Purification and Molecular Characterization of cGMP-dependent Protein Kinase from Apicomplexan Parasites
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m108393200
ISSN1083-351X
AutoresAnne Gurnett, Paul Liberator, Paula M. Dulski, Scott P. Salowe, Robert G. K. Donald, Jennifer Anderson, Judyann Wiltsie, Carmen A. Diaz, Georgiana Harris, Ben Chang, Sandra J. Darkin‐Rattray, Bakela Nare, Tami Crumley, Penny S. Blum, Andrew S. Misura, Tamas Tamas, Mohinder K. Sardana, Jeffrey Yuan, Tesfaye Biftu, Dennis M. Schmatz,
Tópico(s)Synthesis and Biological Evaluation
ResumoThe trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. bothin vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify a ∼120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a parasite signature sequence of nearly 300 amino acids that is not found in mammalian or DrosophilaPKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably different from mammalian enzymes. The activity of both native and recombinantE. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH2-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound 1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with an IC50 of <1 nm. The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. bothin vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify a ∼120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a parasite signature sequence of nearly 300 amino acids that is not found in mammalian or DrosophilaPKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably different from mammalian enzymes. The activity of both native and recombinantE. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH2-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound 1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with an IC50 of <1 nm. cGMP-dependent protein kinase cAMP-dependent protein kinase reverse transcriptase protein kinase inhibitor E. tenella PKG amino-terminal FLAG-tagged EtPKG T. gondii PKG β,γ-methyleneadenosine 5′-triphosphate 1-(2-guanidinoethyl)octahydroazocine unsporulated oocysts Protozoan parasites of the genus Eimeria are the causative agents of the intestinal disease known as coccidiosis. Coccidiosis occurs in several domesticated and wild animal species, but of major economic importance is the impact that Eimeria spp.have on the poultry industry. During acute infections, these parasites cause significant morbidity and mortality in broiler breeds of chicken (reviewed in Ref. 1Williams R.B. Int. J. Parasitol. 1999; 29: 1209-1229Crossref PubMed Scopus (337) Google Scholar). Anticoccidial compounds have been and continue to be used prophylactically in the majority of poultry operations today. The most successful anticoccidials have been the polyether ionophores, a family of compounds that continues to be the industry standard since their introduction nearly 30 years ago (2Croft S.L. Parasitology. 1997; 114 (suppl.): S3-S15Crossref PubMed Google Scholar). Not surprisingly, reports of resistance development due to the extended and constant chemotherapeutic pressure exerted by this class of compounds are not uncommon (3Stephen B. Rommel M. Daugschies A. Haberkorn A. Vet. Parasitol. 1997; 69: 19-29Crossref PubMed Scopus (114) Google Scholar, 4Daugschies A. Gasslein U. Rommel M. Vet. Parasitol. 1998; 76: 163-171Crossref PubMed Scopus (51) Google Scholar). Since that time no novel anticoccidials with efficacy and economic features that approach the ionophore class have been introduced into the poultry industry. The need to identify and develop new drugs for the control of coccidiosis is critically important. In this report we describe the chemotherapeutic efficacy of a novel anticoccidial reagent. Data from biochemical purification and molecular cloning efforts predict that the therapeutic target of this class of compounds in Eimeria is a cGMP-dependent protein kinase (PKG).1 PKG transfers the γ-phosphate of ATP in a cGMP-dependent reaction to serine and/or threonine residues of several cellular proteins (5Kuo J.F. Greengard P. J. Biol. Chem. 