RESUMO
Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.
Assuntos
Antivirais/farmacologia , Biologia Computacional , Cristalografia , Desenho de Fármacos , Genômica , Proteômica , Vírus de RNA/efeitos dos fármacos , RNA Polimerase Dependente de RNA , Replicação Viral/efeitos dos fármacos , Antivirais/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cooperação Internacional , Modelos Moleculares , Vírus de RNA/enzimologia , Vírus de RNA/patogenicidade , Vírus de RNA/fisiologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.
Assuntos
Antimaláricos/química , Ácido Aspártico Endopeptidases/química , Desenho de Fármacos , Plasmodium/enzimologia , Inibidores de Proteases/química , Animais , Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Biologia Computacional/métodos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Plasmodium/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Estrutura Terciária de ProteínaRESUMO
Potassium ion channels enable efficient and selective permeation of K+ ions across nonpolar biological membranes. Here we review the results of recent free energy calculations related to the permeation of monovalent cations through K+ channels and to the channel inhibition by blocker compounds. In particular, the progress in computational studies of the bacterial KcsA channel is discussed.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Potássio/metabolismo , Cátions Monovalentes/metabolismo , Simulação por Computador , Entropia , Modelos Moleculares , Canais de Potássio/química , Canais de Potássio/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
Microscopic molecular dynamics free energy perturbation calculations of the K(+)/Na(+) selectivity in the KcsA potassium channel, based on its experimental three-dimensional structure, are reported. The relative binding free energies for K(+) and Na(+) in the most relevant ion occupancy states of the four-site selectivity filter are calculated. The previously proposed mechanism for ion permeation through the KcsA channel is predicted, in agreement with available experimental data, to have a significant selectivity for K(+) over Na(+). The calculations also show that the individual 'binding site' selectivities are generally not additive and the doubly loaded states of the filter thus display cooperative effects. The only site that is not K(+) selective is that which is located at the entrance to the internal water cavity, suggesting the possibility that internal Na(+) could block outward currents.
Assuntos
Proteínas de Bactérias , Canais de Potássio/química , Potássio/química , Sódio/química , Sítios de Ligação , Cátions Monovalentes , Simulação por Computador , Cristalografia , Modelos Moleculares , Mutação , Canais de Potássio/genética , TermodinâmicaRESUMO
A combination of empirical scoring and conformational sampling for ligand binding affinity prediction is examined. The behaviour of a scoring function with respect to the sensitivity to conformational changes is investigated using ensembles of structures generated by molecular dynamics simulation. The correlation between the calculated score and the coordinate deviation from the experimental structure is clear for the complex of arabinose with arabinose-binding protein, which is dominated by hydrogen bond interactions, while the score calculated for the hydrophobic complex between retinol and retinol binding protein is rather insensitive to ligand conformational changes. For typical ensembles of structures generated by molecular dynamics at 300 K, the variation of the calculated score is considerably smaller than that of the underlying molecular mechanics interaction energies.
Assuntos
Ligantes , Receptores de Droga/química , Algoritmos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteínas de Ligação ao Retinol/química , TemperaturaRESUMO
A series of lipophilic soft drugs structurally related to the nonclassical dihydrofolate reductase (DHFR) inhibitors trimetrexate and piritrexim have been designed, synthesized, and evaluated in DHFR assays, with special emphasis on the inhibition of P. carinii DHFR. The best inhibitors, encompassing an ester bond in the bridge connecting the two aromatic systems, were approximately 10 times less potent than trimetrexate and piritrexim. The metabolites were designed to be poor inhibitors. Furthermore, molecular dynamics simulations of three ligands in complex with DHFR from Pneumocystis carinii and from the human enzyme were conducted in order to better understand the factors determining the selectivity. A correct ranking of the relative inhibition of DHFR was achieved utilizing the linear interaction energy method. The soft drugs are intended for local administration. One representative ester was selected for a pharmacokinetic study in rats where it was found to undergo fast metabolic degradation to the predicted inactive metabolites.
