RESUMEN

Lymphatic filariasis (LF) remains a significant health challenge for populations in developing countries. LF is a parasitic disease transmitted by mosquitoes, mainly caused by the filarial nematode, Wuchereria bancrofti, prevalent in tropical and subtropical regions. Since the present drugs develop complications, including adverse side effects, lack of specificity, and development of drug resistance, the present study focused on developing the potential anti-filariasis drugs targeting crucial proteins for the nematode life cycle. We have identified the therapeutic compounds by targeting the enzyme thioredoxin peroxidase 1 (WbTPx1), which facilitates the conversion of hydrogen peroxide into water, an essential mechanism by which the nematode survives against oxidative stress in the host. This approach might resolve treatment efficacy and activity difficulties at various stages of filarial parasitic worms. We modeled the structure of WbTPx1 and employed the structure-based virtual screening approach, focusing on the dimer interface region of the protein. ADMET prediction profiles of the non-toxic, top-ranked hits with higher docking scores demonstrate higher affinity to the nematode protein than its human homolog. The molecular dynamic simulation studies show WbTPx1-hit complexes' stability and the intactness of hits in the binding site. Further, in vitro validation of identified hits using Setaria digitata, a cattle nematode, showed better IC50 and higher inhibition than the standard drug ivermectin, indicating the potential to inhibit enzyme activity and the development of drug candidates for controlling LF.

RESUMEN

Lymphatic filariasis (LF) is one of the major health problems for the human kind in developing countries including India. LF is caused by three major nematodes namely Wuchereria bancrofti, Brugia malayi, and Brugia timori. The recent statistics of World Health Organization (WHO) showed that 51 million people were affected and 863 million people from 47 countries around worldwide remain threatened by LF. Among them, 90% of the filarial infection was caused by the nematode W. bancrofti. Approved drugs were available for the treatment of LF but many of them developed drug resistance and no longer effective in all stages of the infection. In the current research work, we explored the Glutathione S-transferase (GST) of W. bancrofti, the key enzyme responsible for detoxification that catalyzes the conjugation of reduced GSH (glutathione) to xenobiotic compounds. Initially, we analyzed the stability of the WbGST through 200 ns MD simulation and further structure-based virtual screening approach was applied by targeting the substrate binding site to identify the potential leads from small molecule collection. The in silico ADMET profiles for the top-ranked hits were predicted and the predicted non-toxic lead molecules showed the highest docking score in the range of - 12.72 kcal/mol to - 11.97 kcal/mol. The cross docking of the identified hits with human GST revealed the potential binding specificity of the hits toward WbGST. Through WbGST-lead complex simulation, the lead molecules were observed to be stable and also intactly bound within the binding site of WbGST. Based on the computational results, the five predicted non-toxic molecules were selected for the in vitro assay. The molecules showed significant percentage of inhibition against the filarial worm Setaria digitata which is the commonly used model organism to evaluate the filarial activity. In addition, the molecules also showed better IC50 than the standard drug ivermectin. The identified lead molecules will lay a significant insight for the development of new drugs with higher specificity and lesser toxicity to control and treat filarial infections.

RESUMEN

Lymphatic filariasis (LF), a mosquito-borne parasitic disease caused by nematode Wuchereria bancrofti in tropical and sub-tropical countries. These nematodes are transferred into the human host when the infected mosquito carrying L3 larvae is released into the bloodstream during the blood ingestion process. The host immune system produces ROS (Reactive Oxygen Species) as a primary defence mechanism to remove the invading filarial worms. However, well-defined antioxidant enzymes of the nematodes scavenge the host-produced ROS to escape from oxidative stress. The enzyme peroxiredoxin 6 (Prx6) belongs to the peroxiredoxin family, catalyses hydrogen peroxide (H2O2) into water (H2O). In order to find the inhibitors that inhibit the activity of peroxiredoxin 6 of W. bancrofti. We performed the homology modelling to predict the WbPrx6 three-dimensional structure using the Schrödinger-Prime and the dynamic stability of the modelled WbPrx6 was analyzed by carrying out the molecular dynamic (MD) simulation for the time scale of 200ns. Further, the structure-based virtual screening shortlisted the hit molecules from the ChemBridge database based on the glide score. The potential lead molecules (ID: 10239274, 11112883, 79879205, 58160895, and 42133744) that have better binding and satisfied the ADMET properties were selected for further complex simulation and DFT calculations. The identified compounds interact with the N-terminal region of the thioredoxin domain, which plays a key role in reducing phospholipase A2 activity. Interestingly, upon binding the lead molecule, the fluctuation of the loop region that connects α-IV with the ß-VI plays a vital role in affecting the geometry of the active site, which in turn affects the activity WbPrx6. The outcomes of the present computational studies could help in future drug development and designing of the effective candidate to control Lymphatic filariasis.

