Anticoccidial kinase inhibitors: Identification of protein kinase targets secondary to cGMP-dependent protein kinase

Molecular & Biochemical Parasitology 149 (2006) 86–98

Anticoccidial kinase inhibitors: Identification of protein kinase targets secondary to cGMP-dependent protein kinase夽 Robert G.K. Donald a,∗ , Tanya Zhong a , Helen Wiersma a , Bakela Nare a , Dan Yao b , Anita Lee c , John Allocco a , Paul A. Liberator a b

a Department of Infectious Diseases, Merck & Co., P.O. Box 2000, R80Y-260 Rahway, NJ 07065-0900, USA Department of Drug Metabolism/Labeled Compound Synthesis, Merck & Co., P.O. Box 2000, R80Y-260 Rahway, NJ 07065-0900, USA c Department of Molecular Profiling/Proteomics, Merck & Co., P.O. Box 2000, R80Y-260 Rahway, NJ 07065-0900, USA

Received 16 January 2006; received in revised form 1 May 2006; accepted 4 May 2006 Available online 23 May 2006

Abstract Trisubstituted pyrrole inhibitors of the essential coccidian parasite cGMP dependent protein kinase (PKG) block parasite invasion and show in vivo efficacy against Eimeria in chickens and Toxoplasma in mice. An imidazopyridine inhibitor of PKG activity with greater potency in both parasite invasion assays and in vivo activity has recently been identified. Susceptibility experiments with a Toxoplasma knock-out strain expressing a complementing compound-refractory PKG allele (‘T761Q-KO’), suggest a role for additional secondary protein kinase targets. Using extracts from this engineered T. gondii strain and a radiolabeled imidazopyridine ligand, a single peak of binding activity associated with calmodulin-like domain protein kinase (CDPK1) has been identified. Like PKG, CDPK1 has been implicated in host cell invasion and exhibits sub-nanomolar sensitivity to the compound. Amino acid sequence comparisons of coccidian CDPKs and a mutational analysis reveal that the binding of the ligand to PKG and CDPK1 (but not other CDPK isoforms) is mediated by similar contacts in a catalytic site hydrophobic binding pocket, and can be blocked by analogous amino acid substitutions. Transgenic strains over-expressing a biochemically active but compound-refractory CDPK1 mutant (‘G128Q’) fail to show reduced susceptibility to the compound in vivo, suggesting that selective inhibition of this enzyme is not responsible for the enhanced anti-parasitic potency of the imidazopyridine analog. An alternative secondary target candidate, the ␣-isoform of casein kinase 1 (CK1␣), shows sensitivity to the compound in the low nanomolar range. These results provide an example of the utility of the Toxoplasma model system for investigating the mechanism of action of novel anticoccidial agents. © 2006 Elsevier B.V. All rights reserved. Keywords: cGMP-dependent protein kinase; CDPK; CK1; Toxoplasma; Eimeria

1. Introduction Signal transducing protein kinases are proven therapeutic targets for the treatment of certain cancers and inflammatory diseases. Drugs that block the EGFR tyrosine kinase receptor (Iressa and Tarceva) and the hyperactive Bcr-Abl tyrosine

Abbreviations: CDK, cyclin dependent protein kinase; CDPK, calmodulinlike domain protein kinase; CK1, casein kinase I; IFA, immuno-fluorescence analysis; IP, immuno-precipitation; PKG, cGMP-dependent protein kinase 夽 Note: New T. gondii and E. tenella gene sequence data reported in this paper are available in the Genbank database under accession numbers DQ205646 (TgCDPK3), DQ205647 (EtCDPK2), DQ205648 (EtCDPK1) and AY532161 (EtCK1␣). ∗ Corresponding author. Tel.: +1 732 594 0671; fax: +1 732 594 6708. E-mail address: robert [email protected] (R.G.K. Donald). 0166-6851/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2006.05.003

kinase (Gleevec) are already in clinical use [1–4]. Inhibitors of serine-threonine protein kinases are currently in clinical trials for use as anti-mitotic agents (cyclin dependent kinase, CDK) [5–9] and to inhibit cytokine-mediated inflammatory responses (p38␣ MAP kinase) [10,11]. Protein kinases are also important for the growth of parasitic protozoa and several are under investigation as potential drug targets [12,13]. Clearly, the evolutionary gap between vertebrates and their pathogens presents opportunities for the design of selective compounds that inhibit essential protein kinases of the parasite but not orthologous host cell enzymes. For example, a comparison of the crystal structures of inhibitor-bound Plasmodium falciparum PfPK5 and mammalian CDK has identified variable regions of the catalytic site that may permit significant gains in inhibitor potency and selectivity upon further refinement by medicinal chemistry [14]. The unexpected selectivity of mammalian CDK inhibitors of the

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purvalanol class for an isoform of parasite casein kinase 1 provides another illustration of potentially exploitable differences between parasite and host enzymes [15,16]. The discovery of a potent trisubstituted pyrrole inhibitor (compound 1) of cGMPdependent protein kinase (PKG) with efficacy against Eimeria species and Toxoplasma in animal models, has confirmed that protein kinases are viable therapeutic targets against pathogenic protozoa [17,18]. To understand the basis for inhibitor selectivity as well as the underlying biological mechanism, the PKG target was validated with the use of the Toxoplasma model system [19]. Available evidence suggests that Toxoplasma is a useful surrogate for Eimeria parasites to better understand the mechanism of activity of PKG inhibitors as anticoccidial agents [20,21]. In practical terms, T. gondii tachyzoites are both easy to culture and amenable to genetic manipulation [22,23]. In contrast, Eimeria sporozoites do not propagate beyond a single round of schizogeny in stable cell lines and preparation requires a great deal of effort, involving isolation of oocysts, sporulation and purification on percoll gradients [24]. Significantly, a close correlation between the potency of a variety of PKG inhibitors against T. gondii tachyzoites and Eimeria tenella sporozoites in whole cell in vitro assays has been observed (R. Donald, unpublished data). Toxoplasma has also been a source of active recombinant Eimeria and Toxoplasma PKG enzymes (EtPKG and TgPKG) for use in functional studies [25]. The biochemical properties of these coccidian enzymes are indistinguishable in vitro and they appear to be functionally equivalent in tachyzoites, as the Eimeria enzyme can complement a TgPKG null mutant in trans [19]. Similarity modeling of the PKG catalytic site based on the crystal structure of mammalian p38␣ MAP kinase suggests that unique structural features of the cGMP-dependent protein kinase of coccidian apicomplexans can account for the potency and selectivity of anti-parasitic trisubstituted pyrrole compounds [19,20]. Related to analogous pyrrole or imidazole inhibitors of p38MAP kinase [11,26,27], these compounds exploit a hydrophobic binding pocket that overlaps the ATP binding site. For most protein kinases, access to this potential pocket is blocked by bulky residues. For parasite PKG or for p38␣, but not for vertebrate PKGs or most other MAP kinase family members, a threonine or serine residue at the base of this pocket makes a stabilizing contact with an inhibitor fluorophenyl moiety. Although other ligand–enzyme interactions contribute to the high-affinity binding, introduction of a bulky residue at this ‘gatekeeper’ position is sufficient to prevent compound binding [19,28,29]. Such compound-refractory mutants, in the context of in vitro enzyme assays or expressed in transgenic Toxoplasma strains, have served as useful reagents for pharmacological validation of PKG as well as providing insight into the biochemical role of this enzyme in apicomplexan parasites. Selective inhibition of PKG in susceptible wild-type parasites, but not in compound-refractory transgenic strains, blocks the secretion of adhesins that mediate parasite motility and host cell invasion [30]. Such inhibition is sufficient to confer long-term survival in mice infected with a susceptible wild-type strain in a Toxoplasmosis mouse model [18,19].

