RESUMEN

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC50 9.0±0.2µM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Asunto(s)

Antimaláricos/síntesis química , Endopeptidasa Clp/antagonistas & inhibidores , Plasmodium/efectos de los fármacos , Plasmodium/enzimología , Antimaláricos/química , Antimaláricos/farmacología , Apicoplastos/efectos de los fármacos , Dominio Catalítico , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad

RESUMEN

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-ß treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-ß treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

Asunto(s)

Subtipo H5N1 del Virus de la Influenza A/metabolismo , Gripe Humana/virología , Interferones/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virosis/metabolismo , Células A549 , Animales , Antivirales/farmacología , Modelos Animales de Enfermedad , Perros , Células Epiteliales/virología , Fibroblastos , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Interferón beta , Pulmón/patología , Pulmón/virología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Neutrófilos/patología , Neutrófilos/virología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Genética Inversa , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/fisiología , Virulencia , Factores de Virulencia

RESUMEN

Type I interferons (IFNs) exhibit broad-spectrum antiviral activity, with potential utility against emerging acute virus infections that pose a threat to global health. Recombinant IFN-αs that have been approved for clinical use require cold storage and are administered through intramuscular or subcutaneous injection, features that are problematic for global distribution, storage, and administration. Cognizant that the biological potency of an IFN-α subtype is determined by its binding affinity to the type I IFN receptor, IFNAR, we identified a panel of small molecule nonpeptide compounds using an in silico screening strategy that incorporated specific structural features of amino acids in the receptor-binding domains of the most potent IFN-α, IFN alfacon-1. Hit compounds were selected based on ease of synthesis and formulation properties. In preliminary biological assays, we provide evidence that these compounds exhibit antiviral activity. This proof-of-concept study validates the strategy of in silico design and development for IFN mimetics.

Asunto(s)

Antivirales/farmacología , Virus de la Encefalomiocarditis/efectos de los fármacos , Interferón-alfa/química , Peptidomiméticos/farmacología , Receptor de Interferón alfa y beta/agonistas , Bibliotecas de Moléculas Pequeñas/farmacología , Antivirales/síntesis química , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Linfocitos B/virología , Línea Celular Tumoral , Simulación por Computador , Diseño de Fármacos , Virus de la Encefalomiocarditis/crecimiento & desarrollo , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Modelos Moleculares , Peptidomiméticos/síntesis química , Estructura Secundaria de Proteína , Receptor de Interferón alfa y beta/química , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Proteínas Recombinantes/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad , Interfaz Usuario-Computador

RESUMEN

Falcipain-2 is a papain family cysteine protease and an emerging antimalarial drug target. A pseudo-tripeptide scaffold I was designed using in silico screening tools and the three dimensional structures of falcipain-2, falcipain-3, and papain. This scaffold was investigated at four positions, T1, T2, T3, and T3', with various targeted substitutions to understand the structure-activity relationships. Inhibitor synthesis was accomplished by first obtaining the appropriate dipeptide precursors with common structural components. The pyrrolidine moiety introduced interesting rotamers in a number of synthesized molecules, which was confirmed using high-temperature (1)H NMR spectroscopy. Among the synthesized compounds, 61, 62, and 66 inhibited falcipain-2 activity with inhibition constants (Ki) of 1.8 ± 1.1, 0.2 ± 0.1 and 7.0 ± 2.3 µM, respectively. A group of molecules with a pyrrolidine moiety at the T2 position (68, 70, 71, 72, and 73) also potently inhibited falcipain-2 activity (Ki=0.4 ± 0.1, 2.5 ± 0.5, 3.3 ± 1.1, 7.5 ± 1.9, and 4.6 ± 0.7 µM, respectively). Overall, compound 74 exhibited potent anti-parasitic activity (IC50=0.9 ± 0.1 µM), corresponding with its inhibitory activity against falcipain-2, with a Ki of 1.1 ± 0.1 µM. Compounds 62 and 67 inhibited the growth of the drug resistant parasite Dd2 with better efficacy, and compound 74 exhibited a 7- to 12-fold higher potency against Dd2 and MCamp isolates, than the laboratory strain (3D7). These data suggest that this novel series of compounds should be further investigated as potential antimalarial agents.