1970; 245: 2493-2498Abstract Full Text PDF PubMed Google Scholar). Cyclic GMP is a ubiquitous intracellular messenger that has a role in several aspects of signal transduction that potentially regulate a myriad of physiological processes (reviewed in Ref. 6Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (262) Google Scholar). cGMP also modifies the activity of proteins other than PKG, including cGMP-gated ion channels and cGMP-regulated phosphodiesterases (6Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (262) Google Scholar). Cyclic nucleotide-dependent protein kinases from unicellular organisms such as Paramecium to humans have been biochemically characterized and/or cloned (6Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (262) Google Scholar, 7Miglietta L.A.P. Nelson D.L. J. Biol. Chem. 1988; 31: 16096-16105Abstract Full Text PDF Google Scholar, 8Orstavik S. Natarajan V. Tasken K. Jahnsen T. Sandberg M. Genomics. 1997; 42: 311-318Crossref PubMed Scopus (101) Google Scholar, 9Witczak O. Orstavik S. Natarajan V. Frengen E. Jahnsen T. Sandberg M. Biochem. Biophys. Res. Commun. 1998; 245: 113-119Crossref PubMed Scopus (9) Google Scholar). Members of this group of kinases share sequence homology in both their regulatory and catalytic domains. The most striking feature that distinguishes cAMP-dependent (PKA) from cGMP-dependent protein kinases is that PKA exists as a heterotetramer in its inactive conformation, composed of two identical regulatory and two identical catalytic subunits, while PKG is in most cases a homodimeric enzyme (6Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (262) Google Scholar,10Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (413) Google Scholar). The regulatory and catalytic subunits of PKA are distinct gene products. Activation of PKA by cAMP occurs as a result of a conformational change in the enzyme initiated by the binding of two molecules of the cyclic nucleotide to each regulatory subunit. The conformational change releases the regulatory dimer from the inhibited complex, thereby activating the catalytic dimer (11Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (350) Google Scholar, 12Wilson K.P. Fitzgibbon M.J. Caron P.R. Griffith J.P. Chen W. McCaffrey P.G. Chambers S.P. Su M.S. J. Biol. Chem. 1996; 271: 27696-27700Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Unlike PKA, the nucleotide-binding/regulatory and catalytic domains of PKG are part of the same protein. In the absence of cGMP, PKG assumes a conformation that is autoinhibited (13Wolfe L. Francis S.H. Corbin J.D. J. Biol. Chem. 1989; 264: 4157-4162Abstract Full Text PDF PubMed Google Scholar). Binding of cGMP to two nucleotide-binding domains within each subunit of the homodimer causes an intramolecular conformational change that activates kinase activity. While the two families of enzymes have significant structural differences, the mechanisms of autoinhibition and activation by the respective cyclic nucleotide cofactors are similar. In this report we demonstrate that Apicomplexan parasite PKG has several features that distinguish it from PKG homologues in other organisms. Unlike mammalian and invertebrate PKG enzymes which are typically homodimers, the E. tenella enzyme is a monomeric protein. The E. tenella enzyme has remarkably different cGMP activation kinetics, characterized by a minimal basal kinase activity in the absence of nucleotide cofactor which is induced by as much as 1000-fold upon addition of cGMP. Moreover, the activation profile shows strong cooperativity, a feature that is not as striking in mammalian PKG. The size of the parasite enzyme, demonstrated biochemically and confirmed by cDNA cloning of five Apicomplexan PKGs, is 30–40% larger than most PKG enzymes that have been described. Consistent with the architecture of other PKGs, the nucleotide-binding/regulatory domain of the parasite enzymes is located toward the amino-terminal end of the protein and the catalytic domain is positioned C-terminal. A conserved parasite signature sequence of nearly 300 amino acids resides between the regulatory and catalytic domains of the protein. Within this region we have identified a third nucleotide-binding site, yet another feature that is unique to, but shared among, the collection of Apicomplexan parasite enzymes. Chickens were infected orally with 7.5 × 104Eimeria tenella LS18 sporulated oocysts. The unsporulated oocysts were harvested from the ceca 7 days post-infection and purified according to the method of Schmatz et al. (14Schmatz D.M. Crane M. Murray P.K. J. Protozoology. 