Analogs of 5-methylthioribose, a novel class of antiprotozoal agents

Abstract

Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodiumfalciparum, Giardia lamblia, and Ochromonas malkamensis in vitro. In contrast, these analogs had no effect on cultured mammalian cells. Analogs of MTR represent a potential new class of antiprotozoal drugs.Parasitic protozoa cause some of the most prevalent infectious diseases of humans and produce extensive morbidity and mortality around the world (25). For example, over 600 million people are chronically infected with Plasmodium falciparum and at least 1.5 million malaria-related deaths occur each year (4, 15). Despite intensive research efforts, the development of new and effective antiprotozoal drugs has been difficult because of the close metabolic relationship between protozoa and human cells.5'-Deoxy-5'-methylthioadenosine (MTA) is a naturally occurring nucleoside synthesized from S-adenosylmethionine during the production of the polyamines spermidine and spermine (23). Unlike the polyamines, MTA does not normally accumulate in cells but is rapidly degraded by one of two mechanisms (Fig. 1). In mammals (14), MTA is degraded in one step to adenine and 5-methylthioribose-1-phosphate (MTR-1-P) by MTA phosphorylase (Fig. 1, pathway A). In some other organisms (9), however, MTA is catabolized to MTR-1-P in two steps: (i) catabolism to adenine and MTR via MTA nucleosidase and (ii) conversion of MTR to MTR-1-P in an ATP-dependent reaction catalyzed by MTR kinase (Fig. 1, pathway B). In both instances, MTR-1-P is subsequently recycled to methionine through the intermediate a-ketomethylthiobutyric aci'd (1,11,19). Since MTA nucleosidase and MTR kinase are not present in mammalian cells, we hypothesized that these enzymes represent potential targets for the selective killing of organisms which contain them.To test this hypothesis, cell extracts of P. falciparum, Giardia lamblia, and Ochromonas malhamensis were assayed for MTA phosphorylase (14), MTR kinase (9), and MTA nucleosidase (7) activities. 0. malhamensis, a nonpathogenic protozoan, was studied because it can proliferate by using MTR as a sole source of methionine (21), implying that it contains MTR kinase. Enterobacter aerogenes (8) and Persea americana (avocado fruit; 10) were included as positive controls because each contains MTR kinase activity. Extracts derived from human erythrocytes and liver were also tested. Cell extracts from P. falciparum, G. lamblia, and 0. malhamensis contained significant MTA * Corresponding author. nucleosidase and MTR kinase activities (Table 1). MTA phosphorylase was undetectable in these samples, except in the P. falciparum samples, in which low activity, presumably related to residual...

Thiazole orange: A new dye for Plasmodium species analysis

“…The study corroborates a recent report (7) that employed Giemsa-stained prepa- rations to demonstrate that two strains of €? falciparum were sensitive to both chloroquine and ETR.…”

“…Riscoe and Epitope Inc., Portland, OR). ETR recently has been shown to have plasmodiocidal activity in vitro (7). Aliquots of these cultures were analyzed in a similar fashion.…”

Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

“…This observation would explain why many MTR analogs with bulky extensions at the 5-position have been shown to be weak substrates and/or inhibitors of MTR kinase (3,5). Although the bulkiness of the 5-subsitution does not correlate with their inhibitory concentrations, 5 they are comparable (in the micromolar range) to the K m for MTR observed for the enzyme in many species of bacteria and plants (7,33,46 Proposed Mechanism for Phosphate Transfer-The mechanism of phosphate transfer in a typical kinase is often categorized as either associative or dissociative (54). An associative mechanism for MTR kinase would involve a direct S N 2-like nucleophilic attack of the O-1 hydroxyl oxygen of MTR to the ␥-phosphate of ATP, forming a penta-coordinated trigonal bipyramidal transition state with 50% bond formation between the nucleophilic oxygen and the phosphorus, and 50% bond breakage between the phosphorus and the ADP leaving group.…”

“…In mammalian cells, however, the degradation of MTA and its conversion to MTR 1-phosphate is achieved in a single step by MTA phosphorylase (10). This metabolic difference in the way MTA is removed has been explored and analogs of MTR, synthesized as pro-drugs, have been shown to selectively kill MTR kinase-containing organisms with little effect on mammalian cells (5,11). The absence of a mammalian homolog makes MTR kinase a good target for the design of novel antibiotics.…”

“…MTR kinase is regulated by the environmental methionine level (4) and its expression enables organisms to grow on nonmethionine sulfur sources such as MTR or 5Ј-methylthioadenosine (MTA) (5)(6)(7). MTA is a byproduct and inhibitor of polyamine synthesis (8) and hence is toxic to cells and must be rapidly degraded.…”