RESUMO
Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.
Assuntos
Antivirais/farmacologia , Cisteína Endopeptidases/metabolismo , Isoxazóis/farmacologia , Vírus Norwalk/efeitos dos fármacos , Pirrolidinonas/farmacologia , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Amidas/química , Amidas/farmacologia , Antivirais/química , Linhagem Celular , Cisteína Endopeptidases/química , Citidina/análogos & derivados , Citidina/química , Citidina/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Isoxazóis/química , Simulação de Acoplamento Molecular , Vírus Norwalk/enzimologia , Vírus Norwalk/genética , Papaína/antagonistas & inibidores , Papaína/química , Papaína/metabolismo , Fenilalanina/análogos & derivados , Pirazinas/química , Pirazinas/farmacologia , Pirrolidinonas/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Replicon , Valina/análogos & derivados , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The arenaviruses and hantaviruses are segmented genome RNA viruses that are hosted by rodents. Due to their association with rodents, they are globally widespread and can infect humans via direct or indirect routes of transmission, causing considerable human morbidity and mortality. Nevertheless, despite their obvious and emerging importance as pathogens, there are currently no effective antiviral drugs (except ribavirin which proved effective against Lassa virus) with which to treat humans infected by any of these viruses. The EU-funded VIZIER project (Comparative Structural Genomics of Viral Enzymes Involved in Replication) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the arenaviruses and bunyaviruses. This review highlights some of the major features of the arenaviruses and hantaviruses that have been investigated during recent years. After describing their classification and epidemiology, we review progress in understanding the genomics as well as the structure and function of replicative enzymes achieved under the VIZIER program and the development of new disease control strategies.
Assuntos
Antivirais/farmacologia , Infecções por Arenaviridae/epidemiologia , Arenavirus/efeitos dos fármacos , Descoberta de Drogas/tendências , Infecções por Hantavirus/epidemiologia , Orthohantavírus/efeitos dos fármacos , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/classificação , Arenavirus/genética , Arenavirus/patogenicidade , Genômica , Orthohantavírus/classificação , Orthohantavírus/genética , Orthohantavírus/patogenicidade , Infecções por Hantavirus/tratamento farmacológico , Humanos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
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
High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.
Assuntos
Cristalização , Insulina/química , Microscopia Confocal/métodos , Difusão , Cinética , Modelos QuímicosRESUMO
Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal structure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided structural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Farmacorresistência Bacteriana/genética , Substâncias Macromoleculares , Polienos/metabolismo , Piridonas/farmacologia , Tiazóis/farmacologiaRESUMO
For a long time blood coagulation factor XIII did not get the attention it deserves, and research in the field of transglutaminases was neglected. The interesting results obtained in recent years demonstrate the importance of this protein for the stabilization of a blood clot. The accurate regulation of activation and of localization of factor XIII is not fully understood. Yet the proposed mechanisms suggest a high degree of complexity. In this review, recent biochemical and structural results on factor XIII are discussed.
Assuntos
Fator XIII/metabolismo , Transglutaminases/metabolismo , Fator XIII/química , Fator XIII/genética , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.
Assuntos
Cálcio/metabolismo , Transglutaminases/química , Transglutaminases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Fator XIII/química , Fator XIII/genética , Fator XIII/metabolismo , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Transglutaminases/genéticaRESUMO
Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.
Assuntos
Ligação de Hidrogênio , Proteínas/química , Proteínas de Transporte/química , Glicoproteínas/química , Proteína Associada a Proteínas Relacionadas a Receptor de LDL , Conformação Proteica , Fosfolipases Tipo C/químicaRESUMO
The human pathogen Legionella pneumophila, the etiological agent of the severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits a peptidyl prolyl cis-trans isomerase (PPIase) activity, which appears to be important for infection. Here we report the 2.4 A crystal structure of the Mip protein from L. pneumophila Philadelphia 1 and the 3.2 A crystal structure of its complex with the drug FK506. Each monomer of the homodimeric protein consists of an N-terminal dimerization module, a long (65 A) connecting alpha-helix and a C-terminal PPIase domain exhibiting similarity to human FK506-binding protein. In view of the recent significant increase in the number of reported cases of Legionnaires' disease and other intracellular infections, these structural results are of prime interest for the design of new drugs directed against Mip proteins of intracellular pathogens.