1986; 33: 109-114Crossref PubMed Scopus (27) Google Scholar), and then sporulated by continual agitation for 36 h at 29 °C. For the in vivo studies drug was administered in feed at 50 ppm on day 1. Birds were infected with 3.5 × 103E. tenella sporulated oocysts on day 2 and 3.6 × 103Eimeria acervulina sporulated oocysts on day 3. Medicated feed was continuously available for the duration of infection. The experiment was terminated on day 8 and efficacy was estimated by performing oocyst counts. Conditions for the in vitro culture of parasites and determination of IC50 and or minimal inhibitory concentrations (defined as the lowest concentration (ng/ml) at which parasite growth was fully inhibited) for compounds were conducted according to previously described methods for E. tenella (14Schmatz D.M. Crane M. Murray P.K. J. Protozoology. 1986; 33: 109-114Crossref PubMed Scopus (27) Google Scholar),Toxoplasma gondii (15MacFadden D.C. Seeber F. Boothroyd J.C. Antimicrob. Agents Chemother. 1977; 41: 1849-1853Crossref Google Scholar), and Besnoitia jellisoni(16Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (514) Google Scholar). The Neospora caninum cell based assay (17Howe D.K. Mercier C. Messina M. Sibley L.D. Mol. Biochem. Parasitol. 1997; 86: 29-36Crossref PubMed Scopus (29) Google Scholar) was adapted for use by increasing length of assay from 5 days to 7 days. 2B. Nare, unpublished observations. The N. caninum and T. gondii strains expressing β-galactosidase constructs were obtained from David Sibley (Washington University) and John Boothroyd (Stanford University), respectively. Soluble extracts for binding studies were prepared by vortexing 2 × 109E. tenella unsporulated oocysts with an equal volume of buffer (10 mm HEPES pH 7.4, 1 mm sodium orthovanadate, 20% glycerol, 0.1 mg/ml Bacitracin, and 0.5% Sigma protease inhibitor mixture P8340), and an equal volume of 4-mm glass beads for 20 min. The resulting homogenate was centrifuged (100,000 ×g, 1 h) and the supernatant (S100) used directly.E. tenella protein (10–25 μl) was assayed in 100 μl at a final concentration of 75 mm Tris, pH 7.5, 12.5 mm MgCl2, 1.5 mm EDTA, and 2 nm [3H]Compound 1 (60 Ci/mmol). Nonspecific counts were estimated using a 1000-fold molar excess of unlabeled Compound 1. Samples were incubated for 1 h at 25 °C, and either filtered through Whatman GF/B glass fiber filters (presoaked in 0.6% polyethyleneimine for 1 h at 25 °C), or filtered through prepackaged gel filtration columns (800 μl, Edge Biosystems). The filters were washed with 100 mm NaCl, 10 mmTris, pH 7.4, dried, and radioactivity determined by scintillation counting using Ready-SAFE scintillation mixture. The void volume was collected from the gel filtration columns according to the manufacturers instructions and mixed with Ready-SAFE prior to counting. Oocyst lysates and S100 fractions were prepared as described above. The S100 was dialyzed against 30 mm sodium phosphate, pH 7, 20% glycerol, 1 mm dithiothreitol, 1 mm EDTA, 10 mmsodium fluoride, and 0.1 mm sodium orthovanadate (Buffer A). The dialysate (∼1 g of protein) was applied to a HiLoad 26/10 Q-Sepharose column (Amersham Bioscience) and eluted with a salt gradient (0–1 m NaCl) in Buffer A. Aliquots were taken from fractions for binding assays. Fractions that bound to the column and contained Compound 1 binding activity were dialyzed against Buffer B (30 mm sodium phosphate, pH 7.0, 1 mmdithiothreitol, 10 mm NaF, 1 mm EDTA, 0.1 mm sodium orthovanadate) that is supplemented with 