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Francisca Vicente,
Mercedes de la Cruz,
Jun Lu,
John Allocco,
Janet Sigmund,
John W Phillips,
Stephen Skwish,
Olga Genilloud,
Robert G K Donald,
Michael A Goetz, [......],
Sheo B Singh,
Suzy Lee,
Scott K Smith,
Ronald E Painter,
Deborah L Zink,
Katherine Young,
Russell Onishi,
Jon Polishook, Karen Overbye,
Judyann Wiltsie
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Sheo B Singh,
Michael A Goetz,
Scott K Smith,
Deborah L Zink,
Jon Polishook,
Russell Onishi,
Scott Salowe,
Judyann Wiltsie,
John Allocco,
Janet Sigmund,
Karen Dorso,
Mercedes de la Cruz,
Jesús Martín,
Francisca Vicente,
Olga Genilloud, Robert G K Donald,
John W Phillips
[show abstract]
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ABSTRACT: Emergence of bacterial resistance has eroded the effectiveness of many life saving antibiotics leading to an urgent need for new chemical classes of antibacterial agents. We have applied a Staphylococcus aureus fitness test strategy to natural products screening to meet this challenge. In this paper we report the discovery of kibdelomycin A, a demethylated congener of kibdelomycin, the representative of a novel class of antibiotics produced by a new strain of Kibdelosporangium. Kibdelomycin A is a potent inhibitor of DNA gyrase and topoisomerase IV, inhibits DNA synthesis and shows whole cell antibiotic activity, albeit, less potently than kibdelomycin. Kibdelomycin C-33 acetate and tetrahydro-bisdechloro derivatives of kibdelomycin were prepared which helped define a basic SAR of the family.
Bioorganic & medicinal chemistry letters 10/2012; · 2.65 Impact Factor
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John W Phillips,
Michael A Goetz,
Scott K Smith,
Deborah L Zink,
Jon Polishook,
Russell Onishi,
Scott Salowe,
Judyann Wiltsie,
John Allocco,
Janet Sigmund, [......],
Mercedes de la Cruz,
Jesús Martín,
Francisca Vicente,
Olga Genilloud,
Jun Lu,
Ronald E Painter,
Katherine Young,
Karen Overbye, Robert G K Donald,
Sheo B Singh
[show abstract]
[hide abstract]
ABSTRACT: Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.
Chemistry & biology 08/2011; 18(8):955-65. · 6.52 Impact Factor
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Michael A Goetz,
Chaowei Zhang,
Deborah L Zink,
Marta Arocho,
Francisca Vicente,
Gerald F Bills,
Jon Polishook,
Karen Dorso,
Russell Onishi,
Charles Gill,
Emily Hickey,
Suzy Lee,
Richard Ball,
Stephen Skwish, Robert G K Donald,
John W Phillips,
Sheo B Singh
[show abstract]
[hide abstract]
ABSTRACT: Bacterial resistance to antibiotics, particularly to multiple antibiotics, is becoming a cause for significant concern. The only really viable course of action to counter this is to discover new antibiotics with novel modes of action. We have recently implemented a new antisense-based chemical genetic screening technology to accomplish this goal. The discovery and antibacterial activity of coelomycin, a fully substituted 2,6-dioxo pyrazine, illustrates the application of the Staphylococcus aureus fitness test strategy to natural products discovery.
The Journal of Antibiotics 08/2010; 63(8):512-8. · 1.65 Impact Factor
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Robert G K Donald,
Stephen Skwish,
R Allyn Forsyth,
Jennifer W Anderson,
Tanya Zhong,
Colleen Burns,
Suzy Lee,
Xin Meng,
Lynn LoCastro,
Lisa Wang Jarantow,
Jesus Martin,
Sang Ho Lee,
Ian Taylor,
David Robbins,
Cheryl Malone,
Liangsu Wang,
Carlos S Zamudio,
Philip J Youngman,
John W Phillips
[show abstract]
[hide abstract]
ABSTRACT: The emergence of drug-resistant bacteria coupled with the limited discovery of novel chemical scaffolds and druggable targets inspires new approaches to antibiotic development. Here we describe a chemical genomics strategy based on 245 Staphylococcus aureus antisense RNA strains, each engineered for reduced expression of target genes essential for S. aureus growth. Attenuation of gene expression can sensitize cells to compounds that inhibit the activity of a gene product or associated process. Pools of strains grown competitively in the presence of bioactive compounds generate characteristic profiles of strain sensitivities reflecting compound mechanism of action. Here, we validate this approach with a structurally and mechanistically diverse set of reference antibiotics and, in the accompanying paper in this issue of Chemistry & Biology (Huber et al., 2009), demonstrate its use in the discovery of new cell wall inhibitors.
