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1.
mSphere ; 5(6)2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328352

RESUMO

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Vibrio cholerae/genética , Vibrio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Proteínas de Membrana/genética , Mutagênese , Mutação , Peptídeo Hidrolases/metabolismo , Supressão Genética
2.
RSC Med Chem ; 11(8): 885-895, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33479683

RESUMO

Based on crystal structures of Trypanosoma brucei methionyl-tRNA synthetase (TbMetRS) bound to inhibitors, we designed, synthesized, and evaluated two series of novel TbMetRS inhibitors targeting this parasite enzyme. One series has a 1,3-dihydro-imidazol-2-one containing linker, the other has a rigid fused aromatic ring in the linker. For both series of compounds, potent inhibition of parasite growth was achieved with EC50 < 10 nM and most compounds exhibited low general toxicity to mammalian cells with CC50s > 20 000 nM. Selectivity over human mitochondrial methionyl tRNA synthetase was also evaluated, using a cell-based mitochondrial protein synthesis assay, and selectivity in a range of 20-200-fold was achieved. The inhibitors exhibited poor permeability across the blood brain barrier, necessitating future efforts to optimize the compounds for use in late stage human African trypanosomiasis.

3.
Artigo em Inglês | MEDLINE | ID: mdl-30745384

RESUMO

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.


Assuntos
Antiprotozoários/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Imidazóis/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Piridinas/farmacologia , Animais , Cryptosporidium parvum/genética , Ciclo-Oxigenase 1/efeitos dos fármacos , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Feminino , Células Hep G2 , Humanos , Imidazóis/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piridinas/química
4.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 4): 245-254, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29633973

RESUMO

Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.


Assuntos
Desenho de Fármacos , Metionina tRNA Ligase/química , Metionina tRNA Ligase/metabolismo , Mycobacterium tuberculosis/enzimologia , Catálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica
5.
ACS Infect Dis ; 4(4): 431-444, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29436819

RESUMO

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.


Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Espectrometria de Massas/métodos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Proteínas de Protozoários/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-28848016

RESUMO

Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 µg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Metionina tRNA Ligase/antagonistas & inibidores , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacocinética , Proteínas Sanguíneas/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Escherichia coli/efeitos dos fármacos , Feminino , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Inativação Metabólica , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos , Staphylococcus aureus/efeitos dos fármacos
7.
Bioorg Med Chem Lett ; 27(12): 2702-2707, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28465105

RESUMO

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC50 of 4nM.


Assuntos
Inibidores Enzimáticos/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Metionina/farmacologia , Trypanosoma brucei brucei/enzimologia , Animais , Sítios de Ligação/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Células Hep G2 , Humanos , Metionina/administração & dosagem , Metionina/química , Metionina tRNA Ligase/metabolismo , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade
8.
Biochimie ; 138: 124-136, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28427904

RESUMO

The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNATyr. An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Domínio Catalítico , Leishmania donovani/enzimologia , Modelos Moleculares , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Anticorpos de Cadeia Única , Tirosina/metabolismo , Tirosina-tRNA Ligase/química
9.
ACS Infect Dis ; 3(3): 225-236, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28110521

RESUMO

Human African trypanosomiasis is a neglected tropical disease that is lethal if left untreated. Existing therapeutics have limited efficacy and severe associated toxicities. 2-(2-(((3-((1H-Benzo[d]imidazol-2-yl)amino)propyl)amino)methyl)-4,6-dichloro-1H-indol-1-yl)ethan-1-ol (NEU-1053) has recently been identified from a high-throughput screen of >42,000 compounds as a highly potent and fast-acting trypanocidal agent capable of curing a bloodstream infection of Trypanosoma brucei in mice. We have designed a library of analogues to probe the structure-activity relationship and improve the predicted central nervous system (CNS) exposure of NEU-1053. We report the activity of these inhibitors of T. brucei, the efficacy of NEU-1053 in a murine CNS model of infection, and identification of the target of NEU-1053 via X-ray crystallography.


Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Tripanossomicidas/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Camundongos , Doenças Negligenciadas , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tripanossomicidas/química , Trypanosoma brucei brucei/efeitos dos fármacos
10.
Eur J Med Chem ; 124: 1081-1092, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27788467

RESUMO

A screening hit 1 against Trypanosoma brucei methionyl-tRNA synthetase was optimized using a structure-guided approach. The optimization led to the identification of two novel series of potent inhibitors, the cyclic linker and linear linker series. Compounds of both series were potent in a T. brucei growth inhibition assay while showing low toxicity to mammalian cells. The best compound of each series, 16 and 31, exhibited EC50s of 39 and 22 nM, respectively. Compounds 16 and 31 also exhibited promising PK properties after oral dosing in mice. Moreover, compound 31 had moderately good brain permeability, with a brain/plasma ratio of 0.27 at 60 min after IP injection. This study provides new lead compounds for arriving at new treatments of human African trypanosomiasis (HAT).


