Uptake of NO-releasing drugs by the P 2 nucleoside transporter in trypanosomes

Nitric oxide (NO·) has been identified as a principal regulatory molecule of the immune system and the major cytotoxic mediator of activated immune cells. NO can also react rapidly with a variety of biological species, particularly with the superoxide radical anion O2 at almost diffusion-limited rates to form peroxynitrite anion (ONOO-). ONOOand its proton-catalyzed decomposition products are capable of oxidizing a great diversity of biomolecules and can act as a source of toxic hydroxyl radicals. As a consequence, a strategy for the development of molecules with potential trypanocidal activities could be developed to increase the concentration of nitric oxide in the parasites through NO·-releasing compounds. In this way, the rate of formation of peroxynitrite from NO· and O2 would be faster than the rate of dismutation of superoxide radicals by superoxide dismutases which constitute the primary antioxidant enzymatic defense system in trypanosomes. The adenosine transport systems of parasitic protozoa, which are also in certain cases implicated in the selective uptake of active drugs such as melarsoprol or pentamidine, could be exploited to specifically target these NO·-releasing compounds inside the parasites. In this work, we present the synthesis, characterization and biological evaluation of a series of molecules that contain both a group which would specifically target these drugs inside the parasites via the purine transporter, and an NO-donor group that would exert a specific pharmacological effect by increasing NO level, and thus the peroxynitrite concentration inside the parasite. Correspondence


Introduction
South-American and African trypanosomiasis, in particular African sleeping sickness, a disease caused by parasitic protozoa of the Trypanosoma brucei subgroup, remains a major public health problem, and there is now a great need to develop drugs to replace those to which these parasites have become resistant (1).Protection against active oxygen species is provided in part by an enzymatic defense system which is essential for the survival of aerobic organisms and differs according to species.Both mammalian and protozoan enzymatic systems have in common superoxide dismutases (SODs) that catalyze the dismutation of superoxide radical into hydrogen peroxide and oxygen.
In addition to SODs, the protective mammalian enzymes are various hydroperoxidases such as glutathione peroxidase, catalase, and other hemoprotein peroxidases.In the absence of catalase, the antioxidant defense system in trypanosomes is weak, and essentially based on the presence of a spermidineglutathione conjugate named trypanothione, whose oxidative form is regenerated in its reduced dithiol form by an NADPH-dependent flavoprotein, trypanothione reductase (2).As a consequence, most of the trypanosomes, and parasitic protozoans in general, are susceptible to free oxygen radical-induced oxidative stress and do not tolerate a high concentration of oxygen.This vulnerability to reactive oxygen species could be exploited to design new drugs with trypanocidal activity.
Nitric oxide (NO • ), a key messenger implicated in a wide range of biological processes including cardiovascular (3-5) and neuronal (6) systems, also plays a critical role of protection against parasitic infections as a regulatory molecule and cytotoxic mediator of the immune system (7,8).For example, macrophages from Trypanosoma brucei brucei-infected mice have been shown to produce high levels of nitric oxide (9) and a number of reports have demonstrated that in vitro cytotoxicity against the intracellular form of leishmanias is mediated by NO • (10)(11)(12).Although few physiologic target molecules of NO have been clearly identified, its role in the protective mechanisms would occur through inactivation of critical enzymes and nitrosation of thiols and other nucleophilic residues (13)(14)(15)(16).NO • can also react rapidly with a variety of radical species, like superoxide radical anion O 2 • -.While O 2 • -itself is not an efficient oxidizing agent, together with NO • it can produce the more powerful oxidizing peroxynitrite anion ONOO -at an almost diffusion-limited rate (6.7 nM/s) (17) which depends on the concentrations of both radicals.Peroxynitrite and its proton-catalyzed decomposition prod-ucts are capable of oxidizing a great diversity of biomolecules (18,19) including hemecontaining proteins such as hemoglobin and myeloperoxidase, seleno-proteins such as glutathione peroxidase, DNA or lipids within the cell, or nitrating and nitrosating phenolic compounds such as tyrosines of certain proteins like SODs, and can act as a source of toxic hydroxyl radicals.Peroxynitrite anion, like NO • , seems to play a major role in the protective mechanisms of the host against parasitic infections, and, for example, has been shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes, the causal agent of Chagas disease, inactivating two key enzymes for their energetic metabolism, i.e., succinate dehydrogenase and NADHfumarate reductase (20,21).
Most of parasitic protozoa are unable to synthesize purines de novo and consequently must use specific transporters to obtain them from the hosts for their survival.The African trypanosome Trypanosoma brucei brucei that invades the central nervous system causing the fatal neurologic disorder known as sleeping sickness, possesses two adenosine transporter systems: a P1 type which also transports inosine, and a P2 type which is also able to transport adenine.Both systems have been shown to be implicated in the selective uptake of trivalent melaminophenyl arsenical drugs (22) such as melarsen oxide 1a and melarsoprol 1b (Figure 1), which are still the only drugs of choice for the treatment of the late stage of human African trypanosomiasis, and of pentamidine 2 (23), one of the most frequently administered drugs in the treatment of the early stage of the disease.Moreover, the nitroheterocyclic compound megazol 3 (Figure 1), that has been shown to be active against many microorganisms including Trypanosoma cruzi, might also act as a substrate for carrier protein P2, although the passive diffusion process would remain the major route of entry (Barrett MP, Fairlamb AH, Rousseau B, Perié J and Chauvière G, unpublished results).In view of the analo-Uptake of NO-releasing drugs by trypanosomes gies between adenine (or adenosine), benzamidine and melamine, it has been hypothesized that the amidine motif (N=C-NH 2 ) is the real structural feature for specific P2transporter recognition and uptake (24).
Trypanosoma equiperdum, a non-tsetsetransmissible strain, possesses a transport system similar to that of T. brucei, comprising two adenosine transporters, P1 and P2, the latter also transporting adenine and the melaminophenyl arsenical drug cymelarsen 1c (25) (Figure 1).
In the present paper, we describe the synthesis of a series of drugs 7a-7c which contain both a group that would specifically target these drugs into the parasite via the P2-transporter, and an NO • -donor group that would exert a specific pharmacological effect by increasing the level of NO, and thus the peroxynitrite concentration inside the parasite.The uncommon stability of Snitroso-N-acetylpenicillamine (SNAP) as a solid (26) or in solution suggests that penicillamine derivatives should be good canditates for new stable S-nitrosothiols and could function as useful nitric oxide-releasing compounds.We describe here the effect of melaminyl thionitrite 7a on adenosine transport by T. equiperdum.
Like SNAP compounds, 7a-c were stable as solids and presented a green color when in solution in organic or aqueous media which characterizes the presence of an S-nitroso group.They were fully characterized by mass spectrometry, 1 H/ 13 C-NMR and UV-visible spectroscopies.Decomposition studies were carried out by UV-visible spectroscopy by measuring the disappearance of the characteristic absorbance at 340 nm (e .800 M/cm).7a-c decomposed slowly in sodium phosphate buffers, pH 7.5, with a half-life between 2 and 3 h, comparable to the half-life of SNAP under the same conditions.