Chemistry & biology 09/2009; 16(8):826-36. · 6.52 Impact Factor
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Joann Huber, Robert G K Donald,
Sang Ho Lee,
Lisa Wang Jarantow,
Michael J Salvatore,
Xin Meng,
Ronald Painter,
Russell H Onishi,
James Occi,
Karen Dorso,
Katherine Young,
Young Whan Park,
Stephen Skwish,
Michael J Szymonifka,
Tim S Waddell,
Lynn Miesel,
John W Phillips,
Terry Roemer
[show abstract]
[hide abstract]
ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial and community-acquired pathogen for which few existing antibiotics are efficacious. Here we describe two structurally related synthetic compounds that potentiate beta-lactam activity against MRSA. Genetic studies indicate that these agents target SAV1754 based on the following observations: (i) it has a unique chemical hypersensitivity profile, (ii) overexpression or point mutations are sufficient to confer resistance, and (iii) genetic inactivation phenocopies the potentiating effect of these agents in combination with beta-lactams. Further, we demonstrate these agents inhibit peptidoglycan synthesis. Because SAV1754 is essential for growth and structurally related to the recently reported peptidoglycan flippase of Escherichia coli, we speculate it performs an analogous function in S. aureus. These results suggest that SAV1754 inhibitors might possess therapeutic potential alone, or in combination with beta-lactams to restore MRSA efficacy.
Chemistry & biology 09/2009; 16(8):837-48. · 6.52 Impact Factor
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[show abstract]
[hide abstract]
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 alpha-isoform of casein kinase 1 (CK1alpha), 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.
Molecular and Biochemical Parasitology 10/2006; 149(1):86-98. · 2.55 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296-301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913-22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317-28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300-7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum (PfPKG). Biochemical fractions enriched for a 98kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference (Pf(Tg)PKG) in T. gondii parasites. The protein kinase activity of purified recombinant Pf(Tg)PKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of Pf(Tg)PKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.
Molecular and Biochemical Parasitology 04/2006; 146(1):78-88. · 2.55 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Previous affinity chromatography experiments have described the unexpected binding of an isoform of casein kinase I (CK1) from Leishmania mexicana, Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii to an immobilized cyclin-dependent kinase (CDK) inhibitor (purvalanol B). In order to further evaluate CK1 as a potential anti-parasitic target, two T. gondii CK1 genes were cloned by PCR using primers derived from a putative CK1 gene fragment identified from a T. gondii EST database. The genes are predicted to encode a smaller polypeptide of 38 kDa (TgCK1alpha) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1beta). Enzymatically active recombinant FLAG-epitope tagged TgCK1alpha and TgCK1beta enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1alpha expression was found to be cytosolic, TgCK1beta was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1beta confers this membrane-association. Recombinant TgCK1alpha and TgCK1beta isoforms were also expressed in E. coli and biochemically characterized. A 38kDa native CK1 activity was partially purified from T. gondii tachyzoites by ion-exchange and hydrophobic interaction chromatography with biochemical and serological properties closely resembling those of recombinant TgCK1alpha. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1beta 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1alpha, confirming the existence of potentially exploitable structural differences between host and parasite CK1 enzymes. Since the more cell-permeable aminopurvalanol also inhibits parasite growth, these results provide further impetus to investigate inhibitors of CK1 as anti-parasitic agents.
Molecular and Biochemical Parasitology 06/2005; 141(1):15-27. · 2.55 Impact Factor
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[show abstract]
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ABSTRACT: The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.
International Journal for Parasitology 04/2004; 34(3):369-80. · 3.39 Impact Factor
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ABSTRACT: The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.
Eukaryotic Cell 07/2002; 1(3):317-28. · 3.60 Impact Factor
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Anne M Gurnett,
Paul A Liberator,
Paula M Dulski,
Scott P Salowe, Robert G K Donald,
Jennifer W Anderson,
Judyann Wiltsie,
Carmen A Diaz,
Georgiana Harris,
Ben Chang,
Sandra J Darkin-Rattray,
Bakela Nare,
Tami Crumley,
Penny Sue Blum,
Andrew S Misura,
Tamas Tamas,
Mohinder K Sardana,
Jeffrey Yuan,
Tesfaye Biftu,
Dennis M Schmatz
[show abstract]
[hide abstract]
ABSTRACT: 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. both in 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 approximately 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 Drosophila PKG 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 recombinant E. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH(2)-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 IC(50) of <1 nm.
Journal of Biological Chemistry 06/2002; 277(18):15913-22. · 4.77 Impact Factor
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ABSTRACT: A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial target for the apicomplexan parasite Eimeria tenella [Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald, R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley, T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol. Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in their regulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sites and has distinctive activation properties, including a very large stimulation by cGMP ( approximately 1000-fold) with significant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we found that 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides a maximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP can synergize with the analogue to provide full activation. The results suggest that partial activation is a consequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directed mutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirms that this site is an important functional participant in the allosteric regulation of the kinase and that it exhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activation of Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additional cGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present in the PKGs of higher organisms containing only two allosteric sites.