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Although trisubstituted pyrrole inhibitors of PKG have broad spectrum activity against coccidian parasites, they lack sufficient potency for commercial development. A medicinal chemistry effort centered on an imidazopyridine scaffold has yielded a new series of significantly more potent anticoccidial PKG inhibitors [21]. The activity of a representative lead compound, 4-[7-[(dimethylamino) methyl]-2-(4-fluorophenyl)imidazo[1,2a]pyridin-3-yl]pyrimidin-2-amine, is described and will be referred to herein as compound 2. Compound 2 is somewhat more potent as an inhibitor of PKG enzyme activity compared with the earlier trisubstituted pyrrole lead, but is substantially more potent in both in vitro and in vivo anti-parasitic assays. Further molecular characterization reveals that compound 2 is also a potent inhibitor of two additional parasite protein kinases, CDPK1 and CK1␣. The implications of these results will be discussed in light of precedent for these enzymes as potential therapeutic targets. 2. Materials and methods 2.1. Host cells and parasite cultures RH
HXGPRT strain tachyzoites of T. gondii or derived transgenic lines were maintained by serial passage in confluent monolayers of human foreskin fibroblasts (HFFs) as described [22]. A modified [3 H]-uracil uptake assay [31] adapted for use in 96-well scintillation plates (Cytostar-T, Amersham) was used to measure parasite growth inhibition. Each well, previously seeded with HFF cells, was inoculated with T. gondii tachyzoites (2 × 104 ) in 200 ␮L of growth media containing 2 ␮Ci [5,6]-3 Huracil (Perkin-Elmer NEN) and serially diluted compound. Following incubation at 37 ◦ C for 48 h, tritium-incorporation into host cell monolayers was directly counted in a microplate scintillation counter (MicroBeta, Perkin-Elmer-Wallac). E. tenella sporozoites of the Merck LS18 strain were prepared and purified as described [24,30,32]. 2.2. Parasite invasion and adhesion secretion assays Procedures for measuring the effects of PKG inhibitors on E. tenella sporozoite and T. gondii tachyzoite invasionrelated processes are identical to those detailed in an earlier publication from this group [30]. The extracellular attachment of parasites to pre-fixed Madin Darby bovine kidney (MDBK) or HFF cell monolayers was quantified by IFA with sporozoite or tachyzoite specific antisera. In parallel, parasites were allowed to invade unfixed monolayers. After methanol fixation and IFA, the percentage of intracellular parasites was then calculated by subtraction (total minus extracellular count). A FITC-dextran retention assay [33] that illuminates host cells transiently permeabilized by traversing sporozoites was used to measure parasite mobility. Secreted E. tenella adhesins MIC1 and MIC2 [34] were quantified by Western blot. Relative quantities of protein secreted with each compound treatment were determined by densitometry of scanned images and apparent IC50 values calculated from dose–response plots.

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2.3. Purification of protein kinase targets Small-scale fractionation of T. gondii tachyzoite lysates on a 100 ␮L anion exchange FPLC column (SMART system MonoQ, GE-Pharmacia) was done essentially as described previously [19]. An S100 detergent extract was prepared from a cell culture pellet derived from 10 T-175 cultures (2 × 109 parasites) of strain TgPKG-T761Q-KO. Samples of FPLC fractions (10–20 ␮L) were assayed in a 100 ␮L ligand binding reaction (20 mM Hepes pH 7.4; 1 mM MgCl2 , 1 mM DTT, 0.1 mg/mL BSA) containing tritiated compound 2 (50 nM, 71 Ci/mmol) in the presence or absence of 50 ␮M cold compound 2. Reactions were incubated on ice for 30 min before filtering through a Whatman 24 mm glass fiber (GF/B) disc presoaked in 0.15% PEI and 0.25% TritonX100. Filters were rinsed three times in 3 mL of 0.5% TritonX100, dried and counted in scintillation fluid. No binding activity was detected in flow-through fractions. Fractions with peak binding activity were pooled and subjected to hydrophobic interaction chromatography (HIC) on a 100 ␮L phenyl superose FPLC column. The column was pre-treated with 0.5 mg/mL BSA to reduce non-specific binding. Sample was applied to the column in 1.0 M ammonium sulfate and protein was eluted with a descending gradient (1.0 M → 0). In-gel casein kinase activity of HIC fractions was assayed using a kit (Stratagene). Prior to polymerization, 0.5 mg/mL casein (Sigma) was added to the acrylamide polymerization mix. For large-scale fractionation, 100 T-175 flasks of cultured parasites were harvested (2 × 1010 parasites) and 3 mL of the clarified S100 lysate applied to a 5 mL HiTrapQ FPLC column (GE-Pharmacia). For the second HIC chromatography step, ¨ peak casein kinase fractions were applied to a 1 mL HIC (AKTA system) using the same conditions as for the small-scale purification. Peak fractions of casein kinase activity eluted from the HIC column with ammonium sulfate were pooled and loaded onto a 1 mL casein column. The casein affinity matrix was prepared by coupling a 5 mg/mL solution of casein (1:10 dilution of Sigma 5% solution) to an activated aminolink Plus® agarose according to directions provided in the immobilization kit (Pierce). Bound protein was eluted from the casein column with 0.8 M arginine HCl (Sigma). Recombinant TgCK1␣ and TgCK1␤ expressed in Escherichia coli were purified as described [16]. As heterologous expression of the orthologous EtCK1␣ from E. tenella in E. coli was not successful, native enzyme was purified from unsporulated oocysts (2.7 × 1010 ). The purification scheme is essentially the same as detailed in a related manuscript for the purification of the orthologous CK1 enzyme from Leishmania major promastigotes [35]. Essentially it involved tracking and pooling CK1 activity (with selective phosphopeptide substrate) across three successive chromatography steps: anion exchange (HiLoadQ 26/10; ascending NaCl gradient); HIC (phenyl sepharose; descending ammonium sulfate gradient) and cation exchange (MonoS HR 5/5; ascending NaCl gradient). A single peak of CK1 activity was identified that resolved to a single band on an SDS-PAGE gel stained with silver (not shown). The ∼40 kDa enzyme was excised and digested with trypsin, and the resulting peptides were