1m ammonium sulfate. The dialysate was applied to a butyl-Sepharose column (Amersham Bioscience, two tandem 5-ml columns, pre-treated with bovine serum albumin) and the column was eluted with a reverse salt gradient (from 1 to 0 m(NH4)2SO4 in Buffer B). Fractions were analyzed as above using the Compound 1 binding assay and active fractions dialyzed against Buffer C (10 mm sodium phosphate, pH 7.0, 1 mm dithiothreitol, 10 mmNaF, 0.1 mm sodium orthovanadate). The dialysate was loaded onto two tandem 5-ml hydroxyapatite columns (Bio-Rad) and protein was eluted with a linear salt gradient (in Buffer C) up to 0.4m sodium phosphate. Compound 1 binding fractions from the hydroxyapatite column were pooled, dialyzed against Buffer A, and applied to an anion-exchange MonoQ HR 5/5 column (Amersham Bioscience) which had been equilibrated in Buffer A with 100 mm NaCl. The column was washed with 100 mm NaCl in Buffer A and then eluted with a linear gradient to 1 m NaCl which was started once the unbound proteins had been eluted. The Compound 1 binding activity, which did not bind to the column in 100 mm NaCl, was dialyzed against Buffer A and re-applied to the same column. After unbound proteins had been eluted in the absence of NaCl and the absorbance had returned to baseline, column bound proteins were eluted with a NaCl gradient to 1 m (Fig. 2B) in Buffer A. Fractions were collected and aliquots were assayed for binding activity and also for purity on polyacrylamide gels. In some cases fractions with ligand binding activity were then applied to a Superdex 200 column in Buffer A and analyzed as described above. SDS-polyacrylamide gel electrophoresis was performed on gradient gels (Novex). Western blots were carried out after transfer to nitrocellulose and immunoreactive proteins were detected using the ECL procedure (Amersham Bioscience). Protein sequence analysis of the purified proteins was performed on Coomassie Blue-stained gel slices. The slices were digested with trypsin, peptides separated by high performance liquid chromatography on C18 reverse phase chromatography, and sequence performed on isolated peptides using Edman degradation. DNA manipulations were performed according to standard procedures (18Sambrook J.F. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar). Plasmid DNA was purified either using Promega Wizard Miniprep kits (Promega) or the Qiagen Maxi kit (Qiagen). DNA sequence was obtained using an ABI Prism sequencer and fluorescent sequencing reagents (PerkinElmer Life Science) and analyzed using Vector NTITMSuite software (Informax). Oligonucleotides were purchased from Invitrogen or Integrated DNA Technologies. Enzymes used for the polymerase chain reaction (PCR) were purchased from PerkinElmer Life Science. PCR products were gel purified using Qiaex II (Qiagen) and subcloned directly into the TA-cloning vector pGEM-T Easy (Promega). Gel purified DNA fragments to be used as hybridization probes were random-primer labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Bioscience). Restriction and modifying enzymes were purchased either from Invitrogen or New England Biolabs. Electroporation competent bacterial cells were from Invitrogen. Seven tryptic peptide sequences generated from the purified E. tenella ligand-binding protein were used to design several degenerate oligonucleotides. These were used in coupled reverse transcriptase-PCR reactions with mRNA from E. tenellasporozoites as template. PCR products of interest were cloned and sequenced. A partial cDNA clone generated by RT-PCR was used as a hybridization probe to isolate full-length cDNA clones from anE. tenella unsporulated oocyst cDNA library (19Liberator P. Anderson J. Feiglin M. Sardana M. Griffin P. Schmatz D. Myers R.W. J. Biol. Chem. 1998; 273: 4237-4244Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Clones coding for T. gondii PKG were isolated from a cDNA library obtained from the NIH AIDS Research and Reference Reagent Program (Bethesda, MD, catalog number 1896). Clones were identified by heterologous screening at a reduced hybridization stringency using a portion of the E. tenella cDNA as probe. ACryptosporidium parvum expressed sequence tag containing a fragment of a PKG homologue was identified in a preliminary survey sequence analysis of the Cryptosporidium parvum genome (expressed sequence tag AQ083827 (20Strong W.B. Nelson R.G. Mol. Biochem. Parasitol. 