Assuntos
Antagonistas do Ácido Fólico/síntese química , Pneumocystis/enzimologia , Animais , Ésteres , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/farmacologia , Humanos , Ligantes , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Modelos Moleculares , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase , Trimetrexato/químicaRESUMO
The binding of P1 variants of bovine pancreatic trypsin inhibitor (BPTI) to trypsin has been investigated by means of molecular dynamics simulations. The specific interaction formed between the amino acid at the primary binding (P1) position of the binding loop of BPTI and the specificity pocket of trypsin was estimated by use of the linear interaction energy (LIE) method. Calculations for 13 of the naturally occurring amino acids at the P1 position were carried out, and the results obtained were found to correlate well with the experimental binding free energies. The LIE calculations rank the majority of the 13 variants correctly according to the experimental association energies and the mean error between calculated and experimental binding free energies is only 0.38 kcal/mole, excluding the Glu and Asp variants, which are associated with some uncertainties regarding protonation and the possible presence of counter-ions. The three-dimensional structures of the complex with three of the P1 variants (Asn, Tyr, and Ser) included in this study have not at present been solved by any experimental techniques and, therefore, were modeled on the basis of experimental data from P1 variants of similar size. Average structures were calculated from the MD simulations, from which specific interactions explaining the broad variation in association energies were identified. The present study also shows that explicit treatment of the complex water-mediated hydrogen bonding network at the protein-protein interface is of crucial importance for obtaining reliable binding free energies. The successful reproduction of relative binding energies shows that this type of methodology can be very useful as an aid in rational design and redesign of biologically active macromolecules.
Assuntos
Aprotinina/metabolismo , Simulação por Computador , Tripsina/metabolismo , Animais , Aprotinina/química , Sítios de Ligação , Bovinos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Termodinâmica , Tripsina/química , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismoRESUMO
Organisms that live in constantly cold environments have to adapt their metabolism to low temperatures, but mechanisms of enzymatic adaptation to cold environments are not fully understood. Cold active trypsin catalyses reactions more efficiently and binds ligands more strongly in comparison to warm active trypsin. We have addressed this issue by means of comparative free energy calculations studying the binding of positively charged ligands to two trypsin homologues. Stronger inhibition of the cold active trypsin by benzamidine and positively charged P1-variants of BPTI is caused by rather subtle electrostatic effects. The different affinity of benzamidine originates solely from long range interactions, while the increased binding of P1-Lys and -Arg variants of BPTI is attributed to both long and short range effects that are enhanced in the cold active trypsin compared to the warm active counterpart. Electrostatic interactions thus provide an efficient strategy for cold adaptation of trypsin.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Tripsina/química , Tripsina/metabolismo , Animais , Aprotinina/química , Aprotinina/genética , Aprotinina/metabolismo , Benzamidinas/metabolismo , Catálise , Bovinos , Simulação por Computador , Ligantes , Modelos Moleculares , Mutação/genética , Ligação Proteica , Conformação Proteica , Salmão , Eletricidade Estática , TermodinâmicaRESUMO
Experimental and theoretical studies of the catalytic mechanism in protein tyrosine phosphatases and dual specific phosphatases are reviewed. The structural properties of these enzymes contributing to the efficient rate enhancement of phosphate monoester hydrolysis have been established during the last decade. There are, however, uncertainties in the interpretation of available experimental data that make the commonly assumed reaction mechanism somewhat doubtful. Theoretical calculations as well as analysis of crystal structures point towards an alternative interpretation of the ionisation state in the reactive complex.
Assuntos
Conformação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Animais , Catálise , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genéticaRESUMO
We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.
Assuntos
Proteínas de Bactérias , Bloqueadores dos Canais de Potássio , Tetraetilamônio/metabolismo , Sítios de Ligação , Simulação por Computador , Modelos Moleculares , Mutação Puntual , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação ProteicaRESUMO
Thrombin is an essential protein involved in blood clot formation and an important clinical target, since disturbances of the coagulation process cause serious cardiovascular diseases such as thrombosis. Here we evaluate the performance of a molecular dynamics based method for predicting the binding affinities of different types of human thrombin inhibitors. For a series of eight ligands the method ranks their relative affinities reasonably well. The binding free energy difference between high and low affinity representatives in the test set is quantitatively reproduced, as well as the stereospecificity for a chiral inhibitor. The original parametrisation of this linear interaction energy method requires the addition of a constant energy term in the case of thrombin. This yields a mean unsigned error of 0.68 kcal/mol for the absolute binding free energies. This type of approach is also useful for elucidating three-dimensional structure-activity relationships in terms of microscopic interactions of the ligands with the solvated enzyme.