analyzed by nanoflow HPLC-micro-electrospray ionization tandem mass spectrometry. Collected tandem mass spectra were processed using TurboSEQUEST to search against the cloned E. tenella CKI␣ ORF (Genpept AAS46021). To confirm the identity of the tryptic peptides, an aliquot of the peptides was dried down and esterified in 2 N methanolic HCl, which results in methyl groups (or 14 Da) being added to the acidic groups of every peptide. The resulting methyl esters were analyzed and processed in the same manner as described above. Molecular profiling showed conclusively that the LC-MS/MS profile of the native protein matches the sequence of the cloned 39 kDa EtCK1␣ enzyme. Seven tryptic peptides were positively identified, and five of these peptides were confirmed following methyl ester derivitization. 2.4. Cloning and purification of coccidian CDPK enzymes The TgCDPK1 open-reading frame was PCR-amplified from a tachyzoite cDNA library (#1896, NIH AIDS reference reagent program) using published DNA sequence information [16,36]. Full-length cDNAs corresponding to other coccidian CDPKs were cloned from lambda phage libraries, using DNA probes amplified from gene fragments mined from gene databases. A partial EtCDPK1 gene sequence has been described (CAA96439) [37]. A distinct E. tenella CDPK2 gene sequence was identified from overlapping EST clones found in the Washington University Eimeria EST database (BG561058, BM306800, BE028353) [38]. A novel CDPK3 gene fragment was similarly located in the T. gondii EST database (overlapping TgEST clones 1374436, 33477501 and 33479355) [39]. Full-length E. tenella CDPK1 and CDPK2 cDNA clones were obtained, respectively, from merozoite and unsporulated oocyst stage cDNA libraries using standard procedures [40]. A fulllength T. gondii CDPK3 clone was isolated from the same tachyzoite cDNA library. Prior to expression vector subcloning, N- or C-terminal FLAG epitope tags were added to the TgCDPK1 open-reading frame by PCR with KOD polymerase (Novagen). Amplified gene fragments were cloned into a T. gondii tubulin promoter expression vector as described [16]. For E. coli expression of coccidian CDPKs, DNA fragments were blunt-ligated into the petBlue1 vector and resulting plasmids transformed into strain BL21 RosettaTM (Novagen). Cultures were induced with IPTG (2 mM) for 3 h at 37 ◦ C and cell pellets lysed with BugbusterTM reagent supplemented with a protease inhibitor cocktail (Novagen). Prior to batch adsorption onto 1 mL of agarose beads, NaCl was added to 0.5 M to reduce non-specific binding. Beads were washed three times in 5 mL of TBS (Tris–HCl pH 7.4; 0.5 M NaCl; 1 mM DTT, 10% glycerol; 0.1% NP40) and rinsed once in salt free buffer. Enzymes were then eluted with 0.5 mg/mL FLAG peptide in Hepes buffer (10 mM pH 7.4, 10% glycerol, 1 mM DTT), and washed three times in peptide-free buffer before concentrating in a Biomax-10K filtration unit (Millipore). Protein was measured with a micro-BCA reagent (Pierce) following a TCA-sodium deoxycholate step to remove residual FLAG peptide [41]. Using this single step affinity purification procedure, the overall recovery of recom-

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binant E. tenella and T. gondii CDPK enzymes from 100 mL of culture was ∼100 ␮g. Purity was estimated as greater than 90% from Coomassie blue staining of SDS-PAGE gels. The specific activity of the N-flag recombinant TgCDPK1 enzyme (9 ␮mol min−1 mg−1 ) matches closely the value described previously for the non-tagged E. coli recombinant [36]. TgCDPK1 mutations were introduced into E. coli and T. gondii expression plasmids using a Quickchange mutagenesis kit (Stratagene) and confirmed by DNA sequencing. All oligonucleotides were synthesized by IDT. Enzymes were assayed as described previously with phosphorylated peptide substrates captured on 96-well phosphocellulose filter plates (MAPH-NOB, Millipore) [16,25]. CDPKselective peptide substrates Syntide-2 and CaMKII (281-291) were obtained from Calbiochem. Partially dephosphorylated ␣casein and 5% total milk casein were obtained from Sigma. CK1 enzymes were assayed using the preferred phosphopeptide substrate KRRRALS(p)VASLPGL-OH (custom synthesis by Anaspec). Reference recombinant rat CK1␦ and calmodulin dependent protein kinase type II (CaMKII) enzymes were obtained from New England Biolabs. 2.5. Compounds The synthesis of compound 2 (4-[7-[(dimethylamino) methyl]-2-(4-fluorophenyl)imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine) is described in a related medicinal chemistry paper [21]. To synthesize radiolabeled compound 2, the precursor secondary amine (1.1 mg, 3.16 × 10−3 mmol) was dissolved in DMSO (200 ␮L) and stirred at room temperature under nitrogen. [3 H]-CH3 I (100 mCi in 100 ␮L toluene) was slowly added. The reaction mixture was stirred at room temperature for 1 h. The unreacted methyl iodide and toluene were removed under reduced pressure using a rotary evaporator. The residual was subjected to HPLC purification on Zorbax SBCN at 254 nm, eluted with 70% aqueous (0.1% TFA) and 30% acetonitrile isocratically in 30 min at 5 mL/min. The combined HPLC fractions were concentrated by passing through a Sep-Pak C18 cartridge to afford 10 mCi of tritiated compound 2 in 10 mL ethanol (specific activity, 82.8 Ci/mmol). The purified compound was stored in ethanol with anti-oxidant 2,6-di-tert-butyl-4-methyl-phenol.

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2.6. In vivo efficacy studies All procedures were performed in a facility accredited by the AAALAC and approved by the Merck IACUC. Procedures for the care and use of research animals at Merck Research laboratories meet or exceed all applicable local, national, and international laws and regulations. Efficacy of PKG inhibitors in the mouse toxoplasmosis model has been described [18]. Female C57BL/6J mice were infected by intraperitoneal injection with ∼103 tachyzoites of the respective T. gondii strains. Compound 2 (dissolved in PBS) was dosed at 25 mg/kg body weight by intraperitoneal injection starting 24 h after parasite inoculation and continuing twice daily for 10 days. Mice were monitored twice daily for signs of toxoplasmosis or mortality and moribund mice were promptly euthanized by CO2 gas. 3. Results 3.1. An imidazopyridine PKG inhibitor with potent anti-parasitic activity The structures of the lead trisubstituted pyrrole (compound 1) and imidazopyridine (compound 2) inhibitors of coccidian PKG are shown in Fig. 1. Compound 1 was identified using an empiric whole cell in vitro anti-parasitic screen [17]. It was originally synthesized as part of a glucagon receptor antagonist program [26] and was noted for its activity as an inhibitor of p38␣ MAP kinase (IC50 of 20 nM, [26]). Compound 2 was identified as part of a medicinal chemistry effort aimed at structural optimization of the heterocyclic imidazopyridine core [21]. The initiative was guided by structure–activity relationship (SAR) data from (i) PKG enzyme assays; (ii) whole cell anti-parasitic assays; and (iii) in vivo assays that evaluate compound activity against a spectrum of Eimeria species in chickens. The in vivo activity of compound 2 against four species of Eimeria is 8-fold more potent than compound 1 both with respect to the control of oocyst production and the prevention of intestinal lesions [17,21]. Like compound 1, compound 2 inhibits sporozoite motility and invasion in a variety of assays (Table 1). When sporozoites are allowed to invade Madin Darby bovine kidney cell monolayers in the presence of inhibitor, compound 2 is 4fold more potent than compound 1 in blocking uracil uptake

Fig. 1. The chemical structures of protein kinase inhibitors compound 1 (4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine) and compound 2, (4-[7-[(dimethylamino)methyl]-2-(4-fluorophenyl)imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine). The location of the tritium (T) label on the radioactive ligand is shown.

