RESUMO
The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.
Assuntos
Ebolavirus , Proteínas da Matriz Viral , Ebolavirus/genética , Ebolavirus/metabolismo , Mutação , Oxirredução , Sudão , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , HumanosRESUMO
AIMS: A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. METHODS AND RESULTS: We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wild-type PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated three-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes, and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, ß-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and haemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac haemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. CONCLUSIONS: The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a function. This regulation is important for myocardial adaptation to sustained pressure overload.
Assuntos
Cálcio , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Camundongos , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos Transgênicos , Fosforilação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adrenérgicos/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
In this study, fragment-sized hits binding to Pim-1 kinase with initially modest affinity were further optimized by combining computational, synthetic and crystallographic expertise, eventually resulting in potent ligands with affinities in the nanomolar range that address rarely-targeted regions of Pim-1 kinase. Starting from a set of crystallographically validated, chemically distinct fragments that bind to Pim-1 kinase but lack typical nucleotide mimetic structures, a library of extended fragments was built by exhaustive in silico reactions. After docking, minimization, clustering, visual inspection of the top-ranked compounds, and evaluation of ease of synthetic accessibility, either the original compound or a close derivative was synthesized and tested against Pim-1. For compounds showing the highest degree of Pim-1 inhibition the binding mode was determined crystallographically. Following a structure-guided approach, these were further optimized in a subsequent design cycle improving the compound's initial affinity by several orders of magnitude while synthesizing only a comparatively modest number of derivatives. The combination of computational and experimental approaches resulted in the development of a reasonably potent, novel molecular scaffold for inhibition of Pim-1 that targets specific surface regions, such as the interaction with R122 and P123 of the hinge region, which has been less frequently investigated in similar studies.
Assuntos
Nucleotídeos , Proteínas Proto-Oncogênicas c-pim-1 , Análise por Conglomerados , CristalografiaRESUMO
In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.
Assuntos
Pentosiltransferases/química , Animais , Células Eucarióticas/metabolismo , Feminino , Guanina/metabolismo , Humanos , Camundongos , Nucleosídeo Q , RNA de Transferência/químicaRESUMO
Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.
Assuntos
Dissulfetos/química , Pentosiltransferases/química , Bibliotecas de Moléculas Pequenas/farmacologia , Zymomonas/enzimologia , Sítios de Ligação/efeitos dos fármacos , Ligantes , Modelos Moleculares , Multimerização Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Zymomonas/químicaRESUMO
In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non-covalent interaction inventories are essential to design follow-up synthesis candidates. Two protocols are commonly applied to produce protein-ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment-sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino-acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein-ligand complexes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Ligantes , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Descoberta de Drogas , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tripsina/química , Tripsina/metabolismoRESUMO
Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.
Assuntos
Pentosiltransferases/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ligantes , Modelos Moleculares , Pentosiltransferases/química , Conformação Proteica , Shigella/enzimologiaRESUMO
The incorporation of diamondoid amino acids (DAAs) into peptide-like drugs is a general strategy to improve lipophilicity, membrane permeability, and metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA-containing kinase inhibitors of protein kinaseâ A using a sophisticated molecular dynamics protocol and solid-phase peptide synthesis. By means of a thermophoresis binding assay, NMR, and crystal structure analysis, we determined the influence of the DAAs on the secondary structure and binding affinity in comparison to the native protein kinase inhibitor, which is purely composed of proteinogenic amino acids. Affinity and binding pose are largely conserved. One variant showed 6.5-fold potency improvement, most likely related to its increased side chain lipophilicity. A second variant exhibited slightly decreased affinity presumably due to loss of hydrogen-bond contacts to surrounding water molecules of the first solvation shell.
Assuntos
Aminoácidos/química , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/química , Simulação de Dinâmica MolecularRESUMO
A ligand-binding study is presented focusing on thermodynamics of fragment expansion. The binding of four compounds with increasing molecular weight to protein kinaseâ A (PKA) was analyzed. The ligands display affinities between low-micromolar to nanomolar potency despite their low molecular weight. Binding free energies were measured by isothermal titration calorimetry, revealing a trend toward more entropic and less enthalpic binding with increase in molecular weight. All protein-ligand complexes were analyzed by crystallography and solution NMR spectroscopy. Crystal structures and solution NMR data are highly consistent, and no major differences in complex dynamics across the series are observed that would explain the differences in the thermodynamic profiles. Instead, the thermodynamic trends result either from differences in the solvation patterns of the conformationally more flexible ligand in aqueous solution prior to protein binding as molecular dynamics simulations suggest, or from local shifts of the water structure in the ligand-bound state. Our data thus provide evidence that changes in the solvation pattern constitute an important parameter for the understanding of thermodynamic data in protein-ligand complex formation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Sulfonamidas/química , Termodinâmica , Água/química , Animais , Células CHO , Cricetulus , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Relação Estrutura-AtividadeRESUMO
The human enzyme 17ß-hydroxysteroid dehydrogenase 14 (17ß-HSD14) oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. However, the physiological role of the enzyme remains unclear. We recently described the first class of nonsteroidal inhibitors for this enzyme with compound 1 showing a high 17ß-HSD14 inhibitory activity. Its crystal structure was used as starting point for a structure-based optimization in this study. The goal was to develop a promising chemical probe to further investigate the enzyme. The newly designed compounds revealed mostly very high inhibition of the enzyme and for seven of them the crystal structures of the corresponding inhibitor-enzyme complexes were resolved. The crystal structures disclosed that a small change in the substitution pattern of the compounds resulted in an alternative binding mode for one inhibitor. The profiling of a set of the most potent inhibitors identified 13 (Kiâ¯=â¯9â¯nM) with a good selectivity profile toward three 17ß-HSDs and the estrogen receptor alpha. This inhibitor displayed no cytotoxicity, good solubility, and auspicious predicted bioavailability. Overall, 13 is a highly interesting 17ß-HSD14 inhibitor, which might be used as chemical probe for further investigation of the target enzyme.
Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , 17-Hidroxiesteroide Desidrogenases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
The type II transmembrane serine protease matriptase is a potential target for anticancer therapy and might be involved in cartilage degradation in osteoarthritis or inflammatory skin disorders. Starting from previously described nonspecific thrombin and factor Xa inhibitors we have prepared new noncovalent substrate-analogs with superior potency against matriptase. The most suitable compound 35 (H-d-hTyr-Ala-4-amidinobenzylamide) binds to matriptase with an inhibition constant of 26 nM and has more than 10-fold reduced activity against thrombin and factor Xa. The crystal structure of inhibitor 35 was determined in the surrogate protease trypsin, the obtained complex was used to model the binding mode of inhibitor 35 in the active site of matriptase. The methylene insertion in d-hTyr and d-hPhe increases the flexibility of the P3 side chain compared to their d-Phe analogs, which enables an improved binding of these inhibitors in the well-defined S3/4 pocket of matriptase. Inhibitor 35 can be used for further biochemical studies with matriptase.
Assuntos
Inibidores Enzimáticos/farmacologia , Fator Xa/metabolismo , Serina Endopeptidases/metabolismo , Trombina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores do Fator Xa/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
The human enzymes aldose reductase (AR) and AKR1B10 have been thoroughly explored in terms of their roles in diabetes, inflammatory disorders, and cancer. In this study we identified two new lead compounds, 2-(3-(4-chloro-3-nitrobenzyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (JF0048, 3) and 2-(2,4-dioxo-3-(2,3,4,5-tetrabromo-6-methoxybenzyl)-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (JF0049, 4), which selectively target these enzymes. Although 3 and 4 share the 3-benzyluracil-1-acetic acid scaffold, they have different substituents in their aryl moieties. Inhibition studies along with thermodynamic and structural characterizations of both enzymes revealed that the chloronitrobenzyl moiety of compound 3 can open the AR specificity pocket but not that of the AKR1B10 cognate. In contrast, the larger atoms at the ortho and/or meta positions of compound 4 prevent the AR specificity pocket from opening due to steric hindrance and provide a tighter fit to the AKR1B10 inhibitor binding pocket, probably enhanced by the displacement of a disordered water molecule trapped in a hydrophobic subpocket, creating an enthalpic signature. Furthermore, this selectivity also occurs in the cell, which enables the development of a more efficient drug design strategy: compound 3 prevents sorbitol accumulation in human retinal ARPE-19 cells, whereas 4 stops proliferation in human lung cancer NCI-H460 cells.
Assuntos
Acetatos/química , Aldeído Redutase/antagonistas & inibidores , Inibidores Enzimáticos/química , Uracila/análogos & derivados , Acetatos/metabolismo , Acetatos/farmacologia , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Termodinâmica , Uracila/químicaRESUMO
Determination of structural similarities between protein binding pockets is an important challenge in in silico drug design. It can help to understand selectivity considerations, predict unexpected ligand cross-reactivity, and support the putative annotation of function to orphan proteins. To this end, Cavbase was developed as a tool for the automated detection, storage, and classification of putative protein binding sites. In this context, binding sites are characterized as sets of pseudocenters, which denote surface-exposed physicochemical properties, and can be used to enable mutual binding site comparisons. However, these comparisons tend to be computationally very demanding and often lead to very slow computations of the similarity measures. In this study, we propose RAPMAD (RApid Pocket MAtching using Distances), a new evaluation formalism for Cavbase entries that allows for ultrafast similarity comparisons. Protein binding sites are represented by sets of distance histograms that are both generated and compared with linear complexity. Attaining a speed of more than 20 000 comparisons per second, screenings across large data sets and even entire databases become easily feasible. We demonstrate the discriminative power and the short runtime by performing several classification and retrieval experiments. RAPMAD attains better success rates than the comparison formalism originally implemented into Cavbase or several alternative approaches developed in recent time, while requiring only a fraction of their runtime. The pratical use of our method is finally proven by a successful prospective virtual screening study that aims for the identification of novel inhibitors of the NMDA receptor.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteínas/química , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Sítios de Ligação , Ligantes , NAD/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Curva ROC , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Reprodutibilidade dos TestesRESUMO
Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved (α/ß)8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-NADP(+)-JF0064 complex has been determined at 0.85â Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallographic structure of the corresponding AKR1B10-NADP(+)-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Desenho de Fármacos , Aldo-Ceto Redutases , Ácidos Carboxílicos/química , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Halogênios , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , NADP/química , Proteínas Recombinantes/químicaRESUMO
