Purine Nucleobase Transport in Amastigotes of Leishmania mexicana : Involvement in Allopurinol Uptake

Abstract

Nucleobase and nucleoside transporters play central roles in the biochemistry of parasitic protozoa, as they lack the ability to synthesize purines de novo and are absolutely reliant upon purine salvage from their hosts. Furthermore, such transporters are potentially critical to the pharmacology of these important human pathogens, because they mediate the uptake of purine analogues, as well as some nonpurine drugs, that can be selectively cytotoxic to the parasites. We here report the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [ 3 H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with a K m of 1.6 ؎ 0.4 M and high affinity for adenine, guanine, and xanthine but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol, an antileishmanial hypoxanthine analogue, was apparently taken up by the same transporter. Using Parasitic protozoa of the genus Leishmania are the etiological agents of leishmaniasis. Some 12 million people throughout the world suffer from leishmaniasis, ranging from the disfiguring cutaneous form to often-fatal visceral leishmaniasis. Leishmania species are sandfly-transmitted protozoan parasites. The life cycle is divided into promastigotes and metacyclics in the insect vectors and amastigotes in their mammalian hosts, which are responsible for all clinical manifestations.Most of the currently available antileishmanial drugs have been discovered empirically, as until recently insufficient information was known about the biochemistry, physiology, and molecular biology of these parasites and the interactions with their hosts (23). The limitations of the current treatment for leishmaniasis, as a result of drug resistance and the severe side effects of most of the existing therapeutic agents, and the urgent need for new therapeutic approaches, are well documented (12,56,60).The development of a rational therapeutic strategy for the treatment and prevention of parasitic disease depends on exploitation of fundamental biochemical disparities between parasite and host, such as the inability of protozoan parasites to synthesize purines de novo (5). Current antiprotozoal agents often derive selectivity from selective accumulation by the parasite rather than the host cell (15). The selectivity and the efficacy of purine antimetabolites can be achieved by the cell surface transporters that mediate access to the cell, as substrate recognition by nucleobase transporters is strikingly different in humans and kinetoplastids such as Trypanosoma brucei (58) and Leishmania major promastigotes (2). Purine and pyrimidine antimetabolites have been highly successful against many viral infections as well as malignancies (27) and show great promise against protozoal infections as well (reviewed in references 14, 17, and 23). Allopurinol, a purine nucleobase analogue, is clinically used against various manifestations of leishmaniasis (1,13,39).Whereas purine transporters in prom...

Predictive Computational Models of Substrate Binding by a Nucleoside Transporter

“…This method was used to explain substrate preferences of purine and pyrimidine transporters in T. brucei brucei (32), Leishmania major (33,34), Toxoplasma gondii (35), Leishmania mexicana (36), and the human NBT1 nucleobase transporter (14), human concentrative nucleoside transporters (37), and human equilibrative nucleoside transporters (38) with semi-quantitative models of substrate binding that did not require any structural or genetic information about the transport protein. However, this method still did not allow genuinely quantitative or three-dimensional predictions nor was it suitable for screening of virtual libraries.…”

2,N 6 -Disubstituted Adenosine Analogs with Antitrypanosomal and Antimalarial Activities

“…Evaluation of the compounds for their cytotoxicities for L6 mouse fibroblast cells and for their activities against the chloroquine-and pyrimethamine-resistant line Plasmodium falciparum K1, Trypanosoma brucei rhodesiense strain STIB 900, T. b. brucei strain BS221, the tbat1 Ϫ/Ϫ clone derived from BS221 (23), trypomastigotes of Trypanosoma cruzi strain Tulahuen C4, and amastigotes of Leishmania donovani strain MHOM-ET-67/L82 in primary mouse macrophages was performed at the Swiss Tropical Institute, exactly as described previously (24,30). Additional drug sensitivity assays with culture-adapted bloodstream T. b. brucei s427, Leishmania major promastigotes, and Leishmania mexicana amastigotes were performed at the University of Glasgow, as described previously (1,26,36). Melarsoprol, pentamidine, and DB75 (furamidine) are antitrypanosomal drugs; and chloroquine, mefloquine, and artemisinin are antimalarial drugs.…”

“…By following this rationale, the use of purine derivatives for the treatment of various protozoan infections has been investigated, including leishmaniasis (1,16), African trypanosomiasis (17,34,35), and malaria (18)(19)(20). In previous work we have described the synthesis of a library of di-and trisubstituted 5Ј-carboxamidoadenosine analogs and evaluated their antiprotozoal activities (30).…”

Solution structures of purine base analogues 6-chloroguanine, 8-azaguanine and allopurinol

“…Purine base analogues can also be used to identify interactions of individual functional groups in the natural substrate in the protein complex. For instance, de Koning and co-workers have used several nucleobase analogues in order to understand the interactions various nucleobase transporters make with their nucleobase substrates (Al-Salabi & de Koning, 2005;Wallace, Candlish, & De Koning, 2002). Recent work by Schramm and co-workers utilizing a number of immucillin analogues (analogues of nucleosides where the purine N9 is substituted with a carbon atom and the ribose oxo group is replaced a NH group) shows that change in a single hydrogen-bond interaction can cause a change of up to 10 kJ in binding energy (Kicska et al, 2002).…”