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Table 1 Effect of compounds 1 and 2 on Eimeria parasites in vitro IC50 values, nM

[3 H]-uracil

uptake Attachment Invasion Host-cell wounding Adhesin secretion MIC1(MIC2)

Compound 1

Compound 2

160 ± 50a

45 ± 13 20b 10b 12b 86 (70)

40 ± 10a 600 ± 300a 190 ± 50a 410 (470)a

Details of the E. tenella sporozoite invasion assays are described in [30] and summarized in Section 2.2. a MDBK assay data for compound 1 taken from [30]. b Representative experiment with compound 1 as control.

by intracellular parasites. In contrast, if parasites are allowed to invade prior to treatment, neither compound has a significant effect on intracellular growth even at levels as much as 50-fold the IC50 ([30], not shown). This result confirms that the primary mechanism of action of both inhibitors involves blocking parasite invasion. In IFA-based attachment and invasion assays in which either attached extracellular or intracellular parasites are quantified [30], compound 2 is also significantly more potent than compound 1. The ability of apicomplexan sporozoites to wound host cells through which they may traverse can be measured in a FITC-dextran retention assay [33]. In this assay, compound 2 is at least 10-fold more active than compound 1. The secretion of micronemal adhesins is thought to mediate parasite motility and invasion processes in apicomplexan parasites [42–44]. Compound 2 is ∼5–6-fold more effective at inhibiting the secretion of E. tenella MIC1 and MIC2 adhesins [45]. 3.2. Imidazopyridine potency contributed by activity at secondary protein kinase targets The imidazopyridine compound 2 is ∼2-fold more potent than compound 1 as an inhibitor of E. tenella [21] and T. gondii PKG (Table 2(A)). To determine whether the mechanism of ligand binding is similar, compound sensitivity of selected PKG mutants harboring substitutions at previously defined ‘gateTable 2 Effect of PKG catalytic site mutations on inhibitor potency (A) and anti-parasitic activity (B) Enzyme

Compound 1

(A) Inhibition of PKG enzyme activity (nM, IC50 ) TgPKG (‘wt’) 0.67 ± 0.14 TgPKG T761G 0.26 ± 0.09 (0.4×) TgPKG T761A 0.35 ± 0.02 (0.5×) TgPKG T761Q 21,000 (∼104 ×) TgPKG I824A 5.7 (9×)

Compound 2 0.37 ± 0.02 0.22 ± 0.06 (0.6×) 0.25 ± 0.07 (0.7×) 10,000 (∼104 ×) 5.3 (14×)

(B) Anti-parasitic activity (nM, IC50 for [3 H]-uracil uptake) RH (‘wt’) 235 ± 9 24 ± 8 TgPKG T761Q-KO 4000 ± 1000 (17×) 150 ± 42 (6×) Recombinant protein kinase activities and anti-parasitic efficacy (uracil uptake) were assayed as described [16,25]. Average IC50 values (nM) from several experiments are shown, and were calculated from dose–response curves. The fold increase or decrease in sensitivity of mutants to compound relative to ‘wildtype’ is indicated in parentheses. Compound structures are shown in Fig. 1.

keeper’ residues [19] was compared (Table 2). Computer modeling predicts, and mutational analysis confirms, that the TgPKG T761Q substitution prevents access of both compounds to the catalytic site hydrophobic binding pocket (IC50 ≥ 10 ␮M). Substitution of smaller alanine or glycine residues at this position (T761A or T761G) results in an ∼2-fold increase in sensitivity for both compounds (Table 2(A)). A second conserved residue in the ATP binding pocket of protein kinases forms the basis of a chemical genetics strategy devised by Shokat and collaborators [46–48]. This corresponds to isoleucine 824 for TgPKG (I833 for EtPKG). An alanine substitution here (I824A) reduces the potency of both compound 1 and compound 2 by about 10-fold. Unlike substitutions at T761 that have little effect on enzyme catalytic activity, the I824A mutant is significantly compromised, exhibiting a ∼10-fold lower specific activity (not shown). Since compound 1 and compound 2 are affected in a parallel fashion by mutations that either enhance or reduce inhibitor activity (primarily through steric effects), they appear to exploit the same structural features of the PKG catalytic site for binding. Previous experiments with transgenic T. gondii strains expressing compound-refractory PKG alleles (T761Q/M) have demonstrated that PKG is the primary intracellular target of compound 1 in tachyzoites [19]. In whole cell in vitro assays, a PKG knock-out strain expressing a complementing TgPKG T761Q mutant (‘PKG T761Q-KO’) is ∼20-fold less sensitive than wild-type parasites to compound 1 ([19], Table 2(B)). Compound 2 is 10-fold more potent than compound 1 against wild-type T. gondii parasites, but the engineered strain remains sensitive to this imidazopyridine at sub-micromolar levels (IC50 of 150 nM). Indeed, compound 2 is more potent against this compound-refractory PKG mutant strain than compound 1 is against wild-type parasites. Also, the level of resistance of the refractory ‘PKG T761Q-KO’ strain to compound 2 compared with wild-type parasites is only 6-fold. These results suggest that parasite protein kinase targets in addition to PKG are responsible for the anti-parastitic efficacy of compound 2 at sub-micromolar in vitro levels. 3.3. Identification of CDPK1 as a secondary target In order to identify secondary intracellular targets of compound 2, the same ligand binding assay strategy that led to the discovery of the PKG target in crude lysates of E. tenella and T. gondii was attempted [17,19]. To eliminate “background” binding of radiolabeled compound 2 to PKG, biochemical extracts were generated from a PKG knock-out strain that expresses only the compound-refractory T761Q mutant (‘PKG T761Q-KO’). Detergent S100 supernatants were prepared from 2 × 109 parasites and proteins were fractionated by anion exchange column chromatography (MonoQ). Fractions were assayed in parallel for PKG enzyme activity and binding activity using tritiated compound 2 (Figs. 1 and 2(A)). A single peak of ligand binding activity (fractions 11–12) was detected that was clearly separated from fractions containing PKG activity. Ligand binding to proteins in fractions 11–12 was inhibited by unlabeled compound, suggesting that the interaction is specific. In order to identify an associated protein kinase activity, ligand binding fractions