Asunto(s)

Antimaláricos/farmacología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/clasificación , Inhibidores de Cisteína Proteinasa/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetulus , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Resistencia a Medicamentos/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/crecimiento & desarrollo , Relación Estructura-Actividad

RESUMEN

Small molecules that mimic IFN-α epitopes that interact with the cell surface receptor, IFNAR, would be useful therapeutics. One such 8-amino acid region in IFN-α2, designated IRRP-1, was used to derive 11 chemical compounds that belong to 5 distinct chemotypes, containing the molecular features represented by the key residues Leu30, Arg33, and Asp35 in IRRP-1. Three of these compounds exhibited potential mimicry to IRRP-1 and, in cell based assays, as predicted, effectively inhibited IFNAR activation by IFN-α. Of these, compound 3 did not display cell toxicity and reduced IFN-α-inducible STAT1 phosphorylation and STAT-DNA binding. Based on physicochemical properties' analyses, our data suggest that moieties with acidic pKa on the small molecule may be a necessary element for mimicking the carboxyl group of Asp35 in IRRP-1. Our data confirm the relevance of this strategy of molecular mimicry of ligand-receptor interaction domains of protein partners for small molecule drug discovery.

Asunto(s)

Epítopos/química , Receptor de Interferón alfa y beta/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Ácido Aspártico/química , Línea Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Epítopos/metabolismo , Humanos , Interferón-alfa/metabolismo , Modelos Moleculares , Imitación Molecular , Péptidos/química , Fosforilación/efectos de los fármacos , Conformación Proteica , Estructura Terciaria de Proteína , Receptor de Interferón alfa y beta/química , Factor de Transcripción STAT1/metabolismo

RESUMEN

In multiple sclerosis (MS), myelin basic protein (MBP), critical for the maintenance of myelin compaction and protecting against degradation, is known to contain concentrations of the noncoded amino acid, "citrulline", in abnormal proportions. Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins via the deimination of Arg residues. In the central nervous system, specifically PAD2 and PAD4, are the enzymes responsible for the citrullination. We used in silico screening of commercial libraries to find small molecules that would reversibly inhibit PAD4. An initial set of 10 diverse compounds was selected from the screen, and from these compounds, 3, 4, 6, and 8 showed promising inhibitory activities against PAD4 with Ki in the range of 115-153 µM. Compound 4 was selected to partake in an in vivo MOG EAE mouse model study to evaluate its effect in MS-like conditions. Results from the 24 day pilot mouse study showed an improved clinical outcome for mice being administered compound 4 compared to the control group. In brain, 4 treated mice showed a clear reduction in the CD3 +ve T cells. These results suggest that compound 4 may have potential utility and confirmed that noncovalent inhibitors of PAD enzymes can be developed as potential agents targeting MS pathology.

Asunto(s)

Hidrolasas/antagonistas & inhibidores , Esclerosis Múltiple/tratamiento farmacológico , Animales , Encéfalo/inmunología , Encéfalo/patología , Complejo CD3/metabolismo , Dominio Catalítico , Simulación por Computador , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacología , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Ratones , Modelos Moleculares , Proyectos Piloto , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacología , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Estereoisomerismo , Linfocitos T/metabolismo , Linfocitos T/patología , Tiazoles/síntesis química , Tiazoles/química , Tiazoles/farmacología

RESUMEN

Protein arginine deiminases (PADs) are involved in a number of cellular pathways, and they catalyze the transformation of peptidyl arginine residue into a citrulline as part of post-translational modifications. To understand ligand preferences, a group of probe molecules were investigated against PAD1, PAD2, and PAD4. These probe molecules carried a well-known covalent modifier of the catalytic cysteine residue, 2-chloroacetamidine moiety, which was tethered to an α-amino acid via a carbon linker. The chain length for the linker varied from 0 to 4. Time-dependent assays indicated that 2-chloroacetamidine (2CA) with no linker inhibited all PAD enzymes with a similar trend in the second-order rate constants, although with poor affinity. Among the other three probe molecules, compound 3 with a three-carbon linker exhibited the best second-order rate constants for optimal ligand reactivity with the binding site. These analyses provide insights into the relative patterns of covalent inactivation of PAD isozymes and the design of novel inhibitors targeting PAD enzymes as potential therapeutic targets.

