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1.
Sci Rep ; 13(1): 14382, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37658081

RESUMEN

Novel male contraceptives will promote gender equality in sharing contraceptive responsibility. The sperm-associated protein epididymal protease inhibitor (EPPIN) is a promising target for non-hormonal male contraception. EPPIN interacts with the semen coagulum protein semenogelin-1 (SEMG1) on the sperm surface, leading to transient inhibition of sperm motility after ejaculation. Small organic molecules targeting EPPIN's SEMG1-binding are under development as male contraceptives. Here, we combined computational approaches to uncover key aspects underlying EPPIN binding to SEMG1 and small organic ligands. We generated a human EPPIN model showing a typical arrangement of the WFDC (Whey-acid four disulfide core)-type and Kunitz-type domains, connected by a hinge region. Determining the EPPIN model's intrinsic motion by molecular dynamics simulations and normal mode analysis revealed a conformation, presenting a binding pocket that accommodates SEMG1Glu229-Gln247, EP055, and EP012. EPPIN's residues Phe63 and Lys68 (WFDC domain), Asp71 (hinge region), and Asn113, Asn114, and Asn115 (Kunitz domain) were identified as hot spots for SEMG1, EP055, and EP012 binding. Moreover, hydrophobic and hydrophilic residues in the WFDC and Kunitz domains allow plasma membrane anchoring, orienting the EPPIN binding pocket to the solvent. Targeting EPPIN's essential residues for its biomolecular interactions may improve the rational design of EPPIN ligands as spermiostatic compounds.


Asunto(s)
Anticonceptivos Masculinos , Humanos , Masculino , Anticonceptivos Masculinos/farmacología , Ligandos , Semen , Motilidad Espermática , Anticonceptivos
2.
Cell Rep ; 42(4): 112320, 2023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-37027306

RESUMEN

The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.


Asunto(s)
Receptores de Ghrelina , Sodio , Regulación Alostérica , Sitio Alostérico , Ghrelina/metabolismo , Iones , Receptores de Ghrelina/metabolismo , Transducción de Señal , Sodio/metabolismo
3.
J Chem Theory Comput ; 2023 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-36622950

RESUMEN

Molecular dynamics with excited normal modes (MDeNM) is an enhanced sampling method for exploring conformational changes in proteins with minimal biases. The excitation corresponds to injecting kinetic energy along normal modes describing intrinsic collective motions. Herein, we developed a new automated open-source implementation, MDexciteR (https://github.com/mcosta27/MDexciteR), enabling the integration of MDeNM with two commonly used simulation programs with GPU support. Second, we generalized the method to include the excitation of principal components calculated from experimental ensembles. Finally, we evaluated whether the use of coarse-grained normal modes calculated with elastic network representations preserved the performance and accuracy of the method. The advantages and limitations of these new approaches are discussed based on results obtained for three different protein test cases: two globular and a protein/membrane system.

4.
Front Microbiol ; 13: 1078972, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36620034

RESUMEN

Cyclins are a family of proteins characterized by possessing a cyclin box domain that mediates binding to cyclin dependent kinases (CDKs) partners. In this study, the search for a partner cyclin of the PHO85-1 CDK retrieved PCL-1 an ortholog of yeast Pcls (for Pho85 cyclins) that performs functions common to Pcls belonging to different cyclin families. We show here that PCL-1, as a typical cyclin, is involved in cell cycle control and cell progression. In addition, PCL-1 regulates glycogen metabolism; Δpcl-1 cells accumulate higher glycogen levels than wild-type cells and the glycogen synthase (GSN) enzyme is less phosphorylated and, therefore, more active in the mutant cells. Together with PHO85-1, PCL-1 phosphorylates in vitro GSN at the Ser636 amino acid residue. Modeling studies identified PHO85-1 and PCL-1 as a CDK/cyclin complex, with a conserved intermolecular region stabilized by hydrophobic and polar interactions. PCL-1 is also involved in calcium and NaCl stress response. Δpcl-1 cells are sensitive to high NaCl concentration; on the contrary, they grow better and overexpress calcium responsive genes under high calcium chloride concentration compared to the wild-type strain. The expression of the calcium-responsive CRZ-1 transcription factor is modulated by PCL-1, and this transcription factor seems to be less phosphorylated in Δpcl-1 cells since exhibits nuclear location in these cells in the absence of calcium. Our results show that PCL-1 locates at different cell regions suggesting that it may determine its activity by controlling its intracellular location and reveal an interesting functional divergence between yeast and filamentous fungus cyclins.