Biochemistry 05/2002; 41(13):4385-91. · 3.42 Impact Factor
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ABSTRACT: The cGMP-dependent protein kinase (PKG) of Eimeria tenella and Toxoplasma gondii is the target of a novel coccidiostat that is effective against coccidiosis and toxoplasmosis in animal models. Preparations of native PKG enzyme from Toxoplasma and Eimeria contain a membrane-associated polypeptide (isoform-I) of about 110 kDa and a slightly smaller soluble polypeptide (isoform-II). Expression of T. gondii and E. tenella PKG cDNA clones in Toxoplasma yield similarly sized recombinant polypeptides, which co-migrate on SDS-polyacrylamide gels with the corresponding native isoforms. Results of targeted mutagenesis of potential translational initiation sites suggest that parasite isoform-II is a product of alternative translational initiation from an internal initiator methionine codon. Exclusive expression of isoform-II or isoform-I can be achieved by preventing initiation at the respective primary or secondary sites. Immunofluorescence analysis indicates that recombinant isoform-I localizes primarily to the parasite plasma membrane, while isoform-II remains cytosolic. Mutagenesis and metabolic labeling studies reveal that the observed membrane-association of full-length recombinant PKG is mediated by N-terminal myristoylation and palmitoylation at amino acids G2 and C4. We also confirm the functional significance of a putative third PKG allosteric site, common to apicomplexan PKGs but absent from vertebrate or insect PKGs. In assays with transiently transfected parasites, constructs harboring a mutation at this site express markedly lower levels of cGMP-dependent PKG activity, while a triple mutant bearing mutations in all three sites reduces kinase activity to background levels.
Molecular and Biochemical Parasitology 05/2002; 120(2):165-75. · 2.55 Impact Factor
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[show abstract]
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ABSTRACT: The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) is a potent inhibitor of cyclic GMP-dependent protein kinases from Apicomplexan protozoa and displays cytostatic activity against Toxoplasma gondii in vitro. Compound 1 has now been evaluated against T. gondii infections in the mouse and appeared to protect the animals when given intraperitoneally at 50 mg/kg twice daily for 10 days. However, samples from brain, spleen, and lung taken from infected treated mice revealed the presence of parasites after cessation of administration of compound 1, indicating that a transient asymptomatic parasite recrudescence occurs in all survivors. The ability of mice to control Toxoplasma infection after compound 1 treatment has been terminated suggested that the mouse immune system plays a synergistic role with chemotherapy in controlling the infection. To explore this possibility, gamma interferon (IFN-gamma)-knockout mice were infected with parasites and treated with compound 1, and survival was compared to that of normal mice. IFN-gamma-knockout mice were protected against T. gondii throughout the treatment phase but died during the posttreatment phase in which peak recrudescence was observed in treated immunocompetent mice. These data suggest that an IFN-gamma-dependent immune response was essential for controlling and resolving parasite recrudescence in mice treated with compound 1. In addition, when compound 1-cured immunocompetent mice were rechallenged with a lethal dose of T. gondii, all survived (n = 32). It appears that the cytostatic nature of compound 1 provides an "immunization" phase during chemotherapy which allows the mice to survive the recrudescence and any subsequent challenge with a lethal dose of T. gondii.
Antimicrobial Agents and Chemotherapy 03/2002; 46(2):300-7. · 4.84 Impact Factor
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[show abstract]
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ABSTRACT: Previous affinity chromatography experiments have described the unexpected binding of an isoform of casein kinase I (CK1) from Leishmania mexicana, Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii to an immobilized cyclin-dependent kinase (CDK) inhibitor (purvalanol B). In order to further evaluate CK1 as a potential anti-parasitic target, two T. gondii CK1 genes were cloned by PCR using primers derived from a putative CK1 gene fragment identified from a T. gondii EST database. The genes are predicted to encode a smaller polypeptide of 38 kDa (TgCK1α) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1β). Enzymatically active recombinant FLAG-epitope tagged TgCK1α and TgCK1β enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1α expression was found to be cytosolic, TgCK1β was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1β confers this membrane-association. Recombinant TgCK1α and TgCK1β isoforms were also expressed in E. coli and biochemically characterized. A 38 kDa native CK1 activity was partially purified from T. gondii tachyzoites by ion-exchange and hydrophobic interaction chromatography with biochemical and serological properties closely resembling those of recombinant TgCK1α. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1β 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1α, confirming the existence of potentially exploitable structural differences between host and parasite CK1 enzymes. Since the more cell-permeable aminopurvalanol also inhibits parasite growth, these results provide further impetus to investigate inhibitors of CK1 as anti-parasitic agents.
Molecular and Biochemical Parasitology.
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[show abstract]
[hide abstract]
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 α-isoform of casein kinase 1 (CK1α), 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.
Molecular and Biochemical Parasitology.
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[show abstract]
[hide abstract]
ABSTRACT: Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296–301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913–22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317–28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300–7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum (PfPKG). Biochemical fractions enriched for a 98 kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference (PfTgPKG) in T. gondii parasites. The protein kinase activity of purified recombinant PfTgPKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of PfTgPKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.
Molecular and Biochemical Parasitology.
