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/metabolismoRESUMEN
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 ElectrosprayRESUMEN
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 PirimidinaRESUMEN
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-ActividadRESUMEN
Recent crystal structures of the CorA Mg(2+) transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg(2+)-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg(2+) selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.
Asunto(s)
Proteínas de Transporte de Catión/química , Magnesio/metabolismo , Asparagina/química , Proteínas de Transporte de Catión/metabolismo , Cationes , Cromatografía en Gel , Relación Dosis-Respuesta a Droga , Prueba de Complementación Genética , Iones , Magnesio/química , Modelos Biológicos , Conformación Molecular , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Thermotoga maritima/metabolismoRESUMEN
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 EspecieRESUMEN
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/farmacologíaRESUMEN
Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.
Asunto(s)
Antimaláricos/síntesis química , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Uridina Monofosfato/análogos & derivados , Uridina/análogos & derivados , Animales , Antimaláricos/química , Antimaláricos/farmacología , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Espectrometría de Masas , Methanobacterium/enzimología , Modelos Moleculares , Orotidina-5'-Fosfato Descarboxilasa/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/aislamiento & purificación , Estereoisomerismo , Relación Estructura-Actividad , Uridina/síntesis química , Uridina/química , Uridina/farmacología , Uridina Monofosfato/síntesis química , Uridina Monofosfato/química , Uridina Monofosfato/farmacologíaRESUMEN
Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.
Asunto(s)
Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Orotidina-5'-Fosfato Descarboxilasa/química , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/síntesis química , Calorimetría , Simulación por Computador , Diseño de Fármacos , Cinética , Modelos Moleculares , Termodinámica , Uridina Monofosfato/químicaRESUMEN
The mechanism of proteolysis by serine proteases is a reasonably well-understood process. Typically, a histidine residue acting as a general base deprotonates the catalytic serine residue and the hydrolytic water molecule. We disclose here, the use of an unnatural d-amino acid as a strategic residue in P1 position, designed de novo based on the architecture of the protease catalytic site to impede the catalytic histidine residue at the stage of acyl-enzyme intermediate. Several probe molecules containing d-homoserine or its derivatives at P1 position are evaluated. Compounds 1, 6, and 8-10 produced up to 57% loss of activity against chymotrypsin. More potent and specific inhibitors could be designed with structure optimization as this strategy is completely general and can be used to design inhibitors against any serine or cysteine protease.
Asunto(s)
Aminoácidos/química , Inhibidores de Proteasas/química , Dominio Catalítico , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Orotidine-5'-monophosphate decarboxylase (ODCase) has evolved to catalyze a decarboxylation reaction, most probably via a carbanion species at the C6 position of orotidine-5'-monophosphate. We reveal an unusual biochemical pathway of conversion of 6-cyano-uridine-5'-monophosphate by ODCase to barbiturate-5'-monophosphate via perhaps an electrophilic center at the C6 position, leading to inhibition. This potential of ODCase is very useful in the design of novel inhibitors.