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Animais , Encéfalo/metabolismo , Técnicas de Química Sintética , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Células Hep G2 , Humanos , Metionina tRNA Ligase/química , Metionina tRNA Ligase/metabolismo , Camundongos , Permeabilidade , Conformação Proteica , Relação Estrutura-Atividade , Tripanossomicidas/metabolismo , Tripanossomicidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacos
11.
ACS Infect Dis ; 2(6): 399-404, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27627628

RESUMO

Fluorination is a well-known strategy for improving the bioavailability of drug molecules. However, its impact on efficacy is not easily predicted. On the basis of inhibitor-bound protein crystal structures, we found a beneficial fluorination spot for inhibitors targeting methionyl-tRNA synthetase of Trypanosoma brucei. In particular, incorporating 5-fluoroimidazo[4,5-b]pyridine into inhibitors leads to central nervous system bioavailability and maintained or even improved efficacy.


Assuntos
Inibidores Enzimáticos/química , Metionina tRNA Ligase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/química , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Camundongos , Estrutura Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Piridinas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética
12.
J Med Chem ; 59(13): 6531-46, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27309760

RESUMO

New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses antitoxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human ether-a-go-go-related gene (hERG) inhibitory activity, associated with long Q-T syndrome, and consequently presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These properties make 32 a promising lead for the development of a new antitoxoplasmosis therapy.


Assuntos
Antiprotozoários/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Desenho de Fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Administração Oral , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/química , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Haplorrinos , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Testes de Sensibilidade Parasitária , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Toxoplasma/enzimologia , Toxoplasmose/metabolismo
13.
ACS Med Chem Lett ; 6(12): 1184-1189, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26693272

RESUMO

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure-activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.

14.
Antimicrob Agents Chemother ; 59(11): 7128-31, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26324270

RESUMO

The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs.


Assuntos
Antiprotozoários/farmacologia , Giardia lamblia/efeitos dos fármacos , Metionina tRNA Ligase/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Giardia lamblia/enzimologia , Metronidazol/farmacologia , Trofozoítos/enzimologia
15.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 8): 1684-98, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26249349

RESUMO

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.


Assuntos
Inibidores Enzimáticos/química , Histidina-tRNA Ligase/antagonistas & inibidores , Histidina-tRNA Ligase/química , Bibliotecas de Moléculas Pequenas/química , Trypanosoma cruzi/enzimologia , Sítios de Ligação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/microbiologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Histidina-tRNA Ligase/metabolismo , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/farmacologia , Trypanosoma cruzi/química , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo
16.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 5): 485-99, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25945701

RESUMO

Parasitic protozoa cause a range of diseases which threaten billions of human beings. They are responsible for tremendous mortality and morbidity in the least-developed areas of the world. Presented here is an overview of the evolution over the last three to four decades of structure-guided design of inhibitors, leads and drug candidates aiming at targets from parasitic protozoa. Target selection is a crucial and multi-faceted aspect of structure-guided drug design. The major impact of advances in molecular biology, genome sequencing and high-throughput screening is touched upon. The most advanced crystallographic techniques, including XFEL, have already been applied to structure determinations of drug targets from parasitic protozoa. Even cryo-electron microscopy is contributing to our understanding of the mode of binding of inhibitors to parasite ribosomes. A number of projects have been selected to illustrate how structural information has assisted in arriving at promising compounds that are currently being evaluated by pharmacological, pharmacodynamic and safety tests to assess their suitability as pharmaceutical agents. Structure-guided approaches are also applied to incorporate properties into compounds such that they are less likely to become the victim of resistance mechanisms. A great increase in the number of novel antiparasitic compounds will be needed in the future. These should then be combined into various multi-compound therapeutics to circumvent the diverse resistance mechanisms that render single-compound, or even multi-compound, drugs ineffective. The future should also see (i) an increase in the number of projects with a tight integration of structural biology, medicinal chemistry, parasitology and pharmaceutical sciences; (ii) the education of more `medicinal structural biologists' who are familiar with the properties that compounds need to have for a high probability of success in the later steps of the drug-development process; and (iii) the expansion of drug-development capabilities in middle- and low-income countries.


Assuntos
Antiprotozoários/química , Sistemas de Liberação de Medicamentos/tendências , Desenho de Fármacos , Proteínas de Protozoários/química , Animais , Antiprotozoários/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Previsões , Humanos , Imageamento Tridimensional/métodos , Plasmodium/química , Plasmodium/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Trypanosoma/química , Trypanosoma/efeitos dos fármacos
17.
J Biomol Screen ; 20(1): 122-30, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25163684

RESUMO

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Metionina tRNA Ligase/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Linhagem Celular , Relação Dose-Resposta a Droga , Descoberta de Drogas/normas , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/normas , Humanos , Concentração Inibidora 50 , Doenças Negligenciadas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas , Tripanossomíase Africana/tratamento farmacológico
18.
J Struct Biol ; 187(3): 223-235, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25092625

RESUMO

The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana/química , Estrutura Terciária de Proteína , Vibrio vulnificus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Vibrio vulnificus/genética
19.
Biochimie ; 106: 111-20, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25151410

RESUMO

As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS.


Assuntos
Histidina-tRNA Ligase/química , Histidina/química , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histidina/metabolismo , Histidina-tRNA Ligase/genética , Histidina-tRNA Ligase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma brucei brucei/genética
20.
PLoS One ; 9(3): e92929, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24681759

RESUMO

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.


Assuntos
Neospora/metabolismo , Proteínas Quinases/metabolismo , Toxoplasma/metabolismo , Animais , Cristalografia por Raios X/métodos , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico
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