Computational approaches play an increasingly important role for the analysis and prediction of selectivity profiles. As most of the successfully administered small molecule drugs bind in depressions on the surface of proteins, physicochemical properties of the pocket-exposed amino acids play a central role in ligand recognition during the binding event. Cavbase is an approach to describe binding sites in terms of the exposed physicochemical properties and to compare them independent of their sequence and fold homology. Classification of proteins by means of their binding-site properties is a promising approach to obtain information necessary for selectivity modeling. For this purpose, the workflow clusterScore has been developed to explore the important parameters of a clustering procedure, which will allow an accurate classification of proteins. It has been successfully applied on two diverse and challenging data sets. The predicted number of clusters, as suggested by clusterScore and the subsequent clustering of proteins are in agreement with the EC and Merops classifications. Furthermore, putative cross-reactivity mapped between calpain-1 and cysteine cathepsins on structural level has so far only been described based on ligand data. In a benchmark study using ligand topology, binding site, and sequence information of eleven serine proteases, the emerging clusters indicate a pronounced correlation between the cavity and ligand data. These results emphasize the importance of binding-site information which should be considered for ligand design during lead optimization cycles. The program clusterScore is freely available and can be downloaded from our Web site www.agklebe.de.
Assuntos
Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Algoritmos , Animais , Sítios de Ligação , Análise por Conglomerados , Desenho de Fármacos , Humanos , Camundongos , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos TestesRESUMO
The trypsin-like serine protease plasmin is a target for the development of antifibrinolytic drugs for use in cardiac surgery with cardiopulmonary bypass or organ transplantations to reduce excessive blood loss. The optimization of our recently described substrate-analogue plasmin inhibitors, which were cyclized between their P3 and P2 side chains, provided a new series with improved efficacy and excellent selectivity. The most potent inhibitor 8 binds to plasmin with an inhibition constant of 0.2 nM, whereas K(i) values >1 µM were determined for nearly all other tested trypsin-like serine proteases, with the exception of trypsin, which is also inhibited in the nanomolar range. Docking studies revealed a potential binding mode in the widely open active site of plasmin that explains the strong potency and selectivity profile of these inhibitors. The dialkylated piperazine-linker segment contributes to an excellent solubility of all analogues. Based on their overall profile the presented inhibitors are well suited for further development as injectable antifibrinolytic drugs.
Assuntos
Antifibrinolíticos/farmacologia , Antifibrinolíticos/química , Modelos MolecularesRESUMO
Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC(50) values of 1.7 and 3.0 µM and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine.
Assuntos
Antimaláricos/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/química , Ácidos Graxos/biossíntese , Antimaláricos/síntese química , Antimaláricos/toxicidade , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/enzimologia , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation-siRNA and polycation-pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems.
Assuntos
Inativação Gênica , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Polietilenoimina/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção/métodos , Teste de Materiais , Tamanho da PartículaRESUMO
Designed for gene therapy of chronic diseases, HBP-DEAPA 60 is a non-toxic biodegradable amine modified hyperbranched polyester. This candidate was chosen from a series of hyperbranched polymers for further characterization as it showed the best transfection efficiency and fastest degradation rate. HBP-DEAPA 60/DNA complexes were investigated with regard to stability, uptake and formation to gain a better insight into HBP-DEAPA 60/DNA complex properties. We investigated HBP-DEAPA 60/DNA complex uptake into A 549 cells by FACS and CLSM. Their stability was investigated by a heparin displacement assay as well as by DNAse I assay. Morphology was shown by AFM. HBP-DEAPA 60/DNA complex formation was further characterized in terms of thermodynamic parameters. We studied the conformation of DNA in nano-complexes via circular dichroism (CD) spectroscopy for different NP ratios. Thermodynamic studies showed that binding enthalpies were endothermic; the nano-complex formation was entropically driven. Although PEI/DNA and HBP-DEAPA 60/DNA complexes showed similar behavior with regard to uptake, heparin stability, DNA helicality and their entropically driven complex formation they differ in their binding constant K(a) and in their ability to protect the DNA from DNAse. Concerning K(a) and DNAse stability, HBP-DEAPA/DNA complexes should be further optimized. This shows that different characterization studies are necessary to fully characterize polyplex stability and properties.
Assuntos
Aminas/química , DNA/química , Técnicas de Transferência de Genes , Nanoestruturas/química , Poliésteres/química , Aminas/administração & dosagem , Aminas/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , DNA/administração & dosagem , DNA/farmacocinética , Desoxirribonuclease I/química , Humanos , Nanoestruturas/administração & dosagem , Poliésteres/administração & dosagem , Poliésteres/metabolismo , Polietilenoimina/administração & dosagem , Polietilenoimina/química , Polietilenoimina/metabolismoRESUMO
Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.