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Fig. 2. Purification and identification of a second compound 2 binding protein from T. gondii. (A) An S100 detergent extract derived from transgenic tachyzoites expressing a compound-refractory PKG (‘T761-KO’) was fractionated by MonoQ column chromatography. Fractions were assayed for PKG activity and [3 H]compound 2 binding activity with and without excess non-labeled compound 2. A peak of ligand binding activity (fractions 11–12) is resolved from PKG enzyme activity. Fractions 11 and 12 were pooled and further purified by hydrophobic interaction chromatography (HIC, not shown). (B) Protein fractions with compound 2 binding activity—MonoQ fraction 11 (Q11) and HIC fractions 14–17 were assayed using an in-gel kinase assay. Compound 2 binding activity tracks closely with a 60 kDa protein kinase that phosphorylates casein. The size of this protein is considerably larger than classic casein kinase enzymes that are typically less than 50 kDa (e.g., rat CK1␦). (C) HIC fractions with compound 2 binding activity from a preparative purification were loaded on a casein affinity matrix. A single 57 kDa protein eluted with 0.8 M arginine, is detected on a silver-stained SDS-PAGE gel. Molecular weight was estimated from the mobility of standards (M12, SB; Invitrogen). (D) The identity of the silver-stained 57 kDa protein is confirmed in a replicate immunoblot with isoform-specific CDPK1 antisera. (E) The EtCDPK1 peptide epitope used to elicit the antisera is aligned with related CDPK sequences. The affinity-purified antisera recognizes TgCDPK1 (95% homology), but not other CDPK isoforms TgCDPK3 or CDPK2 (<35% homology, not shown). (F) Summary of the purification steps.

were assayed using a variety of commercially available protein and peptide substrates. A nucleotide independent protein kinase activity with preference for milk casein was identified that was extraordinarily sensitive to inhibition by compound

2 (IC50 < 1 nM, data not shown). This protein kinase activity co-fractionated with compound 2 binding activity after further purification by hydrophobic interaction chromatography. An ingel casein kinase assay of peak fractions from both MonoQ

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(fraction Q11) and HIC columns (fractions 15–16) revealed that the associated kinase has a mobility of ∼60 kDa (Fig. 2(B)). While the compound 2 binding activity co-fractionates with a protein kinase that phosphorylates casein, the 60 kDa size is larger than classic casein kinase enzymes (CK1 or CK2), which are typically less than 50 kDa in molecular weight. Further indication that the compound 2 binding activity is not casein kinase derives from an earlier observation that T. gondii CK1␣ elutes much earlier in MonoQ column chromatography [16]. A second T. gondii isoform, CK1␤, can also be excluded as it does not appear to be expressed in tachyzoites [16]. The purification procedure was scaled up 10-fold (2 × 1010 parasites) to obtain sufficient material for affinity chromatography following preparative anion exchange and HIC chromatographic steps. Active fractions eluting from the HIC column were pooled and loaded onto a casein-agarose affinity column. A single 57 kDa protein, with kinase activity that is sensitive to compound 2, was eluted with 0.8 M arginine (Fig. 2(C and D)). The purified enzyme was sensitive to EGTA, stimulated by calcium and showed a preference for the peptide substrates syntide-2 and CaMII (281–291), all properties that resemble a previously characterized T. gondii calmodulin-like domain protein kinase (TgCDPK1) [36]. For technical reasons (low yield and poor elution from PVDF membrane), attempts to identify the purified compound 2 binding protein by micro-sequencing were not successful. Instead, the identity of the 57 kDa protein purified from tachyzoites was inferred by a comparison of biochemical properties with recombinant TgCDPK1 (Table 3). The purified native enzyme and recombinant TgCDPK1 exhibit similar subnanomolar sensitivity to compound 2, a common preference for a subset of peptide substrates and similar calcium activation kinetics. In addition, affinity purified antisera raised against an isoform-specific coccidian CDPK1 epitope [16] cross-reacted with the purified 57 kDa protein (Fig. 2(D and E)). A comparison of apicomplexan CDPK catalytic site residues that correspond to the PKG inhibitor binding pocket uncovered a high degree of primary sequence conservation (aligned in Fig. 3). The TgCDPK1 enzyme harbors a glycine (G128) at the gatekeeper position (corresponding to T761 of TgPKG), a residue that confers hypersensitivity to pyrrole and imidazopyridine inhibitors for PKG (Table 2). Site-directed mutants of

Table 3 Properties of native and recombinant TgCDPK1

IC50 Compound 1 (nM) IC50 Compound 2 Km ␣-Casein (␮M) Km Syntide-2 Km CaMKII (281–291) Km CK1 phosphopeptide Ka Calcium (␮M)

Native enzymea

FLAG TgCDPK1

20 0.7 3 60 80 IA 5

63 ± 2 0.7 ± 0.5 7 ± 3 (6)b 28 ± 9 (10)b 173 ± 65 (11)b IA 5±2

The Ka values for calcium were determined with substrate syntide-2. ‘IA’ is inactive. a Casein affinity column eluate. b V −1 mg−1 ) for recombinant enzyme are shown in max(app) values (␮mol min parentheses.

Fig. 3. PKG inhibitors exploit an analogous binding pocket in coccidian CDPK1 enzymes. Available CDPK sequences from E. tenella, T. gondii and P. falciparum are aligned (AlignX, Vector NTI software). Conserved and similar residues are shaded, respectively, in grey and dark grey and the definitive threonine “gatekeeper” residue is highlighted in black. The susceptibility (nM, IC50 values) of recombinant coccidian CDPK enzymes to compounds 1 and 2 (using syntide2 peptide as substrate) is indicated. ‘n.d.’, not done. Although not evaluated in vitro, CDPK isoforms of P. falciparum also share homology in this region. Sequence fragments are numbered relative to the initiator methionine and correspond to accession numbers AF333958 (TgCDPK1, [36]); Z71757 (EtCDPK1, [37]); (EtCDPK2, this work); (TgCDPK3, this work); P11275, RnCaMKII or rat calmodulin-dependent protein kinase type II ␣ subunit); X67288, PfCDPK1; Q8ICR0, PfCDPK2; AAF63154, PfCDPK3; Q8IBS5, PfCDPK4.

TgCDPK1 were constructed to test whether increasing bulk at this residue impacts inhibition by these kinase inhibitors. As with the compound-refractory TgPKG T761M/Q mutants, the TgCDPK1 G128T and G128Q mutations had no discernable effect on the catalytic activity of the enzyme in vitro. Values for specific activity (Vmax syntide-2), substrate affinity (Km syntide2) and calcium sensitivity (Ka ) of the mutant and ‘wild-type’ enzymes are within 25% of each other (not shown). Consistent with PKG [19], increasing amino acid bulk at this position in TgCDPK1 from the wild-type G128 to G128T and G128Q decreased sensitivity to both compound 1 and compound 2 (Fig. 3). Other recombinant coccidian CDPKs have been expressed in E. coli and characterized (Table 4). Unlike TgCDPK1, TgCDPK3 was (i) insensitive to EGTA; (ii) not dependent on exogenous calcium; (iii) preferred the CaMKII peptide substrate over syntide-2; and (iv) showed significant activity toward the CK1 phosphopeptide, although only at high concentrations. As predicted from the bulky methionine at the gatekeeper residue in TgCDPK3, activity of this enzyme is not sensitive to inhibition by either compound 1 or compound 2 (Fig. 3). Together these data convincingly illustrate that the compound 2 binding protein purified from T. gondii is not TgCDPK3. Like TgCDPK1, both EtCDPK1 and EtCDPK2 enzymes show a stronger affinity for syntide-2 than the CaMKII peptide, display little or no activity with the CK1 phopshopeptide substrate favored by classic CK1 enzymes and are similarly activated by calcium (Table 4). The gatekeeper residue of EtCDPK1 is threonine and, consistent with wild-type EtPKG and TgPKG, the catalytic activity of EtCDPK1 is inhibited by compound 1 and compound 2 (Fig. 3). In contrast, EtCDPK2 activity is not