5.
Trends Parasitol ; 38(1): 80-94, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34364805

RESUMEN

Parasitic diseases affect millions of individuals worldwide, mainly in low-income regions. There is no cure for most of these diseases, and the treatment relies on drugs that have side effects and lead to drug resistance, emphasizing the urgency to find new treatments. Snake venom has been gaining prominence as a rich source of molecules with antiparasitic potentials, such as phospholipases A2 (PLA2s). Here, we compile the findings involving PLA2s with antiparasitic activities against helminths, Plasmodium, Toxoplasma, and trypanosomatids. We indicate their molecular features, highlighting the possible antiparasitic mechanisms of action of these proteins. We also demonstrate interactions between PLA2s and some parasite membrane components, shedding light on potential targets for drug design that may provide better treatment for the illnesses caused by parasites.


Asunto(s)
Antiparasitarios , Venenos de Serpiente , Antiparasitarios/farmacología , Humanos , Fosfolipasas A2/farmacología , Poliésteres , Venenos de Serpiente/farmacología
6.
Front Cell Infect Microbiol ; 11: 773410, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34858880

RESUMEN

Trypanosoma cruzi faces a variety of environmental scenarios during its life cycle, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible posttranslational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase TcSir2rp3 was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, known to induce reactive oxidative and nitrosactive species in the parasite, associated to that a reduction in the ROS levels was observed. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of TcSODA in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that these parasites presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at a similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible for regulating MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches, we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect to hydrogen-bonding pattern to neighbor residues, suggesting a key participation of this residue to modulate the affinity to O2- . Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection.


Asunto(s)
Mitocondrias/enzimología , Estrés Oxidativo , Sirtuinas , Trypanosoma cruzi , Oxidación-Reducción , Sirtuinas/genética , Sirtuinas/metabolismo , Superóxido Dismutasa/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética
7.
Elife ; 102021 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-34477105

RESUMEN

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.


Asunto(s)
Ghrelina , Receptores Acoplados a Proteínas G , Receptores de Ghrelina , Humanos , Ligandos , Transducción de Señal
8.
Biochimie ; 191: 11-26, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34375717

RESUMEN

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.


Asunto(s)
ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Complejos Multienzimáticos/metabolismo , Neurospora crassa/enzimología , Multimerización de Proteína , ADN de Hongos/genética , Proteínas Fúngicas/genética , Complejos Multienzimáticos/genética , Neurospora crassa/genética
9.
Int J Biol Macromol ; 185: 494-512, 2021 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-34197854

RESUMEN

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10-7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound.


Asunto(s)
Anacardium/química , Ácido Gálico/farmacología , Miotoxicidad/tratamiento farmacológico , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/metabolismo , Venenos de Serpiente/enzimología , Animales , Modelos Animales de Enfermedad , Ácido Gálico/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Miotoxicidad/enzimología , Miotoxicidad/etiología , Inhibidores de Fosfolipasa A2/química , Fosfolipasas A2/química , Tallos de la Planta/química , Proteínas de Reptiles/química , Proteínas de Reptiles/metabolismo , Resonancia por Plasmón de Superficie
10.
Nat Commun ; 12(1): 3938, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34168117

RESUMEN

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.


Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Regulación Alostérica , Sitios de Unión , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína/genética , Transferencia Resonante de Energía de Fluorescencia , Gangliósido G(M3)/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Mutación , Fosfatidilinositol 4,5-Difosfato/química , Conformación Proteica , Receptores de Ghrelina/genética , Transducción de Señal
11.
Sci Rep ; 10(1): 16252, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004851

RESUMEN

The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT, which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results.


Asunto(s)
Bothrops , Venenos de Crotálidos/química , Regulación Alostérica , Animales , Simulación por Computador , Dispersión Dinámica de Luz , Simulación de Dinámica Molecular , Fosfolipasas A2/química , Multimerización de Proteína
12.
Biochem J ; 477(9): 1733-1744, 2020 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-32329788

RESUMEN

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.


Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Glucólisis/fisiología , Lisina/metabolismo , Trypanosoma brucei brucei/metabolismo , Acetilación , Animales , Microcuerpos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo
13.
ACS Omega ; 5(3): 1363-1370, 2020 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-32010806

RESUMEN

Scheelea phalerata Mart. ex Spreng (Arecaceae) is a palm tree found in the Brazilian cerrado. There are no topics related to volatile oils from S. phalerata leaves in the literature. This work determines its chemical composition and evaluates the biological activity under two different seasonal conditions (dry and rainy seasons). The dry essential oil yield was 0.034 ± 0.001% and the rainy essential oil yield was 0.011 ± 0.003%. Both essential oils presented different qualitative and quantitative compositions (99.4 and 98.5%). The main constituents of the dry essential oil were phytol (36.7%), nonadecane (9.7%), linolenic acid (9.1%), (Z)-hex-3-en-1-ol (4.2%), and squalene (4.0%). The main constituents of the rainy essential oil were phytol (26.1%), palmitic acid (18.7%), hexan-1-ol (15.6%), (Z)-hex-3-en-1-ol (9.7%), and oleic acid (4.0%). The antileishmanial activity against promastigotes of Leishmania amazonensis was observed only for the rainy season essential oil (IC50 value of 165.05 ± 33.26 µg mL-1). A molecular docking study showed that alcohols exert a paramount efficacy and that the action of some essential oil compounds may be similar to that of amphotericin B. Still, only the essential oil from the dry season showed moderate antibacterial activity against S. sanguinis (MICs 200-400 µg mL-1). The cytotoxicity against Vero cells was identical (>512) for both essential oils. The novel data here for both chemical characterization and biological activity, in particular, evidence that the action of these compounds is similar to that of amphotericin B, provide valuable information to the drug-discovery field.

14.
Biochimie ; 170: 163-172, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31978419

RESUMEN

Envenoming by snakebite is an important global health issue that has received little attention, leading the World Health Organization to naming it as neglected tropical disease. Several snakebites present serious local symptoms manifested on victims that may not be efficiently neutralized by serum therapy. Phospholipase A2-like (PLA2-like) toxins are present in Viperidae venoms and are responsible for local myotoxic activity. Herein, we investigated the association between BthTX-I toxin and caftaric acid (CFT), a molecule present in plants. CFT neutralized neuromuscular blocking and muscle-damaging activities promoted by BthTX-I. Calorimetric and light-scattering assays demonstrated that CFT inhibitor interacted with dimeric BthTX-I. Bioinformatics simulations indicated that CFT inhibitor binds to the toxin's hydrophobic channel (HCh). According to the current myotoxic mechanism, three different regions of PLA2-like toxins have specific tasks: protein allosteric activation (HCh), membrane dockage (MDoS), and membrane rupture (MDiS). We propose CFT inhibitor interferes with the allosteric activation, which is related to the conformation change leading to the exposure/alignment of MDoS/MDiS region. This is the first report of a PLA2-like toxin fully inhibited by a compound that interacts only with its HCh region. Thus, CFT is a novel candidate to complement serum therapy and improve the treatment of snakebite.


Asunto(s)
Venenos de Crotálidos/toxicidad , Miotoxicidad/tratamiento farmacológico , Bloqueantes Neuromusculares/toxicidad , Fenoles/farmacología , Fosfolipasas A2/química , Animales , Masculino , Ratones , Miotoxicidad/etiología , Fosfolipasas A2/metabolismo , Conformación Proteica
15.
Sci Rep ; 9(1): 17203, 2019 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-31748642

RESUMEN

The World Health Organization recently listed snakebite envenoming as a Neglected Tropical Disease, proposing strategies to significantly reduce the global burden of this complex pathology by 2030. In this context, effective adjuvant treatments to complement conventional antivenom therapy based on inhibitory molecules for specific venom toxins have gained renewed interest. Varespladib (LY315920) is a synthetic molecule clinically tested to block inflammatory cascades of several diseases associated with elevated levels of secreted phospholipase A2 (sPLA2). Most recently, Varespladib was tested against several whole snake venoms and isolated PLA2 toxins, demonstrating potent inhibitory activity. Herein, we describe the first structural and functional study of the complex between Varespladib and a PLA2-like snake venom toxin (MjTX-II). In vitro and in vivo experiments showed this compound's capacity to inhibit the cytotoxic and myotoxic effects of MjTX-II from the medically important South American snake, Bothrops moojeni. Crystallographic and bioinformatics analyses revealed interactions of Varespladib with two specific regions of the toxin, suggesting inhibition occurs by physical blockage of its allosteric activation, preventing the alignment of its functional sites and, consequently, impairing its ability to disrupt membranes. Furthermore, based on the analysis of several crystallographic structures, a distinction between toxin activators and inhibitors is proposed.