2000; 107: 1-32Crossref PubMed Scopus (71) Google Scholar)). To obtain the complete C. parvum PKG gene, a PCR fragment derived from this expressed sequence tag was used to probe a C. parvum genomic library (number 1644, NIH AIDS Research and Reference Reagent Program). A PKG open reading frame spanning two overlapping EcoRI genomic clones was subsequently identified by DNA sequence analysis. An identical un-annotated open reading frame (Contig number 1655) was also located in a BLAST search of the partial C. parvum genome survey data base, made accessible through NCBI by the University of Minnesota C. parvum sequencing project (www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi (21Liu C. Vigdorovitch V. Kapur V. Abrahamsen M. Infect. Immun. 1999; 67: 3960-3969Crossref PubMed Google Scholar)). An alternative strategy was employed to isolate PKG cDNA clones from both Eimeria maxima and Plasmodium falciparumparasites. The first step involved a coupled RT-PCR to generate partial PKG cDNAs for each parasite. Using an alignment of the deduced amino acid sequence from cDNA clones coding for E. tenella and T. gondii PKG proteins, areas of sequence identity within the presumptive cGMP-binding (peptides a, VKFFEML; and b, GEYFGERAL) and catalytic domains (peptides c, RDLKPENI; and d, HYMAPEV) were identified. Total RNA purified from E. maximasporulated oocysts and a trophozoite-enriched preparation of P. falciparum was first converted into cDNA using reverse transcriptase. The respective populations of cDNA products were then used as template in PCR reactions using degenerate oligonucleotide primers from peptides a and d. Primary PCR reaction products were then used as templates in secondary nested PCR reactions using degenerate primers from peptides b and/or c. PCR reaction products produced in the secondary nested reactions were subcloned, DNA sequence confirmed, and then used as hybridization probes to screen E. maxima andP. falciparum cDNA libraries to isolate full-length cDNA clones. PKG cDNA and deduced protein sequence for E. tenella, E. maxima, Toxoplasma gondii, Cryptosporidium parvum, andPlasmodium falciparum have been deposited in GenBankTM with accession numbers AF411961, AF465543,AF413570, AF413571, and AF465544. Sequences were aligned using the modified ClustalW algorithm MultiClustal (22Yuan J. Amend A. Borkowski J. DeMarco R. Bailey W. Liu Y. Xie G. Blevins R. Bioinformatics. 1999; 15: 862-863Crossref PubMed Scopus (33) Google Scholar). The resulting alignment was shaded using Genedoc (available at www.cris.com/∼ketchup/genedoc.shtml) and Adobe Illustrator. Parasite cDNAs encoding PKG open reading frames were modified prior to subcloning by appending an NH2-terminal or COOH-terminal FLAG epitope (Kodak) via PCR amplification (Pfu polymerase, Stratagene). DNA fragments encoding FLAG epitope-tagged PKGs were placed in an expression vector under the control of aToxoplasma α-tubulin promoter (23Soldati D. Boothroyd J.C. Science. 1993; 260: 349-352Crossref PubMed Scopus (298) Google Scholar). Following electroporation, stable transgenic Toxoplasma lines were selected with chloramphenicol (24Kim K. Soldati D. Boothroyd J.C. Science. 1993; 262: 911-914Crossref PubMed Scopus (237) Google Scholar, 25Black M. Seeber F. Soldati D. Kim K. Boothroyd J.C. Mol. Biochem. Parasitol. 1995; 74: 55-63Crossref PubMed Scopus (82) Google Scholar) and clones expressing recombinant PKG were identified by immunofluorescence analysis with FLAG M2 antisera (Sigma). Recombinant FLAG-tagged EimeriaPKG was purified from parasite lysates by FLAG immunoaffinity chromatography as described for the recombinant ToxoplasmaPKG. 