RESUMEN

Orotidine-5'-monophosphate decarboxylase (ODCase) is an interesting enzyme with an unusual catalytic activity and a potential drug target in Plasmodium falciparum, which causes malaria. ODCase has been shown to exhibit unusual and interesting interactions with a variety of nucleotide ligands. Cytidine-5'-monophosphate (CMP) is a poor ligand of ODCase, and CMP binds to the active site of ODCase with an unusual orientation and conformation. We designed N3- and N4-modified CMP derivatives as novel ligands to ODCase. These novel CMP derivatives and their corresponding nucleosides were evaluated against Plasmodium falciparum ODCase and parasitic cultures, respectively. These derivatives exhibited improved inhibition of the enzyme catalytic activity, displayed interesting binding conformations and unusual molecular rearrangements of the ligands. These findings with the modified CMP nucleotides underscored the potential of transformation of poor ligands to ODCase into novel inhibitors of this drug target.

Asunto(s)

Antimaláricos/farmacología , Citidina/química , Malaria Falciparum/tratamiento farmacológico , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/síntesis química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Cinética , Ligandos , Malaria Falciparum/parasitología , Modelos Moleculares , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Plasmodium falciparum/enzimología , Relación Estructura-Actividad , Uridina/análogos & derivados , Uridina/metabolismo

RESUMEN

The importance of the bridge linking the two phenyl moieties of substituted phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) was assessed using a sulfonamide group, which is a bioisostere of sulfonate and ethenyl groups. Forty one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamide (PIB-SA) derivatives were prepared and biologically evaluated. PIB-SAs exhibit antiproliferative activities at the nanomolar level against sixteen cancer cell lines, block the cell cycle progression in G(2)/M phase, leading to cytoskeleton disruption and anoikis. These results were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships. These results evidence that the sulfonate and sulfonamide moieties are reciprocal bioisosteres and that phenylimidazolidin-2-one could mimic the trimethoxyphenyl moiety found in the structure of numerous potent antimicrotubule agents. Finally, compounds 16 and 17 exhibited potent antitumor and antiangiogenic activities on HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membrane similar to CA-4 without significant toxicity for the chick embryos, making this class of compounds a promising class of anticancer agents.

Asunto(s)

Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Antimitóticos/química , Antimitóticos/farmacología , Relación Estructura-Actividad Cuantitativa , Sulfonamidas/química , Sulfonamidas/farmacología , Inhibidores de la Angiogénesis/metabolismo , Animales , Antimitóticos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Membrana Corioalantoides/patología , Colchicina/metabolismo , Humanos , Modelos Moleculares , Conformación Molecular , Sulfonamidas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Bencenosulfonamidas

RESUMEN

Sixty-one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and 13 of their tetrahydro-2-oxopyrimidin-1(2H)-yl analogues (PPB-SOs) were prepared and biologically evaluated. The antiproliferative activities of PIB-SOs on 16 cancer cell lines are in the nanomolar range and unaffected in cancer cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the P-glycoprotein. None of the PPB-SOs exhibit significant antiproliferative activity. PIB-SOs block the cell cycle progression in the G(2)/M phase and bind to the colchicine-binding site on ß-tubulin leading to cytoskeleton disruption and cell death. Chick chorioallantoic membrane tumor assays show that compounds 36, 44, and 45 efficiently block angiogenesis and tumor growth at least at similar levels as combretastatin A-4 (CA-4) and exhibit low to very low toxicity on the chick embryos. PIB-SOs were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships.

Asunto(s)

Arilsulfonatos/síntesis química , Derivados del Benceno/síntesis química , Imidazolidinas/síntesis química , Estilbenos/química , Moduladores de Tubulina/síntesis química , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Arilsulfonatos/química , Arilsulfonatos/farmacología , Derivados del Benceno/química , Derivados del Benceno/farmacología , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Colchicina/metabolismo , Diseño de Fármacos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazolidinas/química , Imidazolidinas/farmacología , Modelos Moleculares , Imitación Molecular , Neovascularización Fisiológica/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , Estilbenos/farmacología , Trasplante Heterólogo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacología

RESUMEN

Fluorinated nucleosides and nucleotides are of considerable interest to medicinal chemists because of their antiviral, anticancer, and other biological activities. However, their direct interactions at target binding sites are not well understood. A new class of 2'-deoxy-2'-fluoro-C6-substituted uridine and UMP derivatives were synthesized and evaluated as inhibitors of orotidine 5'-monophosphate decarboxylase (ODCase or OMPDCase). These compounds were synthesized from the key intermediate, fully protected 2'-deoxy-2'-fluorouridine. Among the synthesized compounds, 2'-deoxy-2'-fluoro-6-iodo-UMP covalently inhibited human ODCase with a second-order rate constant of 0.62 ± 0.02 M(-1) s(-1). Interestingly, the 6-cyano-2'-fluoro derivative covalently interacted with ODCase defying the conventional thinking, where its ribosyl derivative undergoes transformation into BMP by ODCase. This confirms that the 2'-fluoro moiety influences the chemistry at the C6 position of the nucleotides and thus interactions in the active site of ODCase. Molecular interactions of the 2'-fluorinated nucleotides are compared to those with the 3'-fluorinated nucleotides bound to the corresponding target enzyme, and the carbohydrate moieties were shown to bind in different conformations.

Asunto(s)

Flúor/química , Nucleótidos/química , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Sitios de Unión , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Nucleótidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

In recent years, orotidine-5'-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

Asunto(s)

Diseño de Fármacos , Inhibidores Enzimáticos/química , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Animales , Dominio Catalítico , Humanos , Ligandos , Estructura Molecular , Unión Proteica , Nucleótidos de Purina , Nucleótidos de Pirimidina

RESUMEN

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure-activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N'-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on beta(II)-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-beta198 rather than Cys-beta239. In addition, these studies suggest that Cys-beta239 together with Asn-alpha99, Ser-alpha176, Thr-alpha177, Leu-beta246, Asn-beta247, Ala-beta248, Lys-beta252 and Asn-beta256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-beta198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta198 acylation; acylation triggering conformational changes of beta-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.

Asunto(s)

Colchicina/metabolismo , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Sitios de Unión , Línea Celular Tumoral , Colchicina/química , Simulación por Computador , Humanos , Modelos Químicos , Modelos Moleculares , Relación Estructura-Actividad , Tubulina (Proteína)/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacología , Urea/química , Urea/farmacología

RESUMEN

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad

RESUMEN

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.

Asunto(s)

Orotidina-5'-Fosfato Descarboxilasa/química , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico/genética , Cristalografía por Rayos X , Methanobacterium/enzimología , Methanobacterium/genética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Orotidina-5'-Fosfato Descarboxilasa/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Especificidad por Sustrato , Termodinámica , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/metabolismo

RESUMEN

Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus erythematosus, respectively. Specific sequences on the surface of IFN, IFN receptor recognition peptides (IRRPs) mediate the binding and signal transduction when IFN interacts with IFNAR. Structural features of two such IRRPs, IRRP-1 and IRRP-3, were used as templates to design small molecule mimetics. In silico screening was used to identify the molecular structural features mimicking their surface characteristics. A set of 26 compounds were synthesized and their ability to interfere with IFN-IFNAR interactions was investigated. Two compounds exhibited antagonist activity, specifically, blocking IFN-inducible Stat phosphorylation Stat complex-DNA binding. Design principles revealed here pave the way toward a novel series of small molecules as antagonists for IFN-IFNAR interactions.

Asunto(s)

Interferón-alfa/química , Modelos Moleculares , Péptidos/química , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/metabolismo , Línea Celular Tumoral , ADN/metabolismo , Diseño de Fármacos , Furanos/síntesis química , Furanos/química , Furanos/farmacología , Guanidinas/síntesis química , Guanidinas/química , Guanidinas/farmacología , Humanos , Interferón-alfa/metabolismo , Imitación Molecular , Fosforilación , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , Factor de Transcripción STAT1/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/química , Tiofenos/farmacología