Assuntos
Antitrombinas/metabolismo , Modelos Moleculares , Trombina/metabolismo , Antitrombinas/química , Humanos , Ligantes , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Trombina/químicaRESUMO
An overview of the simplified linear interaction energy (LIE) method for calculation of ligand binding free energies is given. This method is based on force field estimations of the receptor-ligand interactions and thermal conformational sampling. A notable feature is that the binding energetics can be predicted by considering only the intermolecular interactions between the ligand and receptor. The approximations behind this approach are examined and different parametrizations of the model are discussed. In general, LIE type of methods appear particularly useful for computational drug lead optimization.
Assuntos
Ligantes , Modelos Lineares , Modelos Moleculares , Humanos , Modelos Biológicos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Eletricidade Estática , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.
Assuntos
Proteínas de Bactérias , Cátions/química , Canais de Potássio/química , Prótons , Sítios de Ligação , Simulação por Computador , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Potássio/química , TermodinâmicaRESUMO
The relative binding affinities to human dihydrofolate reductase of four new potential antifolates, containing ester linkages between the two aromatic systems, were estimated by free energy perturbation simulations. The ester analogue, predicted to exhibit the highest binding affinity to human dihydrofolate reductase, and a reference ester (more structurally related to methotrexate) were synthesized. As deduced from the measured IC(50) values, the calculated ranking of the ligands was correct although a greater difference in affinity was indicated by the experimental measurements. Among the new antifolates the most potent inhibitor exhibited a similar pharmacokinetic profile to methotrexate but lacked activity in a complex antiarthritic model in rat in vivo.
Assuntos
Antagonistas do Ácido Fólico/metabolismo , Metotrexato/análogos & derivados , Metotrexato/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antirreumáticos/síntese química , Antirreumáticos/química , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Disponibilidade Biológica , Feminino , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Masculino , Metotrexato/síntese química , Metotrexato/química , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Ratos , Tetra-Hidrofolato Desidrogenase/química , TermodinâmicaRESUMO
The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu(172). Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 +/- 1. 3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.
Assuntos
Simulação por Computador , Isótopos , Lactoilglutationa Liase/metabolismo , Catálise , Cinética , TermodinâmicaRESUMO
Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol(-1). Other putative permeation pathways can be excluded because they would involve states that are too high in energy.
Assuntos
Canais de Potássio/metabolismo , Proteínas de Bactérias/metabolismo , Permeabilidade , Potássio/metabolismo , Rubídio/metabolismo , Sódio/metabolismo , StreptomycesRESUMO
The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process.
Assuntos
Conformação Proteica , Fosfatases cdc25/química , Sítios de Ligação , Catálise , Ligantes , Modelos MolecularesRESUMO
The reversible folding/unfolding of a short peptide in solution is studied by molecular dynamics simulations. The effects of long-range electrostatic interactions are examined and found to be important both for the equilibrium between folded and unfolded states and the dynamics of the folding process. The neglect of long-range electrostatics leads to an increased population of unfolded states and increased structural fluctuations. When such interactions are taken into account, the peptide unfolds and folds to the experimentally determined structure several times during a 25 ns trajectory, with approximately equal populations of folded and unfolded states in the neighborhood of its proposed melting temperature. The effect of using spherical boundary conditions rather than periodic ones does not appear to have any major effect on the folding dynamics.
Assuntos
Modelos Moleculares , Peptídeos/química , Dobramento de Proteína , Eletricidade Estática , Simulação por Computador , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
Hydrolysis of the phosphoenzyme intermediate is the second and rate limiting step of the reaction catalyzed by the protein tyrosine phosphatases (PTPs). The cysteinyl phosphate thioester bond is cleaved by nucleophilic displacement where an active site water molecule attacks the phosphorus atom. Starting from the crystal structure of the low molecular weight PTP, we study the energetics of this reaction utilizing the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The reactions of the wild-type as well as the D129A and C17S mutants are modeled. For the D129A mutant, which lacks the general acid/base residue Asp-129, an alternative reaction mechanism is proposed. The calculated activation barriers are in all cases in good agreement with experimental reaction rates. The present results together with earlier computational and experimental work now provide a detailed picture of the complete reaction mechanism in many PTPs. The key role played by the structurally invariant signature motif in stabilizing a double negative charge is reflected by its control of the energetics of both transition states and the reaction intermediate.
Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Animais , Domínio Catalítico , Bovinos , Simulação por Computador , Hidrólise , Técnicas In Vitro , Cinética , Modelos Químicos , Modelos Moleculares , Peso Molecular , Mutação Puntual , Conformação Proteica , Proteínas Tirosina Fosfatases/genética , TermodinâmicaRESUMO
Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. Proteins 1999;36:370-379.