Assuntos
Imunofilinas/química , Imunofilinas/metabolismo , Legionella pneumophila/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Tacrolimo/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Doença dos Legionários/microbiologia , Modelos Moleculares , Estrutura Terciária de Proteína , Tacrolimo/química , Zinco/metabolismoRESUMO
Cellosyl is a bacterial muramidase from Streptomyces coelicolor. Similar to other lysozymes, the enzyme cleaves the beta-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine units, but it also exhibits a beta-1,4-N,6-O-diacetylmuramidase activity. The latter enables Cellosyl to degrade the cell walls of Staphylococcus aureus, which are not hydrolyzed by chicken-, goose-, or bacteriophage T4-type lysozymes. The enzymatic activity and amino acid sequence of Cellosyl group it with lysozymes of the Chalaropsis type, for which no detailed structural information has been available so far. The crystal structure of Cellosyl from S. coelicolor has been determined to a resolution of 1.65 A and refined to an R-factor of 15.2%. The enzyme is comprised of a single domain and possesses an unusual beta/alpha-barrel fold. The last strand, beta 8, of the (beta/alpha)(5)beta(3)-barrel is found to be antiparallel to strands beta 7 and beta 1. Asp-9, Asp-98, and Glu-100 are located at the active site. The structure of Cellosyl exhibits a new lysozyme fold and represents a new class of polysaccharide-hydrolyzing beta/alpha-barrels.
Assuntos
Muramidase/química , Streptomyces/enzimologia , Sequência de Aminoácidos , Sequência de Carboidratos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de AminoácidosRESUMO
A diffraction data set has been collected from a blood coagulation factor XIII-Ca(2+) complex crystal at the X-ray diffraction beamline of the ELETTRA synchrotron (Trieste, Italy) at a wavelength of 2.6 A. The data collection could be carried out using the beamline as is, without making any time-consuming changes to the apparatus. Various data-processing schemes have been employed and it has been observed that local or detector scaling procedures are essential for producing the 'best' anomalous differences.
Assuntos
Cristalografia por Raios X/instrumentação , Oxirredutases/química , Raios X , Cristalografia por Raios X/métodos , Análise de Fourier , Paracoccus denitrificans/enzimologia , Conformação ProteicaRESUMO
The ab initio computation of the molecular envelopes of two proteins exclusively from their corresponding diffraction amplitudes demonstrates that an efficient and inherently parallel evolutionary search algorithm can assist in the direct phasing of macromolecules for which almost no a priori structural information is available. The applicability of this evolutionary computational approach is general and should not be limited to the examples described nor to extremes of data resolution, symmetry or structural size.
Assuntos
Evolução Biológica , Cristalografia por Raios X , Termolisina/química , Algoritmos , Modelos TeóricosRESUMO
A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.
Assuntos
Proteínas/química , Bases de Dados Factuais , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.
Assuntos
Antibacterianos/metabolismo , Aurodox/química , Aurodox/metabolismo , Guanosina Difosfato/metabolismo , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Antibacterianos/química , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Leucina , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Thermus thermophilus/metabolismo , TirosinaRESUMO
The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.
Assuntos
Aminoglicosídeos , Escherichia coli , Guanosina Trifosfato/metabolismo , Mutação/genética , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Peptídeos Cíclicos/farmacologia , Aminoacil-RNA de Transferência/metabolismo , Tiazóis/metabolismo , Actinomycetales/química , Adenina/metabolismo , Substituição de Aminoácidos/genética , Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Poli U/genética , Poli U/metabolismo , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/genética , Termodinâmica , Thermus/química , Tiazóis/química , Tiazóis/farmacologiaRESUMO
Hen egg-white lysozyme has been crystallized at slightly alkaline pH using 2-methyl-2,4-pentanediol (MPD) as the precipitant. The crystals are nearly isomorphous to crystals grown at acidic pH using sodium chloride as the precipitant. However, the growth kinetics differ markedly between the two conditions. The major reason for this is a molecule of MPD that binds tightly in between two lysozyme molecules and favors the growth of the crystals along the crystallographic c direction over growth perpendicular to it.
Assuntos
Muramidase/química , Animais , Precipitação Química , Galinhas , Cristalização , Cristalografia por Raios X , Feminino , Glicóis , Concentração de Íons de Hidrogênio , Conformação Proteica , Eletricidade EstáticaRESUMO
Monoclinic factor XIII crystals have been transferred to a solution containing increasing amounts of the precipitant PEG 6000. At a concentration of about 36%(w/v) PEG 6000, a phase transition was observed. The space group of the crystals was preserved on the transition, but half of the 2(1) screw axes were lost, which meant that the unit-cell volume and the content of the asymmetric unit were doubled. The structure of factor XIII in the new crystal form was solved by molecular replacement. About 80% of the changes accompanying the transition can be explained by a rigid-body rotation of half of the factor XIII dimers in the lattice by about 5 degrees. The remaining changes are mostly small interdomain movements of the four domains which constitute one factor XIII monomer.
Assuntos
Fator XIII/química , Cristalização , Cristalografia por Raios X , Dimerização , Humanos , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/químicaRESUMO
Based on the geometrical parameters around seventeen incorrectly assigned trans conformations of peptide bonds in protein structures and their correct cis counterparts, we have devised an algorithm that is capable of detecting these sites. The algorithm was optimized to reliably find all of the seventeen test cases. It can be used to quickly scan an atomic coordinate file or the complete Brookhaven Protein Data Base for more likely candidates for non-Pro cis peptide bonds. Also, it can be of help to guide the crystallographer in intermediate stages of structure determination towards suspect areas.