Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure-activity relationships of apicidin. Part 1.

Apicidin, a natural product recently isolated at Merck, inhibits both mammalian and protozoan histone deacetylases (HDACs). The conversion of apicidin, a nanomolar inhibitor of HDACs, into a series of side-chain analogues that display picomolar enzyme affinity is described within this structure-activity study.

Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure-activity relationships of apicidin. Part 2.

Recently isolated at Merck, apicidin inhibits both mammalian and protozoan histone deacetylases (HDACs). The conversion of apicidin, a nonselective nanomolar inhibitor of HDACs, into a series of picomolar indole-modified and parasite-selective tryptophan-replacement analogues is described within this structure-activity study.

Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents.

Apicidin's indole was efficiently converted into a series of N-substituted quinolone derivatives by indole N-alkylation followed by a two-step, one-pot, ozonolysis/aldol condensation protocol. The new quinolones exhibited good parasite selectivity and potency both at the level of their molecular target, histone deacetylase, and in their whole cell antiproliferative activity in vitro.

Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase.

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-L-tryptophanyl-L -isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin's antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation-deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

Selective labeling of intracellular parasite proteins by using ricin.

Studies focused on the synthesis by intracellular parasites of developmentally regulated proteins have been limited due to the lack of a simple method for selectively labeling proteins produced by the parasite. A method has now been developed in which ricin is employed to selectively inhibit host-cell protein synthesis. Ricin is a heterodimer composed of two subunits, a lectin and a glycosidase, and it binds to terminal galactose residues on the cell surface via the lectin. Following endocytosis of the intact molecule, a disulfide bond linking the two subunits is cleaved, and only the glycosidase subunit enters the cytoplasm, where it inhibits cytoplasmic protein synthesis by catalyzing the cleavage of the 28S rRNA. Due to the loss of the receptor-binding lectin subunit, ricin cannot permeate host-cell mitochondria or intracellular parasites, and, therefore, protein synthesis within these compartments continues uninterrupted. This system has been used to selectively label parasite proteins from Eimeria tenella and Toxoplasma gondii by using the avian cell line DU-24. In these cells, mitochondrial protein synthesis was inhibited by using chloramphenicol. The use of the avian rho0 cell line DUS-3 provided an additional advantage, because these cells lack mitochondrial DNA. Therefore, those proteins radiolabeled with [35S]methionine/cysteine in ricin-treated, parasite-infected rho0 cells are exclusively those of the intracellular parasite. This technique should be applicable for studying protein synthesis by other intracellular parasites.

Selective labelling of proteins synthesized by intracellular parasites using ricin and host cells lacking mitochondrial DNA.

Studies focused on the synthesis of developmentally regulated proteins by intracellular parasites have been limited due to the lack of a simple method for selectively labelling proteins produced by the parasite. A method has now been developed in which ricin, the toxin, is employed to selectively inhibit host cell protein synthesis while protein synthesis by the intracellular parasite is unaffected. Ricin is composed of two subunits, one of which binds to cell surface receptors containing terminal galactose residues while the other subunit enters the cell, inactivates ribosomes and, as a consequence, cytoplasmic protein synthesis. Due to the loss of the receptor-binding subunit, ricin cannot permeate the host cell mitochondria or the intracellular parasite, and therefore protein synthesis within these compartments continues uninterrupted. This system was explored using Eimeria tenella- and Toxoplasma gondii-infected avian rho0 cells. This host cell type was selected because it lacks mitochondrial DNA and supports the intracellular development of E. tenella sporozoites through first-generation merogony. Host mitochondrial proteins are not synthesized when labelling in the presence of ricin because these cells lack mitochondrial DNA. Therefore, those proteins which are radiolabelled with 35S methionine in ricin-treated infected monolayers are exclusively those of the intracellular parasite. Alternatively cells with intact mitochondria can be utilized, and in this case the host mitochondrial protein synthesis can be inhibited by chloramphenicol.

Eimeria tenella: incomplete excystation in the presence of EDTA in a taurodeoxycholate-based medium.

Normally, sporozoites of Eimeria tenella are efficiently excysted in vitro with trypsin and bile salts. However, a one hour treatment at 40 degrees C with a chelator-supplemented excystation medium (purified trypsin and chymotrypsin, taurodeoxycholate and ethylenediaminetetraacetic acid in buffered saline) produced incomplete excystation. The treatment removed the sporocyst plug and left an opened sporocyst containing motile sporozoites, but the release of sporozoites was greatly reduced (less than 12% release). Some of the sporozoites extended a portion of their anterior end through the sporocyst opening then retracted it into the sporocyst. Sporozoites were released when magnesium was added to the chelator-supplemented medium. Manganese was less effective and calcium was ineffective in producing release. Also, sporozoites were released when the incompletely excysted sporocysts were transferred to buffered saline with albumin and this became the basis for a new assay. The assay demonstrated that ethylenediaminetetraacetic acid reduced release in the presence of taurodeoxycholate but not in its absence. Hydrophobic and hydrophilic chelators were tested in the assay. Ethylene-dioxy diethylene-dinitrilotetraacetic acid and 8-hydroxyquinoline were inactive. The chelator 1,10-phenanthroline did not require bile salt to reduce release. The inhibitory effects by phenanthroline were eliminated in the presence of magnesium or manganese, while calcium had no effect. Thus, although certain chelators can inhibit release, a consistent correlation between chelation and inhibition of release has not been established. The application of ethylenediaminetetraacetic acid with taurodeoxycholate as a reversible inhibitor of release is discussed.