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Table 4 Kinetic parameters of EtCDPK and TgCDPK3 isoforms Substrate

␣-Casein Syntide-2 CaMKII (281-291) CK1 phosphopeptide Ka calcium (␮M)

FLAG EtCDPK1

FLAG EtCDPK2

FLAG TgCDPK3

Vmax(app)

Km(app)

Vmax(app)

Km(app)

Vmax(app)

Km(app)

1 2.2 2.4 IA

4 46 175 – 12

15 13 15 1

4 7 74 90 5

25 11 10 7

3 50 6 114 >500

Values for Vmax(app) (␮mol min−1 mg−1 ), Km(app) (␮M) and Ka (␮M) were determined as in Table 3.

sensitive to these inhibitors, consistent with the prediction that its bulky methionine gatekeeper residue prevents compound access to the catalytic site. The calmodulin kinase II enzyme from rat, which has a bulky phenylalanine at the gatekeeper position, is also refractory. Compounds 1 and 2 inhibit the activity of P. falciparum PKG ([49], unpublished) which, like the coccidian PKGs, has a threonine residue at the definitive gatekeeper site. Sequences of four CDPK isoforms from P. falciparum are aligned in Fig. 3. PfCDPK1 and PfCDPK4, with threonine and serine residues at the gatekeeper position, are predicted to be sensitive to these kinase inhibitors. In contrast, isoforms PfCDPK2 and PfCDPK3 harbor a methionine at this critical residue which, by analogy to EtCDPK2 and TgCDPK3 and consistent with the model, should block inhibitor access to the catalytic site. In light of these observations, it will be interesting to explore the pharmacology of these compounds with respect to their putative targets in P. falciparum and their potential effects on the infectivity of sporozoites, merozoites, and sexual stage forms.

recombinant T. gondii enzyme and for the purified E. tenella orthologue, respectively (Table 5). The recombinant TgCK1␤ isoform, which is not expressed in tachyzoites [16], is relatively insensitive to both PKG inhibitors. Compound 2 is also more potent than compound 1 at inhibiting a vertebrate CK1 orthologue, the rat CK1␦ isoform (IC50 ∼ 100 nM). The low nanomolar potency of compound 2 against TgCK1␣ raises the possibility that, like TgCDPK1, this enzyme might contribute to its anti-parasitic efficacy. Nevertheless, compound 2 is ∼20-fold less active against TgCK1␣ than against TgCDPK1, which might explain why the compound 2 binding assay only detected the latter enzyme in the primary MonoQ fractionation step (Fig. 2A). Unlike isoforms of CDPK, the coccidan CK1 enzymes share no significant amino acid homology with PKG in the catalytic site region corresponding to the inhibitor binding pocket.

3.4. CK1 as a potential secondary target

To evaluate the extent to which the efficacy of the imidazopyridine PKG inhibitors is dependent on TgCDPK1, transgenic strains stably expressing recombinant TgCDPK1 or the G128Q mutant allele were generated in both wild-type parasites (RH strain) and in the engineered ‘PKG T761Q-KO’ strain. As with the target identification strategy described earlier (Fig. 2), the use of this transgenic strain (expressing compound-refractory PKG) effectively neutralizes the PKG component of inhibitor efficacy in the parasite, permitting the evaluation of compound activity at targets other than PKG. Tachyzoites stably transformed with TgCDPK1 expression vectors showed an intense cytosolic staining pattern in indirect IFA analysis with anti-FLAG antisera (Fig. 4). Cytosolic staining was observed in transgenic parasites regardless of the location of the epitope tag on the recombinant CDPK1 enzyme (N- or C-terminal, not shown). Among multiple independent transformants, the intensity of FLAG-specific fluorescence was at least 50× the level observed in the transgenic line expressing recombinant TgPKG (compare Fig. 4(A with B)). The level of recombinant PKG enzyme, which localizes to both membrane surfaces and to the cytosol, has been estimated as ∼5-fold higher than for native PKG levels in untransformed parasites [30]. Biochemically active recombinant FLAG TgCDPK1 wild-type and G128Q mutant enzymes were readily recovered in similar yield from transgenic parasites by immuoprecipitation or by affinity purification (not shown). The activity of these

The discovery of a compound 2 ligand binding activity in T. gondii extracts with associated kinase activity that phosphorylates casein, provided initial impetus for the cloning and characterization of two CK1 iso-enzymes from T. gondii [16]. However, the biochemical properties of these classic CK1 isoforms are quite distinct from the kinase activity that was ultimately purified from tachyzoite extracts (Fig. 2, Table 3). Despite their different biochemical properties, both CDPK1 and CK1␣ enzymes share a similar sensitivity to compound 1 (TgCDPK1 IC50 ∼ 60 nM; TgCK1␣; IC50 ∼ 100 nM—Tables 3 and 5). In a manner that parallels inhibition of PKG and CDPK activity, compound 2 is significantly more potent than compound 1 against CK1␣, with IC50 values of 16 nM and 80 nM for the Table 5 Relative sensitivity of CK1 enzymes to PKG inhibitors Enzyme

FLAG TgCK1␣ FLAG TgCK1␤

EtCK1␣ Rat CK1␦

IC50 values, nM Compound 1

Compound 2

100 ∼5000 350 2230

16 ∼5000 80 100

3.5. Use of compound-refractory TgCDPK1 to explore role in vivo efficacy

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Fig. 4. Over-expression of recombinant CDPK1 in intracellular tachyzoites. (A) Anti-FLAG IFA of parental ‘PKG T761Q-KO’ strain, expressing the TgPKGFLAG T761Q mutant in a PKG knock-out background. The characteristic membrane-associated and diffuse cytosolic staining pattern of PKG is apparent in infected vacuoles. (B) Cytosolic staining pattern in a representative ‘FLAG TgCDPK1 G128Q’ transformant. The camera exposure time is 2× shorter than in (A). No significant background staining was observed in wild-type parasite controls.

enzymes expressed in T. gondii was comparable to the respective recombinant proteins produced in E. coli (not shown). The effect of compound 2 on in vitro parasite activity was measured using transgenic T. gondii strains expressing TgCDPK1 in the wild-type or in the ‘PKG T761Q-KO’ genetic background. Parasites over-expressing recombinant TgCDPK1 or the TgCDPK1 G128Q mutant showed no significant difference in susceptibility to compound 2 relative to their respective wild-type or ‘PKG T761Q-KO’ parental strains in uracil uptake assays (not shown). However, small but significant differences among the strains were observed in their ability to adhere to HFF host cell monolayers (Fig. 5). For the parental CDPK strain controls, compound 2 inhibits tachyzoite attachment of wild-type tachyzoites (parental ‘P’, RH strain) with an IC50 of ∼20 nM, but has little effect on parasites expressing the PKG T761Q mutant (‘P/PKG T761Q-KO’). This observation is consistent with the conclusion that PKG is a primary biochemical target of compound 2. Similar results have previously been reported for the activity of compound 1 against these strains but at 10-fold higher compound levels [30]. Unexpectedly, host cell attachment by transformants expressing the CDPK1 G128Q mutant was more