RESUMEN

Protein kinase C (PKC) isozymes are an important class of enzymes in cell signaling and as drug targets. They are involved in specific pathways and have selectivity towards certain ligands, despite their high sequence similarities. Ruboxistaurin is a specific inhibitor of PKC-beta. To understand the molecular determinants for the selectivity of ruboxistaurin, we derived the three-dimensional structures of the kinase domains of PKC-alpha, -betaI, and -zeta using homology modeling. Several binding orientations of ruboxistaurin in the binding sites of these PKC catalytic domains were analyzed, and a putative alternative binding site for PKC-zeta was identified in its kinase domain. The calculated free energy of binding correlates well with the IC(50) of the inhibitor against each PKC isozyme. A residue-based energy decomposition analysis attributed the binding free energy to several key residues in the catalytic sites of these enzymes, revealing potential protein-ligand interactions responsible for ligand binding. The contiguous binding site revealed in the catalytic domain of PKC-zeta provides avenues for selective drug design. The details of structural nuances for specific inhibition of PKC isozymes are presented in the context of the three-dimensional structures of this important class of enzymes.

Asunto(s)

Indoles/metabolismo , Maleimidas/metabolismo , Proteína Quinasa C/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Enlace de Hidrógeno , Indoles/química , Isoenzimas/química , Isoenzimas/metabolismo , Maleimidas/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Quinasa C/química , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Termodinámica

RESUMEN

Orotidine monophosphate decarboxylase (ODCase) generally accepts pyrimidine-based mononucleotides as ligands, but other nucleotides are also known to bind to this enzyme. We investigated the kinetic properties of eight common and endogenous nucleotides with ODCases from three species: Methanobacterium thermoautotrophicum, Plasmodium falciparum, and Homo sapiens. UMP and XMP exhibited higher affinities as compared to the other nucleotides tested. The product of ODCase catalyzed decarboxylation, UMP, displayed inhibition constants (K(i)) of 330 microM against the Mt enzyme and of 210 and 220 microM against the Pf and Hs ODCases, respectively. The K(i) values for XMP were 130 microM and 43 microM, respectively, for Mt and Pf ODCases. Interestingly, XMP's affinity for human ODCase (K(i) = 0.71 microM) is comparable and even slightly better than that of the substrate OMP. Binding of various nucleotides and their structural features in the context of ODCase inhibition and inhibitor design are discussed.

Asunto(s)

Orotidina-5'-Fosfato Descarboxilasa/química , Nucleótidos de Purina/química , Nucleótidos de Pirimidina/química , Animales , Cristalografía por Rayos X , Humanos , Cinética , Ligandos , Methanobacterium/enzimología , Modelos Moleculares , Plasmodium falciparum/enzimología , Unión Proteica , Conformación Proteica , Especificidad de la Especie

RESUMEN

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

Asunto(s)

Antimaláricos/síntesis química , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Plasmodium/efectos de los fármacos , Uridina/análogos & derivados , Uridina/síntesis química , Animales , Antimaláricos/farmacología , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Modelos Moleculares , Plasmodium/enzimología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium vivax/efectos de los fármacos , Relación Estructura-Actividad , Uridina/farmacología , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/síntesis química , Uridina Monofosfato/farmacología

RESUMEN

To decipher the mechanism underlying the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas (CEU) to the colchicine-binding site on beta(II)-tubulin and to design new and selective antimitotic drugs, we developed 3D quantitative structure-activity relationships (3D-QSAR) models using CoMFA and CoMSIA analyses. The present study correlates the cell growth inhibition activities of 56 structurally related CEU derivatives to several physicochemical parameters representing steric, electrostatic, and hydrophobic fields. Both CoMFA and CoMSIA models using two different optimum numbers of components (ONC) 10 and 4, respectively, gave good internal predictions and their cross-validated r2 values were between 0.639 and 0.743. These comprehensive CoMFA and CoMSIA models are useful in understanding the structure-activity relationships of CEU. The two models were compared to the X-ray crystal structure of the complex of tubulin-colchicine and analyzed for similarities between the two modes of analysis. These models will inspire the design of new CEU derivatives with enhanced inhibition of tumor cell growth and targeting specificity of beta(II)-tubulin and the cytoskeleton.

Asunto(s)

Antimitóticos/química , Relación Estructura-Actividad Cuantitativa , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Sitios de Unión , Colchicina , Diseño de Fármacos , Humanos , Modelos Moleculares