RESUMO
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.
Assuntos
Antibacterianos/farmacologia , RNA Antissenso/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/química , Análise por Conglomerados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fenótipo , Interferência de RNA , Staphylococcus aureus/genéticaRESUMO
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.
Assuntos
Antiparasitários/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/enzimologia , Animais , Antiparasitários/química , Antiparasitários/uso terapêutico , Sequência de Bases/genética , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Eritrócitos/parasitologia , Fluoresceína-5-Isotiocianato , Expressão Gênica , Estágios do Ciclo de Vida/fisiologia , Malária Falciparum/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Alinhamento de Sequência , Transfecção/métodosRESUMO
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.
