RESUMO
Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.
Assuntos
Exfoliatinas , Infecções Estafilocócicas , Domínio Catalítico , Exfoliatinas/química , Exfoliatinas/metabolismo , Humanos , Staphylococcus aureus/metabolismoRESUMO
Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.
Assuntos
Antimaláricos/farmacologia , Simulação por Computador , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sítio Alostérico , Antimaláricos/química , Inibidores de Cisteína Proteinase/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum/enzimologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Falcipain-2 (FP-2) is hemoglobinase considered an attractive drug target of Plasmodium falciparum. Recently, it has been shown that peptidomimetic nitriles containing a 3-pyridyl (3Pyr) moiety at P2 display high affinity and selectivity for FP-2 with respect to human cysteine cathepsins (hCats), outperforming other P2-Pyr isomers and analogs. Further characterization demonstrated that certain P3 variants of these compounds possess micromolar inhibition of parasite growth in vitro and no cytotoxicity against human cell lines. However, the structural determinants underlying the selectivity of the 3Pyr-containing nitriles for FP-2 remain unknown. In this work, we conduct a thorough computational study combining MD simulations and free energy calculations to decipher the bases of the selectivity of the aforementioned nitriles. Our results reveal that water bridges involving the nitrogen and one carboxyl oxygen of I85 and D234 of FP-2, respectively, and the nitrogen of the neutral 3Pyr moiety, which are either less prevalent or nonexistent in the other complexes, explain the experimental activity profiles. The presence of crystallographic waters close to the bridging water positions in the studied proteases strongly supports the occurrence of such interactions. Overall, our findings suggest that selective FP-2 inhibitors can be designed by promoting water bridge formation at the bottom of the S2 subsite and/or by introducing complementary groups that displace the bridging water.
Assuntos
Antimaláricos , Peptídeo Hidrolases , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Plasmodium falciparum , ÁguaRESUMO
Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Defensinas/farmacologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Pisum sativum/química , Proteínas de Plantas/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Biópsia , Linhagem Celular , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Defensinas/química , Modelos Animais de Doenças , Feminino , Imunofluorescência , Glucosilceramidas/metabolismo , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental , Camundongos , Modelos Moleculares , Proteínas de Plantas/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.
Assuntos
Inibidores de Cisteína Proteinase/química , Modelos Moleculares , Proteínas de Protozoários/antagonistas & inibidores , Quinolinas/química , Cisteína Endopeptidases , Desenho de Fármacos , Ligantes , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Falcipain-2 (FP-2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide-based inhibitors of FP-2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP-2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near-native ligand conformations when applied to a validation set of protein-ligand structures. Overall, we proposed substrate-like binding modes of the studied compounds fulfilling the structural requirements for FP-2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666-1683. © 2017 Wiley Periodicals, Inc.