Asunto(s)
Acetatos/farmacología , Indoles/farmacología , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/química , Proteínas de Reptiles/química , Toxinas Biológicas/antagonistas & inhibidores , Animales , Bothrops , Cristalografía por Rayos X , Cetoácidos , Simulación de Dinámica Molecular , Fosfolipasas A2/metabolismo , Conformación Proteica , Proteínas de Reptiles/metabolismo
16.
Sci Rep ; 9(1): 510, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30679550

RESUMEN

Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America, Bothrops snakes are responsible for the majority of the snakebite envenomings that are not efficiently treated by conventional serum therapy. Thus, the search for simple and efficient inhibitors to complement this therapy is a promising research area, and a combination of functional and structural assays have been used to test candidate ligands against specific ophidian venom compounds. Herein, we tested a commercial drug (acetylsalicylic acid, ASA) and a plant compound with antiophidian properties (rosmarinic acid, RA) using myographic, crystallographic and bioinformatics experiments with a phospholipase A2-like toxin, MjTX-II. MjTX-II/RA and MjTX-II/ASA crystal structures were solved at high resolution and revealed the presence of ligands bound to different regions of the toxin. However, in vitro myographic assays showed that only RA is able to prevent the myotoxic effects of MjTX-II. In agreement with functional results, molecular dynamics simulations showed that the RA molecule remains tightly bound to the toxin throughout the calculations, whereas ASA molecules tend to dissociate. This approach aids the design of effective inhibitors of PLA2-like toxins and, eventually, may complement serum therapy.


Asunto(s)
Aspirina , Cinamatos , Venenos de Crotálidos , Depsidos , Fosfolipasas A2 Grupo II , Simulación de Dinámica Molecular , Animales , Aspirina/química , Aspirina/farmacología , Cinamatos/química , Cinamatos/farmacología , Venenos de Crotálidos/química , Venenos de Crotálidos/toxicidad , Cristalografía por Rayos X , Depsidos/química , Depsidos/farmacología , Fosfolipasas A2 Grupo II/química , Fosfolipasas A2 Grupo II/toxicidad , Masculino , Ratones , Estructura Cuaternaria de Proteína , Ácido Rosmarínico
17.
Sci Rep ; 8(1): 10317, 2018 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-29985425

RESUMEN

Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.


Asunto(s)
Fosfolipasas A2/metabolismo , Proteínas de Reptiles/metabolismo , Suramina/química , Animales , Sitios de Unión , Bothrops , Venenos de Crotálidos/metabolismo , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Fosfolipasas A2/química , Estructura Cuaternaria de Proteína , Proteínas de Reptiles/química , Dispersión del Ángulo Pequeño , Suramina/metabolismo , Termodinámica
18.
Chem Biol Interact ; 280: 8-14, 2018 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-29208359

RESUMEN

One of the promising fields for improving the effectiveness of antimicrobial agents is their combination with efflux pump inhibitors (EPIs), which besides expanding the use of existing antibiotics. The goal of this research was to evaluate a series of aminoguanidine hydrazones (AGH's, 1-19) as antibacterial agents and NorA efflux pump inhibitors in Staphylococcus aureus strain SA-1199B. Molecular modeling and docking studies were also performed in order to explain at the molecular level the interactions of the compounds with the generated NorA efflux pump model. The MICs of the antibiotic and ethidium bromide were determined by microdilution assay in absence or presence of a subinhibitory concentration of aminoguanidine hydrazones and macrophages viability was determined through MTT assay. Bioinformatic software Swiss-Model and AutoDock 4.2 were used to perform modeling and docking studies, respectively. As results, all AGH's were able to potentiate the action for the antibiotic norfloxacin, causing MIC's reduction of 16-fold and 32-fold to ethidium bromide. In the cell viability test, the concentration of 10 µg/mL showed better results than 90% and the concentration of 1000 µg/mL showed the lowest viability, reaching a maximum of 50% for the analyzed aminoguanidine hydrazones. Molecular docking studies showed that both norfloxacin and derivative 13 were recognized by the same binding site of NorA pump, suggesting a competitive mechanism. The present work demonstrated for the first time that AGH derivatives have potential to be putative inhibitors of NorA efflux pump, showing a promising activity as an antibacterial drug development.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidrazonas/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Staphylococcus aureus/metabolismo , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Guanidinas/química , Hidrazonas/síntesis química , Hidrazonas/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Norfloxacino/farmacología , Estructura Terciaria de Proteína , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad
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