ABSTRACT
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.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Histone Deacetylase Inhibitors , Peptides, Cyclic/pharmacology , Animals , Antiprotozoal Agents/pharmacology , Biological Factors/pharmacology , Cattle , Cell Line , Combinatorial Chemistry Techniques , Eimeria tenella/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fusarium/chemistry , HeLa Cells , Humans , Microbial Sensitivity Tests , Peptides, Cyclic/chemical synthesis , Plasmodium falciparum/drug effects , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Histone Deacetylase Inhibitors , Peptides, Cyclic/pharmacology , Animals , Antiprotozoal Agents/pharmacology , Biological Factors/pharmacology , Cattle , Cell Division/drug effects , Cell Line , Combinatorial Chemistry Techniques , Eimeria tenella/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fusarium/chemistry , HeLa Cells , Humans , Indoles/chemistry , Microbial Sensitivity Tests , Peptides, Cyclic/chemical synthesis , Plasmodium falciparum/drug effects , Structure-Activity Relationship , Tryptophan/chemistryABSTRACT
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.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors , Indoles/chemical synthesis , Peptides, Cyclic/chemistry , Quinolones/chemical synthesis , Animals , Antimalarials/chemical synthesis , Antimalarials/chemistry , Antimalarials/pharmacology , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Binding, Competitive , Cell Division/drug effects , Cell Extracts , Chickens , Eimeria tenella/cytology , Eimeria tenella/drug effects , Eimeria tenella/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HeLa Cells , Histone Deacetylases/metabolism , Humans , In Vitro Techniques , Indoles/chemistry , Indoles/pharmacology , Liver/metabolism , Plasmodium falciparum/drug effects , Quinolones/chemistry , Quinolones/pharmacology , Structure-Activity RelationshipABSTRACT
The characterization of protein synthesis by eukaryotic intracellular parasites is inherently difficult because it may represent only a small fraction of host cell protein synthesis. Here, Mark Carrington, Yael Shochat and Anne Gurnett describe a method for overcoming this problem through the use of the toxin ricin to inhibit host cell protein synthesis specifically.
ABSTRACT
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.
Subject(s)
Antiprotozoal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Eukaryota/drug effects , Histone Deacetylase Inhibitors , Malaria/drug therapy , Peptides, Cyclic/pharmacology , Plasmodium berghei , Animals , Eimeria tenella/drug effects , Female , Humans , Kinetics , Mice , Mice, Inbred BALB C , Neospora/drug effects , Peptides, Cyclic/therapeutic use , Plasmodium falciparum/drug effects , Protein Binding , Protozoan Infections/drug therapy , Structure-Activity Relationship , Toxoplasma/drug effectsABSTRACT
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.
Subject(s)
Eimeria tenella/metabolism , Protozoan Proteins/biosynthesis , Ricin , Toxoplasma/metabolism , Animals , Autoradiography , Cell Line , Chickens , Coccidiosis/parasitology , Cysteine/metabolism , Eimeria tenella/growth & development , Eimeria tenella/isolation & purification , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Humans , Male , Methionine/metabolism , Molecular Weight , Protozoan Proteins/analysis , Protozoan Proteins/isolation & purification , Skin , Sulfur RadioisotopesABSTRACT
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.
Subject(s)
Eimeria tenella/metabolism , Mitochondria/metabolism , Protozoan Proteins/metabolism , Ricin/pharmacology , Toxoplasma/metabolism , Animals , Isotope Labeling/methods , MethionineABSTRACT
The variable domain of the trypanosome variant surface glycoprotein (VSG) ILTat 1.24 has been shown by X-ray crystallography to resemble closely the structures of VSG MITat 1.2, despite their low sequence similarity. Specific structural features of these VSGs, including substitution of carbohydrate for an alpha-helix, can be found in other VSG sequences. Thus antigenic variation in trypanosomes is accomplished by sequence variation, not gross structural alteration; the extensive sequence differences among VSGs may be required for another reason, such as the avoidance of recognition by helper T cells. Additionally, VSG sequences are found to define families, within a VSG superfamily, which have evolved in the trypanosome genome.
Subject(s)
Trypanosoma brucei brucei/chemistry , Variant Surface Glycoproteins, Trypanosoma/chemistry , Amino Acid Sequence , Animals , Antigenic Variation , Computer Simulation , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Trypanosoma brucei brucei/immunology , Variant Surface Glycoproteins, Trypanosoma/classification , X-Ray DiffractionABSTRACT
The intact variant surface glycoprotein (VSG) ILTat 1.24 from Trypanosoma brucei has been crystallized. An amino-terminal domain of the protein comprising two thirds of the sequence had been crystallized previously after proteolytic digestion. Now intact VSG crystals have been grown from 50 mM-Mes (pH 6.5) containing 62% (w/v) saturated ammonium sulfate. The crystals are demonstrated to contain the intact VSG by h.p.l.c. gel filtration and reaction with an antibody to the inositol phosphate oligosaccharide on the VSG carboxy terminus. The space group of the crystals is P6(2)22 (or P6(4)22) with unit cell dimensions a = b = 184 A and c = 214 A. Preparative isoelectric focusing may have facilitated crystallization.
