Toxoplasma gondii is capable of exogenous folate transport. A likely expansion of the BT1 family of transmembrane proteins.

Folates are key elements in eukaryotic biosynthetic processes. The protozoan parasite Toxoplasma gondii possesses the enzymes necessary for de novo folate synthesis and has been suggested to lack alternative mechanisms for folate acquisition. In this paper, we present a different view by providing evidence that Toxoplasma is capable of salvaging exogenous folates. By monitoring uptake of radiolabeled folates by parasites in axenic conditions, our studies revealed a common folate transporter that has a high affinity for folic acid. Transport of this compound across the parasite plasma membrane is rapid, biphasic, temperature dependent, bi-directional, concentration dependent and specific. In addition, morphological evidence demonstrates that fluorescent methotrexate, a folate analog, is internalized by Toxoplasma and shows localization reminiscent to the mitochondrion. The presence of putative folate transporter genes in the Toxoplasma genome, which are homologous to the BT1 family of proteins, suggests that Toxoplasma may encode proteins involved in folate transport. Interestingly, genome analysis suggests that the BT1 family of proteins exists not only in Toxoplasma, but in other Apicomplexan parasites as well. Altogether, our results not only have implications for current therapeutic regimens against T. gondii, but they also allude that the folate transport mechanism may represent a novel Apicomplexan target for the development of new drugs.

A blasticidin S-resistant Plasmodium falciparum mutant with a defective plasmodial surface anion channel.

Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltage-dependent ion channel induced on the host membrane after infection, may play a central role in these permeability changes. Here, we identified a functional PSAC mutant through in vitro selection with blasticidin S. Resistance to blasticidin S was generated during culture and correlated with significant reductions in permeability to multiple solutes, consistent with uptake via a common pathway. Single channel recordings revealed marked changes in PSAC gating with the addition of a subconductance state not present in wild-type channels. The channel's selectivity profile and pharmacology also were significantly altered. Eventual loss of the mutant phenotype upon removal of selective pressure and slower growth of mutant parasites suggest that PSAC serves an important role in intracellular parasite survival. These findings provide solid evidence for the uptake of diverse solutes via PSAC and implicate one or more parasite genes in expression of this channel.

Nonconserved residues Ala287 and Ser290 of the Cryptosporidium hominis thymidylate synthase domain facilitate its rapid rate of catalysis.

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency approximately 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is approximately 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

Effects of conformation on the stereochemistry of solvent attack on benzylic beta-hydroxycarbocations: mechanisms of epoxide hydrolysis reactions.

The rates and products from the acid-catalyzed and the pH-independent reactions of two diastereomeric 6-methoxy-trans-1,2,3,4,4a,10a-hexahydrophenanthrene 9,10-oxides (5b and 7b), along with their cis and trans chlorohydrins, have been determined in dioxane/water solutions. The mechanisms of the acid-catalyzed hydrolysis of 5b and 7b involve rate-limiting formation of benzylic carbocations (6b and 8b), which have sufficient lifetimes to be trapped by azide ion. Each carbocation is stabilized by the 6-methoxy group and held in single conformation by the adjacent trans-fused cyclohexane ring. The stereochemistry of the attack of water on each carbocation is independent of whether the precursor is an epoxide, a cis chlorohydrin, or a trans chlorohydrin, and the major diol hydrolysis product from each compound results from the axial attack of a solvent molecule on the carbocation intermediate. The hydrolysis of the trans chlorohydrin formed from the reaction of 5b with HCl exhibits a common ion rate depression. The major product from the pH-independent reaction of 5b is a trans diol, and the major product from the pH-independent reaction of 7b is an isomeric ketone. The rate of the pH-independent reaction of 7b is >10(4) times faster than that of 5b.

Eosin B as a novel antimalarial agent for drug-resistant Plasmodium falciparum.

4',5'-Dibromo-2',7'-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC(50)) of 180 microM. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC(50) of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.

Synthesis and hydrolysis of a cis-chlorohydrin derived from a benzo[a]pyrene 7,8-diol 9,10-epoxide.

(+/-)-7beta,8alpha-Dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-1) undergoes reaction with anhydrous HCl in dioxane to yield predominantly ( approximately 94%) a single chlorohydrin. This chlorohydrin was assigned structure 9, in which the chloro goup at C-10 is located cis to the C-9 hydroxyl group, on the basis of its (1)H NMR spectrum. This result is in contrast to the reaction of a diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxide (DE-2) with HCl, which yields only trans-chlorohydrin 8. The hydrolysis of cis-chlorohydrin 9 in 10:90 dioxane-water solutions yields the same ratio of tetrols ( approximately 89% cis/11% trans) as that formed by acid-catalyzed hydrolysis of DE-1. This result again contrasts with the hydrolysis of trans-chlorohydrin 8, which undergoes hydrolysis to give tetrols in a ratio different from that from acid-catalyzed hydrolysis of DE-2. A marked common ion rate depression in the hydrolysis of cis-chlorohydrin 9 is observed, which shows that hydrolysis proceeds via an intermediate carbocation that has a sufficient lifetime to be trapped by external chloride ion. The observation that DE-1 reacts with HCl to give mainly the cis-chlorohydrin is rationalized by quantum chemical calculations that suggest that the cis-chlorohydrin is more stable than the epimeric trans-chlorohydrin.

Chloride ion catalyzed conformational inversion of carbocation intermediates in the hydrolysis of a benzo[a]pyrene 7,8-diol 9,10-epoxide.

A highly efficient procedure for converting 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (1) to its trans-9,10-chlorohydrin (5) with excellent yield and purity by the reaction of anhydrous HCl in THF has been developed. The rate of reaction of 5 has been determined as a function of sodium chloride concentration in 1:1 dioxane-water solutions. A large common ion rate depression for the reaction of the chlorohydrin was observed, and the rate data are fit to a mechanism in which all of the tetrol products are formed by the reaction of water with the C-10 carbocation intermediate. Yet, the cis/trans ratio of tetrols from the reaction of the carbocation intermediate from the hydrolysis of chlorohydrin 5 is different than the cis/trans tetrol ratio from the acid-catalyzed hydrolysis of diol epoxide 1, which hydrolyzes via a carbocation with the same connectivity as that formed in the hydrolysis of 5. To rationalize these results, it is proposed that the S(N)1 reaction of chlorohydrin 5 yields a different distribution of carbocation conformations than that formed from the reaction of 1 with H(+). The energy barrier for the inversion of these carbocation conformations must be large relative to the energy barriers for the reaction of each carbocation conformation with water. In solutions containing sufficient concentrations of chloride ion, however, a lower energy pathway via a halohydrin exists for the interconversion of the carbocation conformations. Thus, chloride ion catalyzes the interconversion of these two carbocation conformations.