Assuntos
Antimaláricos/química , Cisteína Endopeptidases/química , Malária Falciparum/tratamento farmacológico , Peptídeos/química , Animais , Antimaláricos/uso terapêutico , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Humanos , Malária Falciparum/parasitologia , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES: The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS: We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS: Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS: Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/química , Naftoquinonas/química , Naftoquinonas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Análise de Sequência de ProteínaRESUMO
Over 110 years after the first formal description of Chagas disease, the trypanocidal drugs thus far available have limited efficacy and several side effects. This encourages the search for novel treatments that inhibit T. cruzi targets. One of the most studied anti-T. cruzi targets is the cysteine protease cruzain; it is associated with metacyclogenesis, replication, and invasion of the host cells. We used computational techniques to identify novel molecular scaffolds that act as cruzain inhibitors. First, with a docking-based virtual screening, we identified compound 8, a competitive cruzain inhibitor with a Ki of 4.6 µM. Then, aided by molecular dynamics simulations, cheminformatics, and docking, we identified the analog compound 22 with a Ki of 27 µM. Surprisingly, despite sharing the same isoquinoline scaffold, compound 8 presented higher trypanocidal activity against the epimastigote forms, while compound 22, against the trypomastigotes and amastigotes. Taken together, compounds 8 and 22 represent a promising scaffold for further development of trypanocidal compounds as drug candidates for treating Chagas disease.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Cisteína Endopeptidases/farmacologia , Doença de Chagas/tratamento farmacológico , Proteínas de ProtozoáriosRESUMO
Endostatin is a potent antiangiogenic protein derived from the noncollagenous domain 1 (NC1) of collagen XVIII. The mechanism by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid N-terminal fragment of murine endostatin has antitumor, antimigration, and antipermeability activities comparable to the full soluble protein. To understand how this peptide can exert such elaborate function, we performed structural analysis using molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here, we show that the N-terminal peptide of murine endostatin is able to assume a well-defined structure, folding into a zinc-dependent ß-hairpin conformation. Analyzing the folding mechanism, we were able to understand why the N-terminal peptide of human endostatin with the same length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and of a shorter form of the human peptide with 25 residues. Finally, we show that the ß-hairpin conformation assumed by the zinc-bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis inhibitors: the integrin-binding portion of the NC1 domain of collagen IV. Indeed, our docking simulations show that arresten, canstatin, and the endostatin peptide bind to the same spot of αVß3 integrin, suggesting similar interactions via a common binding site on this receptor.
Assuntos
Endostatinas/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Sítios de Ligação , Colágeno Tipo IV , Colágeno Tipo XVIII/química , Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
Cellulases from thermophiles are capable of cleaving sugar chains from cellulose efficiently at high temperatures. The thermo-resistant Cel9A-68 cellulase possesses two important domains: CBM and a catalytic domain connected by a Pro/Ser/Thr rich linker. These domains act cooperatively to allow efficient catalysis. Despite exhaustive efforts to characterize cellulase binding and mechanism of action, a detailed description of the cellulose intrinsic flexibility is still lacking. From computational simulations we studied the temperature influence on the enzyme plasticity, prior to substrate binding. Interestingly, we observed an enhancement of collective motions at high temperatures. These motions are the most representative and describe an intrinsic hinge bending transition. A detailed analysis of these motions revealed an interdomain approximation where D459 and G460, located at the linker region, are the hinge residues. Therefore, we propose a new putative site for mutagenesis targeting the modulation of such conformational transition that may be crucial for activity.
Assuntos
Carboidratos/química , Celulase/química , Domínio Catalítico , Celulase/metabolismo , Temperatura Alta , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , TermodinâmicaRESUMO
During their life cycle, Leishmania parasites display a fine-tuned regulation of the mRNA translation through the differential expression of isoforms of eukaryotic translation initiation factor 4E (LeishIF4Es). The interaction between allosteric modulators such as 4E-interacting proteins (4E-IPs) and LeishIF4E affects the affinity of this initiation factor for the mRNA cap. Here, several computational approaches were employed to elucidate the molecular bases of the previously-reported allosteric modulation in L. major exerted by 4E-IP1 (Lm4E-IP1) on eukaryotic translation initiation factor 4E 1 (LmIF4E-1). Molecular dynamics (MD) simulations and accurate binding free energy calculations (ΔGbind ) were combined with network-based modeling of residue-residue correlations. We also describe the differences in internal motions of LmIF4E-1 apo form, cap-bound, and Lm4E-IP1-bound systems. Through community network calculations, the differences in the allosteric pathways of allosterically-inhibited and active forms of LmIF4E-1 were revealed. The ΔGbind values show significant differences between the active and inhibited systems, which are in agreement with the available experimental data. Our study thoroughly describes the dynamical perturbations of LmIF4E-1 cap-binding site triggered by Lm4E-IP1. These findings are not only essential for the understanding of a critical process of trypanosomatids' gene expression but also for gaining insight into the allostery of eukaryotic IF4Es, which could be useful for structure-based design of drugs against this protein family.