Fig. 5. Transgenic expression of compound-insensitive CDPK1 enhances the ability of compound 2 to block parasite attachment to HFF host cells. Extracellular tachyzoites were pre-treated with the indicated levels of compound 2 for 15 min at 37 ◦ C and then incubated with prefixed host cell monolayers for 20 min. After washing to remove unattached parasites, parasites were quantified by IFA with FITC-conjugated anti-SAG1 antisera (Biodesign) and numbers compared with untreated controls. Data from parental RH and PKG T761Q-KO strains (‘P’) and two derived independent transgenic clones (‘1’ and ‘2’) expressing the CDPK1 G128Q mutant are shown. Results are from a representative experiment yielding comparable results (n = 3).

sensitive to compound 2 (across multiple doses) than the attachment of the corresponding parental strains. CDPK1 G128Q strains, both in the parental ‘wild-type’ and ‘PKG T761Q-KO’ backgrounds, strains were evaluated in the toxoplasmosis mouse model. As previously reported for compound 1 [18], compound 2 administered twice daily for 10 days protects mice (80% long-term survival) from an otherwise lethal infection of wild-type parasites (Fig. 6). Compound 2 is more potent than compound 1 in this model, affording this level of protection at a dose of 25 mg/kg (Fig. 6). The three transgenic T. gondii strains (PKG T761Q-KO, RH::CDPK1 G128Q and PKG T761Q-KO::CDPK1 G128Q) are as virulent as the parental wild-type strain, with mice succumbing within 10 days of parasite injection when not treated (Fig. 6, dotted lines).

Fig. 6. Transgenic expression of compound-insensitive CDPK1 potentiates the anti-parasitic efficacy of compound 2 in a murine toxoplasmosis model. Groups of 10 mice were infected with 103 tachyzoites of representative T. gondii strains. Beginning the following day, mice were treated twice daily with 25mg/kg of compound 2 for 10 consecutive days. Mice infected with parasites overexpressing the CDPK1 G128Q mutant, either in the parental wild-type or in the PKG T761Q-KO background (open symbols), respond better to compound 2 treatment than mice infected with the respective T. gondii strains expressing wild-type CDPK (closed symbols).

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Significantly, mice infected with the TgPKG T761Q-KO strain harboring the PKG allele that is insensitive to compound 2 are not completely refractory to treatment, with some mice surviving as long as 28 days. In contrast, this strain is completely resistant to compound 1 treatment and all infected mice succumb to acute infection within 10 days of the infection [18]. Consistent with this result is the hypothesis that secondary protein kinase targets contribute to the efficacy of the imidazopyridine class of compounds. If inhibition of TgCDPK does contribute to the in vivo efficacy of compound 2, then the T. gondii strains carrying the G128Q CDPK mutant allele (with in vitro enzyme activity that is insensitive to compound 2, Fig. 3) would be predicted to respond less well to treatment with compound 2, either in the wild-type or TgPKG T761Q-KO background. However, in comparison to the respective parental strains, the CDPK1 G128Q transgenic strains showed a marked increase in responsiveness to compound 2. The long-term survival of compound 2 treated mice increased from 80% for the wild-type RH strain to 100% for mice infected with the CDPK1 G128Q transformed wildtype strain. Similarly, 30% of mice infected with the TgPKG T761Q-KO::CDPK1 G128Q strain of parasites displayed longterm survival (>80 dpi) in response to compound 2 while mice infected with parental strain TgPKG T761Q-KO parasites and treated with compound 2 succumbed to infection by Day 29. These results are surprising, but are consistent with the behavior of the same strains in response to compound 2 in the in vitro attachment assays (Fig. 5). 4. Discussion Toxoplasma has proven to be a powerful system for validating the mechanism of action of anticoccidial trisubstituted pyrrole inhibitors such as compound 1. Compound-refractory PKG mutant alleles have been used both in in vitro assays and in transgenic tachyzoites to validate mechanism of action and to measure the degree of selectivity of inhibitors at this primary target [19,30]. With these reagents in hand, we set out to characterize the activity of an imidazopyridine inhibitor of PKG (compound 2) that shows substantial gains in potency in a chicken coccidiosis efficacy model [21]. Compound 2 is a more potent inhibitor of PKG activity and is significantly more active than compound 1 at blocking the invasion of E. tenella sporozoites or T. gondii tachyzoites into their respective host cells in vitro. Catalytic site mutations in PKG that impact inhibitor potency have similar affects on both compounds, suggesting a common mechanism of binding. The most discernable difference between the two compounds is the more potent activity of compound 2, as an inhibitor of both PKG and secondary kinase targets. Inhibition of other parasite protein kinases was first suggested in dose–response titrations with the compoundrefractory PKG knock-out strain (‘PKG T761Q-KO’, Table 2), and subsequently confirmed with the identification of compound 2 sensitive CDPK1 and CK1␣ enzymes from E. tenella and T. gondii (Tables 3–5; Figs. 2 and 3). CDPK enzymes have been a source of intrigue to parasitologists as transducers of calcium signaling and as potential drug targets [12,50–52]. They are found only in plants and in

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some protozoa and contain a unique C-terminal calmodulin-like domain with four functional EF hands that bind calcium. The proximal catalytic domain is related to vertebrate calmodulin dependent kinases (CaMK), which contain a distinct C-terminal activating domain that binds exogenous Ca2+ -calmodulin. This domain structure has led to speculation that the plant and protozoan CDPK enzyme has arisen through the fusion of an ancestral CaMK gene with a calmodulin gene [53,54]. In plants CDPK enzymes constitute a large gene family, members of which are spatially and temporally controlled throughout development [55]. They play a role in different aspects of calcium signaling, including stress responses and plantmycorrhizal symbiosis [52]. Calcium signaling is important for apicomplexan parasite motility, invasion and stage differentiation [51,56–58]. TgCDPK1 has been implicated in parasite invasion in studies using KT5926, a small molecule which inhibits both TgCDPK1 enzyme activity and parasite attachment at similar levels (IC50 ∼ 100 nM) [36]. This compound also inhibits the secretion of the MIC2 micronemal adhesin, which is essential for motility and invasion in T. gondii [59,60], but does not inhibit PKG [30]. A role in parasite invasion has also been proposed for the E. tenella orthologue EtCDPK1, based on morphological evidence from IFA staining of invading sporozoites [37]. In this case the enzyme was found to be associated with apical components of the cytoskeleton and some of the antigen appears to be left behind on the host cell membrane at the point of entry. Another isoform, CDPK4, has been shown to be essential for ookinete infectivity and mosquito transmission of P. berghei [61]. Biochemical purification of TgCDPK1 using the [3 H]compound 2 ligand binding assay predicts that amino acid residues critical for inhibitor binding to PKG may be conserved in CDPK enzymes. A convincing parallel structure activity relationship is apparent for both inhibitors, particularly with respect to amino acid side chain bulk at the ‘gatekeeper’ residue. Comparative evaluation of wild-type and site-directed mutants of TgPKG and TgCDPK1 illustrates that glycine in the definitive gatekeeper position confers hypersensitivity to pyrrole and imidazopyridine kinase inhibitors. Enzymes with a larger threonine residue at this position are characterized by catalytic activity that is moderately sensitive to the inhibitors, while substitution to glutamine results in TgPKG and TgCDPK1 activity that is not inhibited by either compound (Fig. 3, Table 2). Inhibition of the catalytic activity of other CDPK isoforms further supports the hypothesis that larger residues at this position block inhibitor access to the pocket. Enzymes with bulky methionine (TgCDPK3 and EtCDPK2) or phenylalanine (rat CaMK2) amino acids at the gatekeeper position are not inhibited by either compound, while EtCDPK1 activity (with a threonine gatekeeper residue) is sensitive. Together the parallel inhibition of PKG and CDPK isoforms demonstrates that compounds 1 and 2 interact with similar residues in the respective binding pockets. Equally important is the observation that the activity of only one CDPK isoform from T. gondii and one isoform from E. tenella is sensitive the pyrrole and imidazopyridine kinase inhibitors. The discovery that compound 2 is a sub-nanomolar inhibitor of both TgPKG and TgCDPK1 initially provided a plausible

