Mutations on RBD of SARS-CoV-2 Omicron variant result in stronger binding to human ACE2 receptor.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to more than 270 million infections and 5.3 million of deaths worldwide. Several major variants of SARS-CoV-2 have emerged and posed challenges in controlling the pandemic. The recently occurred Omicron variant raised serious concerns about reducing the efficacy of vaccines and neutralization antibodies due to its vast mutations. We have modelled the complex structure of the human ACE2 protein and the receptor binding domain (RBD) of Omicron Spike protein (S-protein), and conducted atomistic molecular dynamics simulations to study the binding interactions. The analysis shows that the Omicron RBD binds more strongly to the human ACE2 protein than the original strain. The mutations at the ACE2-RBD interface enhance the tight binding by increasing hydrogen bonding interaction and enlarging buried solvent accessible surface area.

New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding.

Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure-activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33% success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in the present work.

A community-randomized evaluation of the effect of intermittent preventive treatment in infants on antimalarial drug resistance in southern Tanzania.

BACKGROUND: Intermittent preventive treatment in infants (IPTi) is the administration of sulfadoxine-pyrimethamine (SP) at 2, 3, and 9 months of age to prevent malaria. We investigated the influence of IPTi on drug resistance. METHODS: Twenty-four areas were randomly assigned to receive or not receive IPTi. Blood collected during representative household surveys at baseline and 15 and 27 months after implementation was tested for SP and resistance markers. RESULTS: The frequency of SP in blood was similar in the IPTi and comparison areas at baseline and at 15 months. dhfr and dhps mutations were also similar at baseline and then increased similarly in both arms after 15 months of SP-IPTi. First-line treatment was switched from SP to artemether-lumefantrine before the final survey, when SP positivity fell among infants in comparison areas but increased in IPTi areas. This was accompanied by an increase in dhfr but not dhps mutations in IPTi areas (P = .004 and P = .18, respectively). CONCLUSIONS: IPTi did not increase drug pressure or the selection on dhfr and dhps mutants, when SP was the first-line malaria treatment. Introduction of artemether-lumefantrine was followed by an increase in dhfr mutations, consistent with weak selection attributable to SP-IPTi, but not by an increase in dhps mutations, suggesting a fitness cost of this mutation.

Computational insights into differential interaction of mammalian angiotensin-converting enzyme 2 with the SARS-CoV-2 spike receptor binding domain.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to the receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals, predicted the structures of ACE2-RBD complexes by homology modeling, and refined the complexes using molecular dynamics simulation. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The analysis outcomes suggest that the ACE2 of bovine, cat, and panda form strong binding interactions with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse, and civet, the ACE2 proteins interact weakly with RBD.

Direct experimental observation of blue-light-induced conformational change and intermolecular interactions of cryptochrome.

Cryptochromes are blue light receptors that mediate circadian rhythm and magnetic sensing in various organisms. A typical cryptochrome consists of a conserved photolyase homology region domain and a varying carboxyl-terminal extension across species. The structure of the flexible carboxyl-terminal extension and how carboxyl-terminal extension participates in cryptochrome's signaling function remain mostly unknown. In this study, we uncover the potential missing link between carboxyl-terminal extension conformational changes and downstream signaling functions. Specifically, we discover that the blue-light induced opening of carboxyl-terminal extension in C. reinhardtii animal-like cryptochrome can structurally facilitate its interaction with Rhythm Of Chloroplast 15, a circadian-clock-related protein. Our finding is made possible by two technical advances. Using single-molecule Förster resonance energy transfer technique, we directly observe the displacement of carboxyl-terminal extension by about 15 Å upon blue light excitation. Combining structure prediction and solution X-ray scattering methods, we propose plausible structures of full-length cryptochrome under dark and lit conditions. The structures provide molecular basis for light active conformational changes of cryptochrome and downstream regulatory functions.

Structure-Based Functional Modification Study of a Cyanobacterial Chloride Pump for Transporting Multiple Anions.

Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (SyHR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (MrHR), can take up divalent ions, such as SO42-, as well as chloride ions. Here, the crystal structure of MrHR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9 Å. The detailed structure of MrHR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B-C loop. Structural and functional analyses of MrHR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how SyHR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing SyHR, the only molecule known to transport both sulfate and chloride ions.

Computer aided protein engineering to enhance the thermo-stability of CXCR1- T4 lysozyme complex.

