-parasite activity and selectivity index.
Section snippets
Cloning and expression of trypanosome alternative oxidase without mitochondrial targeting sequence (ΔMTS-TAO)
PCR amplification of ΔMTS-TAO and full length (fl) rTAO gave bands on agarose gel corresponding to 915 bp and 990 bp, respectively (Fig. S1A), while the PCR amplification of these TAO genes from the pET101-NHis6SUMO plasmid, i.e. products NHis6SUMO-ΔMTS-TAO and NHis6SUMO-fl-TAO gave bands corresponding to 1.26 kb and 1.34 kb, respectively (Fig. S1B). These PCR products were confirmed by sequence analysis to be the correct DNA fragments, and in the correct orientation.
Plasmids with either the
Discussion
The TAO gene contains 990 nucleotides, encoding a protein of 330 amino acids including the N-terminal 25 amino acid residue Mitochondrial Targeting Signal (MTS). For the majority of mitochondrial proteins, their transport into the mitochondria relies on two key fundamentals: (i) the presence of an MTS in the protein sequence and (ii) the presence of specific translocators in the mitochondrial membrane domain that recognize the specific signals [27]. Three main types of MTS have been found in
Conclusion
The reported procedure for the high yield expression of ΔMTS-TAO that is substantially more soluble, stable, and active, than fl-TAO is a helpful tool for the development of new TAO inhibitor drug candidates. We have successfully developed a class of potent and selective new hits active against human (T. brucei spp.) and veterinary (T. congolense) African trypanosomes, and confirmed their designed mode of action as inhibition of TAO. This was accomplished by efficiently targeting the compounds
Chemistry
Anhydrous solvents were purchased to Aldrich/Fluka in SureSeal™ bottles and used as received. Thin Layer chromatography (TLC) was performed on silica gel 60 F254 aluminum TLC plates (MERCK). Medium pressure silica chromatography was performed on a FlashMaster Personal system using FlashPack SI prepacked columns (2, 5, 10, 20, and 50 g). Melting points were measured with a Reichert-Jung Thermovar apparatus and are uncorrected. LC-MS spectra were recorded on a WATERS apparatus integrated with a
Acknowledgements
This work was funded by the Spanish Ministerio de Economia y Competitividad (SAF2015-66690-R). This investigation also received financial support (ID No. B40103 to EOB) from TDR, the Special Programme for Research and Training in Tropical Diseases (co-sponsored by UNICEF, UNDP, the World Bank and WHO). G. U. Ebiloma was supported by a TET-Fund studentship from the government of Nigeria and by a Mac Robertson Travel Scholarship from the College of Medical, Veterinary and Life Sciences of the
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These authors contributed equally.