Key points are not available for this paper at this time.
The trisubstituted pyrrole 4-2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-ylpyridine (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-ylpyridine (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). the and catalytic of PKG are of the In the of cGMP, PKG a that is L. S.H. Corbin J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of cGMP to two within each of the an conformational change that kinase the two of have significant the of and by the cyclic nucleotide are In this report we that Apicomplexan parasite PKG has several features that from PKG in other mammalian and PKG which are the E. tenella enzyme is a The E. tenella enzyme has remarkably different cGMP characterized by a kinase activity in the of nucleotide cofactor which is by as as 1000-fold of the a feature that is not as striking in mammalian PKG. The of the parasite enzyme, biochemically and by cloning of Apicomplexan is than most PKG that have been with the of other the of the parasite is the amino-terminal of the protein and the catalytic is A parasite signature sequence of nearly 300 amino acids the regulatory and catalytic of the this we have identified a third feature that is but the of Apicomplexan parasite enzymes. with tenella The unsporulated oocysts from the and to the of M. J. PubMed Scopus Google and by for the in was in on with tenella oocysts on and oocysts on was for the of The was on and efficacy was by for the in vitro of parasites and of IC50 and or as the which parasite growth was for compounds to for E. tenella M. J. PubMed Scopus Google gondii Google and Biol. 1994; PubMed Scopus Google Scholar). The M. Biochem. Parasitol. 1997; PubMed Scopus Google was for by of from to The and T. gondii from and for binding by tenella unsporulated oocysts with an of 1 and inhibitor and an of for The was 1 and the used tenella protein was in a of and 1 using a 1000-fold of Compound for 1 and in for 1 or The with and by using The was from the to the and with to lysates and as The was 30 1 1 and The of was to a and with a in A. from for binding that to the and Compound 1 binding activity 1 1 that is with The was to a two with and the was with a reverse 1 to in as using the Compound 1 binding and 1 The was two and protein was with a to Compound 1 binding from the and to an which been in A with The was with in A and with a to 1 which was the proteins been The Compound 1 binding which not to the in was A and to the proteins been in the of and the to proteins with a to 1 in A. and for binding activity and also for on In cases with binding activity to a in A and as was on to and proteins using the sequence of the proteins was on The with by on reverse and sequence on using to standard J.F. A Scholar). was using or the sequence was using an and and using from or used for the reaction from Science. using and into the to be used as with and from or from from the E. tenella protein used to several used in reverse with from E. as of cloned and A by was used as a to full-length from tenella unsporulated P. J. M. M. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). for T. gondii PKG from a from the and identified by a using a of the E. tenella as expressed sequence a of a PKG was identified in a sequence of the sequence Nelson Biochem. Parasitol. PubMed Scopus Google the PKG a from this expressed sequence was used to a and A PKG open reading frame two was identified by sequence identical open reading frame was also in a of the by the of V. V. M. 1999; PubMed Google alternative was to PKG from both Eimeria and The first a to PKG for each an of the deduced amino sequence from for E. tenella and T. gondii PKG of sequence within the cGMP-binding and and catalytic and from E. oocysts and a of P. was first into using reverse The of used as in using from a and reaction used as in using from and/or reaction in the sequence and used as to E. to full-length PKG and deduced protein sequence for E. E. have been in with and using the J. A. J. W. Y. 1999; PubMed Scopus Google Scholar). The was using and PKG open reading to by an NH2-terminal or in an the control of Science. PubMed Scopus Google Scholar). with K. Science. PubMed Scopus Google Scholar, M. K. Biochem. Parasitol. 1995; PubMed Scopus Google and recombinant PKG identified by with FLAG-tagged was from parasite lysates by as for the recombinant S. S. and P. in in to the on amino sequence of the EtPKG full-length and to to was activity was using a and enzyme was with a reaction is as cGMP, 1 or The reaction was to for 1 and with the of to a of was on or on In both cases with was by and from in reaction 1 cGMP, and of and of and the two ATP and each and their was while the other was and the inhibitor was and its initiated with of enzyme for the and for 30 in a 30 The by the of of a The was with and with 1 on a for The was with and and a The in of and in a the for each the was to the using for two for for are with their standard from the Activation also as that cGMP was and ATP and Data was to the using for the for by the cGMP, and the are with their standard from the kinase using the to the that protein was used as the and cGMP was to in the kinase with on was to the the was with 1 The or was with an of and to the which was with of the The was with of 1 with of was to the of J. Biol. Chem. Full Text PDF PubMed Google Scholar). The trisubstituted 4-2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-ylpyridine (Compound the in vitro development of several intracellular Apicomplexan parasites including E. T. and B. Compound 1 is E. tenella and E. in a of in the and is also in a of B. J. P. PubMed Scopus Google Scholar). identify the molecular target of this a binding was using a tritiated of Compound 1 as binding to an of E. be with with an IC50 of not The protein was from the E. tenella by using a that is in is a single of binding activity in of the The to this