RESUMO
Protein flexibility is essential for enzymatic function, ligand binding, and protein-protein or protein-nucleic acid interactions. Normal mode analysis has increasingly been shown to be well suited for studying such flexibility, as it can be used to identify favorable structural deformations that correspond to functional motions. However, normal modes are strictly relevant to a single structure, reflecting a particular minimum on a complex energy surface, and are thus susceptible to artifacts. We describe a new theoretical framework for determining "consensus" normal modes from a set of related structures, such as those issuing from a short molecular dynamics simulation. This approach is more robust than standard normal mode analysis, and provides higher collectivity and symmetry properties. In an application to HIV-1 protease, the low-frequency consensus modes describe biologically relevant motions including flap opening and closing that can be used in interpreting structural changes accompanying the binding of widely differing inhibitors.
Assuntos
Protease de HIV/química , HIV-1 , Simulação de Dinâmica Molecular , Humanos , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
Lunasin is a 43-amino acid peptide from seeds and grains with bioavailability in humans and potent chemotherapeutic action against several cancer cell lines. Here, we investigate new information about the physicochemical and structural properties of lunasin using circular dichroism (CD), fluorescence spectroscopy, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), size exclusion chromatography (SEC), molecular dynamics (MD), and bioinformatics. CD analysis and disorder prediction obtained by PONDR indicate that lunasin has a mostly unordered structure. Double wavelength [θ]222nm x [θ]200nm plot data suggests that lunasin is an intrinsically disordered peptide (IDP) in a pre-molten globule-like (PMG-like) state, while CD spectrum deconvolution and MD simulation indicate small ß-strand content. The presence of residual structure was supported by loss of CD signal at 222 nm after treatment with urea and by increasing fluorescence emission upon bis-ANS binding. Lunasin also demonstrated stability to heating up to the temperature of 100 °C, as verified by CD. MD and CD analyses in the presence of TFE and MoRFpred prediction indicated the helix propensity of lunasin. ESI-IMS-MS data revealed that lunasin shows a propensity to form disulfide bonds at the conditions used. MD data also indicated that disulfide bond formation affects the adopted structure, showing a possible role of aspartyl-end in structure stabilization and compaction. In conclusion, our data support a characterization of lunasin as a peptide with an intrinsic disorder in a PMG-like state and reveal new aspects about its structural stability and plasticity, as well as the effects of disulfide bond formation and electrostatic attractions.
Assuntos
Antineoplásicos Fitogênicos/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas de Soja/química , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/isolamento & purificação , Dissulfetos , Humanos , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Simulação de Dinâmica Molecular , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas de Soja/isolamento & purificação , Glycine max/química , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Ureia/químicaRESUMO
Trypanosoma cruzi is the causative agent of Chagas disease, a neglected infection affecting millions of people in tropical regions. There are several chemotherapeutic agents for the treatment of this disease, but most of them are highly toxic and generate resistance. Currently, the development of allosteric inhibitors constitutes a promising research field, since it can improve the accessibility to more selective and less toxic medicines. To date, the allosteric drugs prediction is a state-of-the-art topic in rational structure-based computational design. In this work, a simulation strategy was developed for computational discovery of allosteric inhibitors, and it was applied to cruzain, a promising target and the major cysteine protease of T. cruzi. Molecular dynamics simulations, binding free energy calculations and network-based modelling of residue interactions were combined to characterize and compare molecular distinctive features of the apo form and the cruzain-allosteric inhibitor complexes. By using geometry-based criteria on trajectory snapshots, we predicted two main allosteric sites suitable for drug targeting. The results suggest dissimilar mechanisms exerted by the same allosteric site when binding different potential allosteric inhibitors. Finally, we identified the residues involved in suboptimal paths linking the identified site and the orthosteric site. The present study constitutes the first approximation to the design of cruzain allosteric inhibitors and may serve for future pharmacological intervention. Here, no major effects on active site structure were observed due to compound binding (modification of distance and angles between catalytic residues), which indicates that allosteric regulation in cruzain might be mediated via alterations of its dynamical properties similarly to allosteric inhibition of human cathepsin K (HCatK). The current findings are particularly relevant for the design of allosteric modulators of papain-like cysteine proteases.