Asunto(s)

RESUMEN

COVID-19 pandemic causative SARS-CoV-2 coronavirus is still rapid in progression and transmission even after a year. Understanding the viral transmission and impeding the replication process within human cells are considered as the vital point to control and overcome COVID-19 infection. Non-structural Protein 1, one among the proteins initially produced upon viral entry into human cells, instantly binds with the human ribosome and inhibit the host translation process by preventing the mRNA attachment. However, the formation of NSP1 bound Ribosome complex does not affect the viral replication process. NSP1 plays an indispensable role in modulating the host gene expression and completely steals the host cellular machinery. The full-length structure of NSP1 is essential for the activity in the host cell and importantly the loop connecting N and C-terminal domains are reported to play a role in ribosome binding. Due to the unavailability of the experimentally determined full-length structure of NSP1, we have modelled the complete structure using comparative modelling and the stability and conformational behaviour of the modelled structure was evaluated through molecular dynamics simulation. Interestingly, the present study reveals the significance of the inter motif loop to serves as a potential binding site for drug discovery experiments. Further, we have screened the phytochemicals from medicinal plant sources since they were used for several hundred years that minimizes the traditional drug development time. Among the 5638 phytochemicals screened against the functionally associated binding site of NSP1, the best five phytochemicals shown high docking score of -9.63 to -8.75 kcal/mol were further evaluated through molecular dynamics simulations to understand the binding affinity and stability of the complex. Prime MM-GBSA analysis gave the relative binding free energies for the top five compounds (dihydromyricetin, 10-demethylcephaeline, dihydroquercetin, pseudolycorine and tricetin) in the range of -45.17 kcal/mol to -37.23 kcal/mol, indicating its binding efficacy in the predicted binding site of NSP1. The density functional theory calculations were performed for the selected five phytochemicals to determine the complex stability and chemical reactivity. Thus, the identified phytochemicals could further be used as effective anti-viral agents to overcome COVID-19 and as well as several other viral infections.

Asunto(s)

RESUMEN

Galectin-3 is an important protein in molecular signalling events involving carbohydrate recognition, and an understanding of the hydrogen-bonding patterns in the carbohydrate-binding site of its C-terminal domain (galectin-3C) is important for the development of new potent inhibitors. The authors are studying these patterns using neutron crystallography. Here, the production of perdeuterated human galectin-3C and successive improvement in crystal size by the development of a crystal-growth protocol involving feeding of the crystallization drops are described. The larger crystals resulted in improved data quality and reduced data-collection times. Furthermore, protocols for complete removal of the lactose that is necessary for the production of large crystals of apo galectin-3C suitable for neutron diffraction are described. Five data sets have been collected at three different neutron sources from galectin-3C crystals of various volumes. It was possible to merge two of these to generate an almost complete neutron data set for the galectin-3C-lactose complex. These data sets provide insights into the crystal volumes and data-collection times necessary for the same system at sources with different technologies and data-collection strategies, and these insights are applicable to other systems.

Asunto(s)

RESUMEN

Hemoglobin is an honorary enzyme, a two-way respiratory carrier, transporting oxygen from the lungs to the tissues and facilitating the return transport of carbon dioxide. Hemoglobin has high affinity for oxygen and low affinity for carbon dioxide and other substances in the arterial circulation, whereas in the venous circulation these relative affinities are upturned. The oxygen affinity of hemoglobin increases with the fall in temperature and decreases with the increase in pH and 2, 3-bisphosphoglycerate; point mutations also affect the tetrameric arrangement and alter the oxygen affinity. Though several studies have revealed the specific reasons for the adaptation of increased oxygen affinity of avian hemoglobins at high-altitudes, further structural insights on hemoglobins from high oxygen affinity species are required to understand the detailed oxygen adaptation at the molecular level. Herein, we describe the structural investigation of hemoglobin from emu (Dromaius novaehollandiae), a high oxygen affinity bird. Hemoglobin from emu was purified using anion-exchange chromatography, crystallized and determined the structure in the oxy form at a resolution of 2.3 Å; the R-factor of the model was 19.2%. The structure was compared with other oxy hemoglobins of high oxygen affinity avian species; significant changes are noted at intra-subunit contacts which provide the clues for increased oxygen affinity of emu hemoglobin.

Asunto(s)

RESUMEN

The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the ß-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

Asunto(s)

RESUMEN

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins.

Asunto(s)

RESUMEN

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of ß-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

Asunto(s)

RESUMEN

How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn(2+) first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn(2+) ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of ∼10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.

Asunto(s)

RESUMEN

The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn(2+) loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a nonnative Zn(2+) ion with microM affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn(2+) ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features.

Asunto(s)