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.

Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.

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.

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella.

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4.

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.

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.

Amylopectin synthase of Eimeria tenella: identification and kinetic characterization.

A soluble enzyme amylopectin synthase (UDP-glucose-alpha 1,4-glucan alpha-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form alpha-1,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella. UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km), with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing alpha-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-alpha-1,4-glucosetransferase EC 2.4.1.11).