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Catepsina K/química , Catepsina K/efeitos dos fármacos , Desenho Assistido por Computador , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Falcipain-2 (FP-2) is a Plasmodium falciparum cysteine protease that has been extensively targeted to identify novel antimalarials. Remarkably, previous reports have shown that FP-2 can be allosterically modulated and, for a particular noncompetitive chalcone inhibitor, the existing lines of experimental evidence can guide the prediction of its unknown binding mode to the enzyme in a reliable fashion. In this work, we propose a structure of FP-2 in complex with the aforementioned compound that fulfills all of the experimental data, by employing a combination of molecular modeling tools, such as pocket volume measurements, docking, molecular dynamics (MD) simulations, and free energy calculations. Our results show that the studied inhibitor binds a transient pocket occluded in all of the available FP-2 crystal structures and lying in a region previously characterized as a potential allosteric site in related cysteine proteases. In addition, we detected in silico the occurrence of significant community reorganization in FP-2, increased signal transmission between the allosteric pocket and the active site, and change in loop motions and residue pKa values upon the compound binding, thus providing insight into the uncharacterized allosteric mechanism. Overall, this study yields valuable predictions for the design of novel allosteric inhibitors against FP-2 and other cysteine proteases.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/enzimologia , Trypanosoma cruzi/enzimologia , Sítios de Ligação/efeitos dos fármacos , Cisteína Endopeptidases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , TermodinâmicaRESUMO
BACKGROUND: Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica. METHODOLOGY/PRINCIPAL FINDINGS: Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity. CONCLUSIONS/SIGNIFICANCE: The present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.
Assuntos
Catepsina L/antagonistas & inibidores , Desenho Assistido por Computador , Descoberta de Drogas/métodos , Fasciola hepatica/efeitos dos fármacos , Criação de Animais Domésticos , Animais , Sítios de Ligação/genética , Catepsina L/metabolismo , Fasciola hepatica/enzimologia , Fasciolíase/diagnóstico , Fasciolíase/tratamento farmacológico , Fasciolíase/parasitologia , Humanos , Estágios do Ciclo de Vida , Simulação de Dinâmica Molecular , Testes de Sensibilidade Parasitária , Zoonoses/tratamento farmacológico , Zoonoses/parasitologiaRESUMO
The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.
Assuntos
Mutação , Proteínas Proto-Oncogênicas c-kit/química , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Regulação Alostérica , Sequência de Aminoácidos , Antineoplásicos/química , Benzamidas/química , Células Eucarióticas/metabolismo , Células Eucarióticas/patologia , Humanos , Mesilato de Imatinib , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Piperazinas/química , Análise de Componente Principal , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/química , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Assuntos
Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ativadores de Plasminogênio/química , Plasminogênio/química , Yersinia pestis/enzimologia , Sequência de Aminoácidos , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
The drugs against tropical neglected diseases, especially Chagas' Disease, were launched more than 30 years ago, and the development of resistance requires the discovery of new and more effective chemotherapeutic agents. Trypanosoma cruzi has a redox enzyme called trypanothione reductase which was successfully inhibited for peptide derivatives (McKie et al., Amino Acids, 2001, 20: 145). This work aims at studying the mechanism of inhibition of this enzyme through molecular dynamics simulations and evaluating the behavior of some derivatives when inhibiting this protein. We should affirm that any particular molecular dynamics analysis tools (Hbond pattern, 3-D root-mean-square deviation, solvent accessible surface area, etc.) cannot be used apart from the others to justify completely these peptides inhibitory patterns. Based on our results, we reproduced the experimental data and, moreover, we discriminated against a new site in enzyme aperture, which can assist the development of powerful inhibitors against trypanothione reductase enzyme.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Domínio Catalítico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , NADH NADPH Oxirredutases/química , Trypanosoma cruzi/químicaRESUMO
BACKGROUND Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.