3Donald, R. G. K., Allocco, J., Nare, B., Singh, S. B., Salowe, S. P., Wiltsie, J., and Liberator, P. (2002)Eukaryotic Cell, in press. Antisera were raised in rabbits to the following peptides based on amino acid sequence of the EtPKG full-length clone: EDTQAEDARLLGHLEKREKT (TR3) and EEDEGIELEDEYEWDKDF (TR6). Both peptides were conjugated to Keyhole Limpet hemocyanin prior to immunization. Antibody production was performed at Covance Research Laboratories, Denver, PA. Kinase activity was detected using a peptide substrate and [γ-33P]ATP. An aliquot containing enzyme (1 μl) was mixed with a reaction mixture (10 μl) whose composition is as follows: 25 mm HEPES pH 7.4, 10 mm MgCl2, 20 mmβ-glycerophosphate, 5 mm β-mercaptoethanol, 10 μm cGMP, 1 mg/ml bovine serum albumin, 400 μm Kemptide, or 9 μm myelin basic protein, 2 μm [γ-33P]ATP (0.1 mCi/ml). The reaction was allowed to proceed for 1 h at room temperature and then terminated with the addition of phosphoric acid to a final concentration of 25 mm. Labeled peptide was captured on P81 filters or on Millipore 96-well plates (MAPH-NOB). In both cases filters were washed with 75 mm phosphoric acid, dried, and33P-labeled phosphopeptide was detected by liquid scintillation counting. AMP-PCP and guanethidine (1-(2-guanidinoethyl)octahydroazocine) were obtained from Sigma. Kinase assays were performed in 50-μl reaction volumes containing 25 mm HEPES pH 7.0, 10 mmMgCl2, 20 mm β-glycerophosphate, 1 mm dithiothreitol, 0.1 mg/ml bovine serum albumin, 10 μm cGMP, between 2 and 12 μCi of [γ-33P]ATP, and varying levels of ATP, peptide substrate (biotinyl-e-aminocaproyl-GRTGRRNSI-OH), and inhibitor. For the two substrate pattern, ATP and peptide were each varied at four concentrations between 0.5 and 3 times their respectiveKm values. For inhibition patterns, one substrate concentration was fixed at 10 μm while the other was varied at four concentrations between 0.5 and 3 times itsKm; the inhibitor concentration was varied between 0 and 3 times its Ki. Reactions were initiated with 5 μl of enzyme (or buffer for the background) and incubated for 30 min in a heating block at 30 °C. The assays were terminated by the addition of 25 μl of 8 m guanidine-HCl solution (Pierce) before spotting 15 μl onto a SAM2®streptavidin membrane (Promega). The membrane was washed twice with 1m NaCl and twice with 1 m NaCl, 1% H3PO4 on a rotating mixer for 20 min. The membrane was then rinsed successively with water and ethanol and dried under a heat lamp. The individual assays were then separated, placed in scintillation vials containing 2 ml of Ultima Gold mixture (Packard), and counted in a Packard TriCarb 2500 liquid scintillation counter. After subtracting the appropriate background for each assay point, the data was fit to the appropriate equation using GraFit (Erithacus Software): v =Vmax[A][B]/(KA[B] + KB[A] +KIAKB + [A][B]) for two substrate variation;v = Vmax/(1 +Km(1 + [I]/Kis)/[S]) for competitive inhibition; v =Vmax/(1 + [I]/Kii +Km(1 + [I]/Kis)/[S]) for noncompetitive inhibition. Km andKi values are reported with their standard errors derived from the fit. Activation experiments were also performed as described above except that cGMP concentration was varied and ATP and peptide concentrations were fixed at 20 μm. Data was fit to the following modified Hill equation using Kaleidagraph (Synergy Software): V = V0 + (Vmax − V0)/(1 + (K50/[cGMP])h). Values for K50, the concentration for half-maximal activation by the allosteric activator cGMP, and h, the Hill coefficient, are reported with their standard errors derived from the fit. In-gel kinase assays were performed using the In-gel Protein Kinase Assay Kit (Stratagene, number 206020) according to the manufacturer’s recommendation, except that myelin basic protein was used as the substrate and cGMP was added to 20 μm in the kinase assay buffer along with [γ-32P]ATP. Chromatography on cGMP-agarose was performed according to the manufacturers instructions (Biolog, A019). Briefly the 0.6-ml column was equilibrated with Buffer G (50 mm HEPES pH 7.4, 