Biological evaluation
Cymelarsen 1c, SNAP and thionitrite 7a were tested on T. equiperdum E1 for their ability to inhibit the uptake of [2-3 H]adenosine via the transporter P2 in the presence of saturating concentration of inosine which was required to inhibit P1 transporter.The compounds were also tested for their in vitro toxicity on the same strain (LD 100 after 18 h).The data reported in Table 1 show that the melaminyl derivative 7a efficiently inhibits adenosine transport in T. equiperdum in the presence of inosine, suggesting a specific interaction with the P2 transporter with a K i of 0.5 µM which is equal to the K M values of adenosine that enters the parasite through both adenosine transporters P1 (K M = 0.6 µM) and P2 (K M = 0.7 µM).In comparison, the uptake of cymelarsen via P2 is less efficient (K i = 41 µM) and SNAP has no affinity for this transporter (K i >100 mM).

Discussion
Chemical reagents that release NO • under physiological conditions are good candidates to mimic the activity of NO-synthase (29), an NADPH-dependent flavo-hemoprotein that produces NO • from L-arginine in many types of cells by a two-step oxidation reaction.A potential therapeutic application lies in their possible use as vasodilators or as drugs for the treatment of angina, and a chemical application is their use as a depot for NO • gas which is difficult to handle due to its high reactivity and toxicity as a free radical.Naturally occurring thionitrites like S-nitroso-albumine (30) or S-nitrosoglutathione (31) are currently postulated to be carriers of NO • , but synthetic S-nitrosothiols are usually relatively unstable and spontaneously decompose in solution to yield quantitatively NO • and the corresponding thiyl radical, which dimerizes to give the disulfide, as primary products (32)(33)(34)(35).In spite of the presence of amino groups, the three thionitrites of this study, like the well-known SNAP, exhibit significant stability in the solid form and in solution to permit full structural characterization or a therapeutic use, and are capable to slowly generate NO • under physiological conditions with half-life times of several hours.
Conversion of penicillamine into its corresponding thietanone for coupling with  The thionitrite 7a, which possesses a P2 recognition motif, strongly inhibits the adenosine uptake by the transporter P2.Despite a specific interaction with this transporter, these data do not indicate if compound 7a is really transported by P2.Under the same conditions, the melaminophenyl arsenical drug cymelarsen that has been shown to enter through the trypanosomal P2 transporter, has a higher K i value, and SNAP does not compete with adenosine for transport.Both compound 7a and SNAP present a weak in vitro antiparasitic activity against Trypanosoma equiperdum which was quantitatively related to the amount of NO produced by the decomposition of these thionitrites, thus suggesting the direct toxic effect of NO • on trypanosomes.However, this activity may also be due to the relatively rapid homolytic breakdown of the thionitrites under physiological conditions with half-life times of 2.5 h.
In summary, we have prepared a series of molecules with potential trypanocidal activities that contain both a P2-transporter recognition motif and a group that acts as an NO donor.Preliminary affinity data indicate a specific interaction of compound 7a with P2.The toxicity of this melaminyl compound could be attributed mainly to its ability to yield nitric oxide, but its rapid decomposition might in part explain its poor in vitro activity.The biological activities of compounds 7b and 7c are under study.

Table 1 -
Inhibition of adenosine transport by T. equiperdum in the presence of inosine and in vitro toxicity of the same strain.