is a of activity in the from the first in the the of binding activity the on of proteins in that to the of binding activity from are by in Two proteins with molecular of and are with the of binding of a of and was to for the and but was not to the two proteins not The Compound 1 and protein kinase that have been in the S. M.J. P.R. 1994; PubMed Scopus Google Scholar, J. A. B. B. S. A. B. J. P. 1998; PubMed Scopus Google to for and kinase activity the in the binding the purification protein kinase activity was not in the from the in of the binding both the and proteins for sequence from the protein but no sequence was from the of the was used as a to in of nucleotide as well as protein but no significant on the first and used in in with E. tenella as of from and a that was in a reaction with a from with the The from this was cloned and its has an open reading frame that is identical to both PKG and the regulatory of PKA of from the Compound 1 binding protein and their within the open reading frame deduced from the E. tenella residues in the deduced amino sequence from the in a new on the that the protein is a cyclic nucleotide-dependent protein enzyme activity in the was in the of cyclic cAMP a not cGMP stimulated kinase activity by Compound 1 inhibited the cGMP-dependent activity with an IC50 of nm. are consistent with the that the activity of Compound 1 is in to of of native and recombinant expressed is the of cGMP for PKG to PKG expressed gondii using is the of cGMP for PKG to PKG expressed gondii in a new using of nucleotide-dependent kinase in of E. tenella by is in using the binding was a of nucleotide-dependent kinase activity that was not by this was a single of PKG activity in this purification and this activity was inhibited by Compound 1 of The of binding activity with the PKG activity not In two of PKA activity in this of these from the cGMP activity and the cAMP activity in these was to Compound 1 of that the protein is parasite PKG, kinase The in in this The in that kinase activity was in the of in the of activity was in the of kinase activity was inhibited by Compound 1 not activity was in the to the protein that with E. tenella PKG the The of Compound 1 binding activity ion as in was characterized using of PKG activity is with binding activity A of the cGMP an of the protein these the that the protein from E. tenella is a PKG. A of the of native E. tenella PKG was using a of both an with of for ATP and 1 for the that EtPKG in with protein a both are to the enzyme to the of with used to the binding in the ATP was a inhibitor ATP and with to was with the and are consistent with the of to the enzyme. Compound 1 was also to be a potent and inhibitor by a of the of PKG to using in a new PKG to using The as a was used as a to an E. tenella unsporulated and several to The in this group is in with a deduced open reading frame of amino of for a protein of nearly has a and a also each of for the open reading of the from the protein are within this open reading The deduced amino sequence of the full-length protein most to the cGMP-binding in the amino-terminal of the protein and to the catalytic the of the PKG from E. T. and P. have also been and the deduced amino of the parasite proteins are in The parasite PKG proteins are in the amino-terminal share sequence their The parasite proteins are also in with a is that the parasite PKG proteins are than the enzyme and other that have been in the is a PKG J. Biol. Chem. Full Text PDF PubMed Google that is from to for a A of parasite and PKG in to a of nearly 300 amino acids the and catalytic that is found in the parasite PKG but is from The signature sequence in the parasite proteins for the of the in to other of the amino sequence in this has identified a third cGMP-binding which is in the enzyme. of the E. tenella PKG with an amino-terminal has been in the Apicomplexan parasite T. S. S. and P. in S. and K. in and recombinant PKG enzyme are in as for ATP and in of native and recombinant enzyme activity by Compound 1 is also The activity of both in the of cyclic nucleotide was and to The which represents the of the and the be with This is which has enzyme activity in the of nucleotide The cGMP nucleotide cofactor both native EtPKG and and to a by of activity in a cGMP a for both native and recombinant with of and The cyclic nucleotide-dependent of native and recombinant EtPKG are and are different from mammalian PKG J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google two from the deduced E. tenella PKG sequence. In an from E. each two proteins and of these with the protein The also with a protein in the binding the purification purification a is this protein is not in the or in is to a The protein that with the protein not with E. tenella was also by of the with a single was This protein with the in the In a the into the while the protein was found in the of these that the protein is Since each of the proteins to and is from a not that two isoforms of E. tenella PKG. The soluble E. tenella PKG isoform has been in and to amino-terminal sequence The NH2-terminal sequence with serine within the open reading frame deduced from the this a is to which in is in a translation initiation the soluble PKG isoform be the of an translation initiation The membrane-associated PKG which is to be the amino P. Biochem. Parasitol. PubMed Scopus Google has not been The membrane-associated and soluble isoforms of native PKG are to by Compound with IC50 of and is inhibited by Compound 1 with an IC50 of nm. most PKG are is not for catalytic activity M. T. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is by a that is the amino-terminal of PKG proteins T. T. J. U. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, S.H. Corbin J.D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). no such be within the parasite PKG sequence. EtPKG as a soluble protein from and by with an molecular and not Since the soluble native protein the amino acids from the we a role for these in the full-length recombinant also as a not This that the of the amino residues of the open reading frame is not to cause to the molecular of the membrane-associated isoform of PKG are by the of the proteins with and are not The in this the activity of Compound both in and animal Compound 1 is to known protein kinase S. M.J. P.R. 1994; PubMed Scopus Google Scholar, J. A. B. B. S. A. B. J. P. 1998; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. PubMed Scopus Google and we that this a kinase in the PKG. other protein kinase Compound 1 is with to protein kinases the that inhibitor a of Compound 1 binding activity a single of binding activity was the purification binding with or not be using the we that PKG the and Compound in E. tenella. The of Compound 1 to parasite enzyme activity is a for development as an The of parasite and PKG corresponds to the catalytic is of the catalytic of mammalian PKG using the for the catalytic of has to several residues for ATP binding and catalytic activity Dostmann W. A. W. P. Biophys. PubMed Scopus Google Scholar). of these residues is in the parasite PKG the amino of residues for ATP binding and catalytic Compound 1 is a inhibitor of parasite enzyme PKG, by and ion is inhibited by Compound of and J. In the of cyclic PKG in an L. S.H. Corbin J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, W. V. PubMed Scopus Google in which the amino-terminal of the protein with the catalytic with this of mammalian PKG activity be nucleotide of cGMP to the isoform kinase activity to with both native and recombinant tenella PKG have kinase activity in the of nucleotide of cGMP, the of of parasite kinase activity is to 1000-fold and the of that cyclic nucleotide is a The deduced amino sequence of PKG from Apicomplexan parasite A and contains a of nearly 300 amino acids that not with mammalian or in this of the parasite proteins is a third cGMP-binding a feature that distinguishes the parasite from other The for nucleotide binding is not striking PKG PKG PKG and two residues are for both cAMP and cGMP binding to PKA and PKG, J. Taylor S.S. PubMed Scopus Google Scholar, Corbin J.D. PubMed Scopus Google Scholar). The of the PKA regulatory (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 to a that with the of the nucleotide This is residues by a that with the of the cyclic The amino a serine or threonine in PKG but not a with the amino group of in of mammalian kinases to this on the cGMP cAMP binding of PKG and PKA L. Corbin J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of nucleotide with each of the parasite cGMP-binding we that each to the of the parasite kinases P. Biochem. Parasitol. PubMed Scopus Google that are two isoforms of parasite PKG a feature of mammalian cGMP-dependent of E. tenella A. and J. W. gondii that PKG is for by a single gene in these The two found in for but distinct PKG In cases a is also to isoform W. V. PubMed Scopus Google Scholar). of E. tenella and T. gondii identified that the of the two parasite of the full-length EtPKG and each two proteins with that with the native isoforms P. Biochem. Parasitol. PubMed Scopus Google Scholar). sequence of the soluble native isoform of E. tenella PKG has identified as the first amino This is to which in is in a initiation a exists in that the soluble PKG isoform result from a initiation an This is by P. Biochem. Parasitol. PubMed Scopus Google which that isoform be by amino that and The NH2-terminal amino sequence of EtPKG and deduced from the share a signal that both of these expressed as are and P. Biochem. Parasitol. PubMed Scopus Google Scholar). have to that is of of the PKG of mammalian is for of this PKG of this isoform is critically to regulate a E. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The two of E. tenella PKG that be have different in by their that the molecular isoform is and the molecular isoform is be that the isoform with the we have not found this to be the The contains an of the of the in the of are to of the soluble isoform of the isoform with these proteins that are with (reviewed in Ref. 1997; Scopus Google and have been found in soluble biochemical E. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the E. tenella is to an that the isoform is found in the the parasite is in and to ion in the two isoforms and in this The most for this result is that the isoform in the of to to the and is to be for the binding activity identified in the the amino-terminal of are not well molecular has that this of the protein has several domains. The T. T. J. U. Sci. U. S. A. 1994; PubMed Scopus Google found in most is for of this family of This is from each of the parasite the of a PKG has been to a role in the of PKG with cellular proteins 1994; PubMed Scopus Google Scholar). is that PKG, by NH2-terminal or by is in to cGMP M. J. PubMed Scopus Google Scholar). report in this which that native soluble E. tenella PKG as a enzyme. Since native soluble EtPKG the NH2-terminal amino residues of the full-length be that the full-length protein is a of its and with full-length PKG is to by The is a full-length protein that is not but the first amino acids of the open reading This recombinant EtPKG in with the native soluble PKG, that the amino acids of EtPKG not a on these we that both parasite PKG isoforms are The of for cGMP-dependent protein kinases in has in the several years A. J. J. Sci. PubMed Google Scholar, J. 1997; PubMed Scopus Google Scholar). A role for PKG has been in distinct processes including the control of intestinal and P. A. A. P. M. Sci. U. S. A. PubMed Scopus Google Scholar). several of these processes not have in the The biochemical role of PKG in the parasite is is to that on kinase to the of new or chemotherapeutic S. for the of Compound 1 and for reading of the the of and for to and for a to
Gurnett et al. (Wed,) studied this question.