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explanation for its increased anti-parasitic potency. If both protein kinase activities are essential for parasite invasion, dual inhibition might be expected to confer effective control at lower doses. However, experiments with transgenic parasite lines expressing compound-refractory TgCDPK1 have failed to establish any link between inhibition of this target and the efficacy of compound 2 in T. gondii. Expression of an inhibitor-refractory CDPK1 would be expected to reduce the anti-parasitic efficacy of this compound if the mechanism of action is dependent on this target (see Table 2). Remarkably, transgenic expression of TgCDPK1 G128Q augments the potency of compound 2 in parasite attachment assays and in the mouse model (Figs. 5 and 6). The effect is observed in two strain backgrounds, T. gondii parasites wild-type at the genomic PKG locus as well as a transgenic strain expressing a compound-refractory PKG mutant (‘PKG T761Q-KO’). From these data we conclude that TgCDPK1 is not essential for parasite attachment and invasion in vitro, or parasite virulence in vivo. It may be that the compound 2-insensitive CDPK isoforms (CDPK2 or CDPK3 in T. gondii and CDPK2 in E. tenella) provide functionally redundant activity. Similarly, the inhibition of T. gondii invasion by KT5926 [36] is not likely to be due to inhibition of TgCDPK1 alone. Instead the anti-parasitic activity of KT5926 activity could be due to inhibition of (i) multiple CDPK family members; (ii) related calmodulin activated kinases (e.g., CaMKII, Fig. 3); or (iii) other essential protein kinases. KT5926 has been reported to inhibit myosin light chain kinases in animal cells [62–64]. Inhibition of a parasite orthologue of this enzyme might interfere with the function of the actin-myosin motor that drives motility and invasion [43,65]. The potentiation of compound 2 efficacy in TgCDPK1 G128Q strains is harder to explain. It might be a consequence of calcium signal transduction pathway desensitization induced by artificial over-expression (e.g., sequestration of intracellular calcium by the CDPK1 calmodulin domain). Alternatively, this unexpected phenotype might reflect a dominant-negative effect exerted on other protein kinases as a result of elevated levels of CDPK1. Ultimately, more definitive evidence for the role of individual CDPK enzymes in parasite biology awaits the construction and evaluation of T. gondii strains where individual isoforms are conditionally expressed [66]. Such an approach has been adapted for use in T. gondii and successfully applied to validate the role of myosin A in parasite invasion [65]. Parasite CK1 enzymes have not yet been genetically validated as essential targets in parasitic protozoa, but are worthy of consideration based on their susceptibility to anti-parasitic purvalanol compounds in vitro [15,16]. Aminopurvalanol shows selectivity toward the CK1␣ isoform found in tachyzoites (IC50 50 nM, [16]), but is not as potent as PKG inhibitors against the parasite (IC50 350 nM). Furthermore, purvalanol compounds have generally poor pharmacokinetic properties [67] and are therefore unlikely to be effective in vivo. The CK1␣ isoform is sensitive to both trisubstituted pyrrole and imidazopyridine protein kinase inhibitors (Table 5). In a manner similar to their relative potency as PKG and CDPK inhibitors, compound 2 is 4–8-fold more potent than compound 1 against both TgCK1␣ and EtCK1␣ enzymes.

The most compelling evidence linking the anti-parasitic efficacy of these kinase inhibitors with the CK1 target comes from L. major [35]. Unlike apicomplexans, kinetoplastid parasites lack both PKG and CDPK enzymes but promastigote cultures are sensitive to sub-micromolar levels of compound 2 and other structurally related imidazopyridines. Trisubstituted pyrroles such as compound 1 also show efficacy, but are less potent. CK1 isoform 2 has been purified from L. major promastigotes using a [3 H]-imidazopyridine ligand binding assay [35], analogous to the strategy described in this manuscript. L. major CK1 isoform 2 shares greatest homology with the coccidian CK1␣ enzyme. Since CK1 enzymes do not share the same catalytic site binding pocket as PKG and CDPK1, ligand binding must involve different, and as yet unidentified, amino acid contacts. The potential for development of resistance in parasites, especially in the context anticoccidial or antimalarial compounds, represents an enormous challenge to the discovery of novel drug candidates. Anti-parasitic compounds that act at multiple essential protein kinase targets should help to minimize the onset of resistance. Although the identity of all relevant intracellular targets of imidazopyridine inhibitors is probably not yet fully realized, the example set by compound 2 provides evidence that potent broad spectrum multi-functional compounds can indeed be found. This is clearly a double edged sword—increasing the spectrum of activity against parasite kinases may also result in off-target activity against essential host cell enzymes and associated safety issues. In this regard, the imidazopyridine scaffold has been tainted with the discovery of associated genotoxicity [21]. As a result, the identification of safer compounds with similar biochemical and anti-parasitic properties will require a medicinal chemistry program focused on a different structural core. Acknowledgements We thank Tesfaye Biftu for providing compounds 1 and 2, and Yui S. Tang for designing the synthesis of radiolabeled compound 2. We are also grateful to Jennifer Crohn for editorial help. References [1] Barker AJ, Gibson KH, Grundy W, et al. Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett 2001;11(14):1911–4. [2] Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001;19(13):3267–79. [3] Lydon NB, Druker BJ. Lessons learned from the development of imatinib. Leuk Res 2004;28(Suppl. 1):S29–38. [4] Duensing A, Heinrich MC, Fletcher CD, Fletcher JA. Biology of gastrointestinal stromal tumors: KIT mutations and beyond. Cancer Invest 2004;22(1):106–16. [5] Blagden S, de-Bono J. Drugging cell cycle kinases in cancer therapy. Curr Drug Targets 2005;6(3):325–35. [6] Meijer L, Raymond E. Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials. Acc Chem Res 2003;36(6):417–25.

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