Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase

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.     

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.     

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.     

A new class of prolylcarboxypeptidase inhibitors, part 1: discovery and evaluation

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.     

The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase

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.     

Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae

The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the approximately 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.     

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure–activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.     

Kibdelomycin A,a congener of kibdelomycin,derivatives and their antibacterial activities

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.     

A new class of prolylcarboxypeptidase inhibitors, part 2: the aminocyclopentanes

A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.