Subject(s)
Trypanosoma brucei brucei/analysis , Variant Surface Glycoproteins, Trypanosoma/chemistry , Ammonium Sulfate , Animals , Antibodies/immunology , Chromatography, High Pressure Liquid , Humans , Inositol Phosphates/immunology , Oligosaccharides/immunology , Solubility , Variant Surface Glycoproteins, Trypanosoma/immunology , X-Ray DiffractionABSTRACT
The isolation in our laboratories of several antigens of interest from sporulated oocysts of Eimeria species by bioselective adsorption on matrices containing immobilized antigen-specific immunoglobulins IgG was initially unsuccessful. The preparations serving as source materials for these antigens contained low levels of the zwitterionic sulfobetaine detergent, Zwittergent 3-12. Since usually immunoaffinity processes are carried out in the presence of various detergents, we were surprised, subsequently, to find this detergent to be the cause of the problem in that it prevented antigen-antibody binding. These findings led us to study the potential role of Zwittergent 3-12 as an eluting agent from matrices holding bioselectively adsorbed materials. The results of seven case studies are presented in this paper and include experiments with beta-D-galactosidase adsorbed biospecifically and bioselectively on matrices via either specific antibody or inhibitor analogue. In all cases, Zwittergent 3-12 proved to be an effective desorbing agent.
Subject(s)
Betaine/analogs & derivatives , Chromatography, Affinity , Animals , Antigens, Protozoan/isolation & purification , Blotting, Western , Eimeria/immunology , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Surface-Active AgentsABSTRACT
Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.
Subject(s)
Antigens, Protozoan/immunology , Antigens, Surface/immunology , Glycoproteins/immunology , Membrane Proteins/immunology , Trypanosoma brucei brucei/immunology , Animals , CD55 Antigens , Carbohydrates/immunology , Epitopes , Immunosorbent Techniques , Variant Surface Glycoproteins, TrypanosomaABSTRACT
Membrane-form variant surface glycoprotein of Trypanosoma brucei can be prepared in the presence of para-chloromercuriphenylsulphonic acid. The membrane-bound enzyme that usually cleaves a lipid from this glycoprotein, thus producing the soluble variant surface glycoprotein, is inhibited by a range of sulphydryl reagents. The effect of such inhibitors, both on cell lysates and on semi-purified enzyme, reveals that the enzyme may have a sulphydryl at or near its active site. Fatty acid analysis and isoelectric point measurements of membrane form and soluble form are presented.
Subject(s)
Glycoproteins/isolation & purification , Membrane Proteins/isolation & purification , Trypanosoma brucei brucei/analysis , 4-Chloromercuribenzenesulfonate/pharmacology , Animals , Electrophoresis, Polyacrylamide Gel , Fatty Acids/analysis , Isoelectric Focusing , Trichloroacetic Acid , Trypanosoma brucei brucei/enzymology , Variant Surface Glycoproteins, TrypanosomaABSTRACT
The solution properties of the membrane form and soluble form of variant surface glycoproteins from Trypanosoma brucei have been compared. Solution cross-linking studies established that both forms are dimers, although dissociation of membrane-form variant surface glycoprotein can be promoted by certain ionic and zwitterionic detergents. Sedimentation coefficients were measured under a range of conditions, and the results were comparable with the results of solution cross-linking. Stokes radii were measured by gel filtration, allowing a value for the frictional coefficient to be calculated. The two forms show no differences other than those consistent with binding of detergent micelles to the hydrophobic moiety present on membrane form surface glycoprotein. This validates the use of soluble variant surface glycoprotein in X-ray crystallography experiments.
Subject(s)
Glycoproteins/analysis , Membrane Proteins/analysis , Trypanosoma brucei brucei/analysis , Animals , Cross-Linking Reagents , Detergents , Micelles , Solubility , Trypanosoma brucei brucei/immunology , Variant Surface Glycoproteins, TrypanosomaABSTRACT
The amino acid sequences of the myoglobins of two rodents, the casiragua and the house mouse, have been determined. The myoglobin of casiragua differs from that of viscacha (another hystricomorph) at 6 positions. Mouse myoglobin differs from that of mole-rat (another myomorph) at 17 positions, whereas casiragua and mouse differ at 22 positions. Mouse myoglobin possesses several features unique among all known myoglobins (Gly 31, Cys 66, Thr 74 and Glu 113) and one substitution unique among known mammalian myoglobins (Glu 53).
Subject(s)
Mice/metabolism , Myoglobin/analysis , Rodentia/metabolism , Amino Acid Sequence , Animals , Peptide Fragments/analysis , Species SpecificityABSTRACT
An N-terminal signal peptide has been identified on a precursor of the variant surface glycoprotein (VSG) of Trypanosoma brucei, synthesised both in vitro and in vivo. An ordered cyanogen bromide (CNBr) cleavage map was constructed for both mature VSG and for precursor VSG, and this allowed a size estimate of the signal peptide to be made by comparison of the N-terminal CNBr peptides produced from each. The signal peptide contains 30-40 amino acids. Analysis of the precursor VSGs produced when nascent polypeptide chains on T. brucei polysomes were completed in the absence of reinitiation showed that the signal peptide is removed before translation is complete and that the same translational start signal is utilised in the trypanosome and in the rabbit reticulocyte protein synthesis system.