RESUMO
Electron transfer between the title compounds and their radical cations, which were generated by photoinduced electron transfer from the sulfides to excited 2,4,6-triphenylpyrylium cations, was investigated by time-resolved measurements of chemically induced dynamic nuclear polarization (CIDNP) in acetonitrile. The strongly negative activation entropies provide evidence for an associative-dissociative electron exchange involving dimeric radical cations. Despite this mechanistic complication, the free energies of activation were found to be well reproduced by the Marcus theory of electron transfer, with the activation barrier still dominated by solvent reorganization.
RESUMO
The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mutação , Dobramento de Proteína , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Escherichia coli/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.
Assuntos
Mycoplasma/enzimologia , Peptidilprolil Isomerase/química , Proteínas de Ligação a Tacrolimo/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Primers do DNA/química , Escherichia coli/enzimologia , Histidina/química , Ligação de Hidrogênio , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mycoplasma/genética , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 µM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.
Assuntos
Inibidores Enzimáticos/síntese química , Mycobacterium tuberculosis/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Timidilato Sintase/químicaRESUMO
Using NMR spectroscopy we show that the cellular prion protein constitutes a target for binding of various acridine and phenothiazine derivatives. We unambiguously map the quinacrine binding site of recombinant human prion protein to residues Tyr225, Tyr226, and Gln227 of helix alpha3, which is located near the "protein X" epitope. The millimolar dissociation constant of the complex suggests that in vivo inhibition of prion propagation occurs after 10000-fold concentration of quinacrine within endolysosomes.
Assuntos
Antimaláricos/química , Príons/química , Quinacrina/química , Sítios de Ligação , Cloroquina/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fragmentos de Peptídeos/química , Fenotiazinas/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Diversity and robustness of NMR based screening methods make these techniques highly attractive as tools for drug discovery. Although not all screening techniques discussed here may be applicable to any given target, there is however a good chance that at least one of the described methods will prove productive in finding several medium affinity ligands. A comparison of each of the methods is given in Table 1. For drug targets of molecular weight < 30 kDa SAR by NMR appears to be the method of choice since it yields detailed information about the location of the binding site. It remains to be seen whether 15N-1H-TROSY based screening techniques will prove useful for larger protein targets, especially considering the added effort needed for spectral assignment and the increased complexity due to spectral overlap. Nevertheless, with the application of new cryo-cooled NMR probes, 15N-1H-HSQC based screening can now be considered a high throughput method. Ligand-based NMR screening methods can be used for protein targets of virtually any size, but are restricted in the ligand's binding affinity range. Because sufficient ligand-protein dissociation rates are needed, only binding of ligands with low (milimolar) to intermediate (micromolar) affinities is detectable. It is expected that cryo-cooled NMR probe technology will also advance ligand detected NMR screening to the high throughput level. Certainly protein and ligand concentrations can be lowered drastically and experiment times can be shortened with increased sensitivity. However, spectral overlap will be of major concern when mixtures of up to 100 compounds are to be screened. For such applications only techniques for which the signals of bound ligands survive will be useful, and sophisticated software will be needed to deconvolute the spectra of multiple bound ligands. Although only ligands with medium to low affinities can be found, ligand based NMR screening has been used as an effective prescreening tool for assay based high throughput screening. Identifying a large ensemble of medium affinity ligands may not only aid in building a binding site pharmacophore model (see Chapter 11), but also may yield crucial information for overcoming tissue availability, toxicity, or even intellectual property related problems. Although NMR based screening is only one of the more recent additions to the bag of tools used in drug discovery [1, 2], its simplicity and wide range of application (including protein-protein and protein-nucleic acid interactions) has attracted much attention. Advances in NMR instrumentation and methodology have already paved the road for NMR based screening to become a high throughput technique. In addition to this, NMR is exceptional in the amount of detailed structural [table: see text] information it can provide. Not only can NMR readily reveal the binding site (15N-1H-HSQC screening) or the conformation of the bound ligand (transfer NOE), but it can also supply information that enables precise docking of the ligand to the protein's binding pocket (isotope-filtered NOESY). NMR data can therefore provide a natural connection between experimental HTS and combinatorial chemistry techniques with computational methods such as 3D-database searching (see Chapter 10), virtual screening (docking) and structure-based ligand design (see also Chapter 8).
Assuntos
Espectroscopia de Ressonância Magnética , Farmacologia/instrumentação , Animais , Difusão , Desenho de Fármacos , Humanos , Ligantes , Receptores de Droga/química , Relação Estrutura-AtividadeRESUMO
Members of the fibroblast growth factor receptor tyrosine kinase family (FGFR1-4) play an important role in many signalling cascades. Although tightly regulated, aberrant activity of these enzymes may lead to, or become features of, disease pathologies including cancer. FGFR isoforms have been the subject of drug discovery programmes, with a number of kinase-domain inhibitors in pre-clinical and clinical development. Here, we present the first (83% complete) backbone resonance assignments of apo-FGFR1 kinase.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Sequência de Aminoácidos , Humanos , Estrutura Terciária de ProteínaRESUMO
Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.
Assuntos
L-Lactato Desidrogenase/antagonistas & inibidores , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios Enzimáticos , Humanos , Isoenzimas/antagonistas & inibidores , Lactato Desidrogenase 5 , Espectroscopia de Ressonância Magnética , Malonatos/síntese química , Malonatos/química , Malonatos/farmacologia , Modelos Moleculares , Estrutura Molecular , Niacinamida/química , Ácido Oxâmico/análogos & derivados , Ácido Oxâmico/síntese química , Ácido Oxâmico/química , Ácido Oxâmico/farmacologia , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieAssuntos
Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Cristalografia por Raios X , Dimetil Sulfóxido , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Fenilalanina/química , Plasmídeos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
MDM2 and MDM4 are proteins involved in regulating the tumour suppressor p53. MDM2/4 and p53 interact through their N-terminal domains and disrupting this interaction is a potential anticancer strategy. The MDM2-p53 interaction is structurally and biophysically well characterised, whereas equivalent studies on MDM4 are hampered by aggregation of the protein. Here we present the NMR characterization of MDM4 (14-111) both free and in complexes with peptide and small-molecule ligands. MDM4 is more dynamic in its apo state than is MDM2, with parts of the protein being unstructured. These regions become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism; this might influence rational design of MDM4 inhibitors.
Assuntos
Proteína Supressora de Tumor p53/metabolismo , Humanos , Ligantes , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desenho de Fármacos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas/metabolismo , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.
Assuntos
Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Quinases/química , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Relação Estrutura-AtividadeRESUMO
Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.