10% glycerol, 10 mmsodium fluoride, 0.1 mm sodium orthovanadate, 1 mm EDTA). The sample (crude S100 or purified protein) was mixed with an equal volume of Buffer G and applied to the column, which was then washed with 10 ml of the same buffer. The column was then washed with 10 ml of Buffer G containing 1 mm GMP. Proteins were eluted with 10 ml of Buffer G containing 15 mmcGMP. Phase separation was performed according to the method of Bordier (27Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). The trisubstituted pyrrole, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1, Fig. 1A), prevents the in vitro development of several intracellular Apicomplexan parasites including E. tenella, T. gondii, N. caninum, and B. jellisoni (Fig.1B). Moreover, Compound 1 is orally active against E. tenella and E. acervulina in parasite-infected chickens at a dose of 50 ppm in the feed (Fig. 1C) and is also efficacious in a mouse model of Toxoplasmosis (28Nare B. Allocco J. Liberator P. Donald R.G.K. Antimicrob. Agents Chemother. 2002; 46: 300-307Crossref PubMed Scopus (52) Google Scholar). To identify the molecular target of this compound, a binding assay was developed using a tritiated version of Compound 1 as ligand. Saturable binding to an S100 extract of E. tenellacan be competed with unlabeled ligand with an IC50 of 10 nm (data not shown). The ligand-binding protein was purified from the E. tenella S100 fraction by conventional chromatography using a four-column protocol that is summarized in Fig.2A. There is only a single peak of binding activity detected in three of the four column profiles. The one exception to this is a small amount of activity detected in the flow through from the first column in the series, HiLoad Q-Sepharose. Fig. 2B illustrates the profile of binding activity following the final chromatographic step on MonoQ anion exchange. Gel electrophoresis of proteins in fractions that correspond to the peak of binding activity from MonoQ are visualized by silver staining in Fig.2C. Two proteins with apparent molecular weights of 120,000 and 150,000 are coincident with the peak of binding activity. Subsequent gel filtration chromatography of a pool of fractions 34 and 35 was able to enrich for the 120- and 150-kDa pair, but was not able to resolve the two proteins (data not shown). The structural similarity between Compound 1 and protein kinase inhibitors that have been described in the literature (29Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3175) Google Scholar, 30Lisnock J. Tebben A. Frantz B. O'Neill E.A. Croft G. Li S.J. O'Keefe B. Hacker C. de Laszlo S. Smith A. Libby B. Liverton N. Hermes J. LoGrasso P. Biochemistry. 1998; 37: 16573-16581Crossref PubMed Scopus (112) Google Scholar) prompted us to assay for and detect kinase activity (using the peptide substrate Kemptide) in the ligand binding fractions during the purification protocol. However, protein kinase activity was marginal (data not shown), even in the final fractions from the MonoQ column pictured in Fig. 2C. Following electrophoretic resolution of the column purified ligand binding activity, both the 120- and 150-kDa proteins were submitted for sequence analysis. Seven tryptic peptide sequences were generated from the 120-kDa protein (Table I), but no sequence information was retrieved from the larger protein. Each of the peptides was used as a query to search in silicotranslations of nucleotide data bases as well as protein data bases, but no significant matches were found. Several degenerate oligonucleotides were designed based on the first four peptides and used in various combinations in coupled RT-PCR reactions with E. tenella sporozoite mRNA as template. One primer set consisting of oligonucleotides from peptides 3 and 4 generated a PCR product that was successfully nested in a secondary reaction with a second oligonucleotide from peptide 3 along with the original peptide 4 oligonucleotide. The primary PCR product from this series was cloned and its
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