CXCR1, a member in G-protein coupled receptor (GPCR) family, binds to chemokine interleukin-8 (IL-8) specifically and transduces signals to mediate immune and inflammatory responses. Despite the importance of CXCR1, high-resolution structure determination is hindered by the challenges in crystallization. It has been shown that properly designed mutants with enhanced thermostability, together with fusion partner proteins, can be useful to form crystals for GPCR proteins. In this study, in silico protein design was carried out by using homology modeling and molecular dynamics simulations. To validate the computational modeling results, the thermostability of several mutants and the wild type were measured experimentally. Both computational results and experimental data suggest that the mutant L126W has a significant improvement in the thermostability. This study demonstrated that in silico design can guide protein engineering and potentially facilitate protein crystallography research.

Identification of Drug Binding Sites and Action Mechanisms with Molecular Dynamics Simulations.

Identifying drug binding sites and elucidating drug action mechanisms are important components in a drug discovery process. In this review, we briefly compared three different approaches (sequence- based methods, structure-based methods and probe-based molecular dynamics (MD) methods) to identifying drug binding sites, and concluded that probe-based MD methods are much more advantageous in dealing with flexible target macromolecules and digging out druggable macromolecule conformations for subsequent drug screening. The applications of MD simulation to studying drug-target interactions were demonstrated with different types of target molecules, including lipid membrane, protein and DNA. The results indicate that MD simulations with enhanced sampling methods provide a powerful tool to determine free energy profiles/surfaces and identify important intermediate states, which are essential for the elucidation of drug action mechanisms. The future development of methods in MD simulations will benefit and speed up the drug discovery processes.

Using molecular dynamics for the refinement of atomistic models of GPCRs by homology modeling.

Despite GPCRs sharing a common seven helix bundle, analysis of the diverse crystallographic structures available reveal specific features that might be relevant for ligand design. Despite the number of crystallographic structures of GPCRs steadily increasing, there are still challenges that hamper the availability of new structures. In the absence of a crystallographic structure, homology modeling remains one of the important techniques for constructing 3D models of proteins. In the present study we investigated the use of molecular dynamics simulations for the refinement of GPCRs models constructed by homology modeling. Specifically, we investigated the relevance of template selection, ligand inclusion as well as the length of the simulation on the quality of the GPCRs models constructed. For this purpose we chose the crystallographic structure of the rat muscarinic M3 receptor as reference and constructed diverse atomistic models by homology modeling, using different templates. Specifically, templates used in the present work include the human muscarinic M2; the more distant human histamine H1 and the even more distant bovine rhodopsin as shown in the GPCRs phylogenetic tree. We also investigated the use or not of a ligand in the refinement process. Hence, we conducted the refinement process of the M3 model using the M2 muscarinic as template with tiotropium or NMS docked in the orthosteric site and compared with the results obtained with a model refined without any ligand bound.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa.

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

New insights into the stereochemical requirements of the bradykinin B1 receptor antagonists binding.

Bradykinin (BK) is a nonapeptide involved in several pathophysiological conditions including among others, septic and haemorrhagic shock, anaphylaxis, arthritis, rhinitis, asthma, inflammatory bowel disease. Accordingly, BK antagonists have long been sought after for therapeutic intervention. Action of BK is mediated through two different G-protein coupled receptors known as B1 and B2. Although there are several B1 antagonists reported in literature, their pharmacological profile is not yet optimal so that new molecules need to be discovered. In the present work we have constructed an atomistic model of the B1 receptor and docked diverse available non-peptide antagonists in order to get a deeper insight into the structure-activity relationships involving binding to this receptor. The model was constructed by homology modeling using the chemokine CXC4 and bovine rhodopsin receptors as template. The model was further refined using molecular dynamics for 600ns with the protein embedded in a POPC bilayer. From the refinement process we obtained an average structure that was used for docking studies using the Glide software. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. The results of the docking study underline the role of specific receptor residues in ligand binding. The results of this study permitted to define a pharmacophore that describes the stereochemical requirements of antagonist binding, and can be used for the discovery of new compounds.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

Designing type II topoisomerase inhibitors: A molecular modeling approach.

Nosocomial infections are produced by pathogens with the ability to persist in hospital environments and with the propensity to develop resistance to diverse antimicrobials. In order to tackle resistance, it has been pointed as good strategy to select resilient drug targets that are evolutionally constrained to design drugs less susceptible to develop resistance. Molecular modeling can help to fulfill this goal by providing a rationalization of the observed resistance at the molecular level and, suggesting modifications on existing drugs or in the design of new ones to overcome the problem. The present report focus on type II topoisomerases, a clinical validated target for antibacterials and describe diverse modes of intervention including, inhibition of their ATPase function, stabilization of the cleavage complex or prevention of DNA strand hydrolysis. Moreover, the origin of resistance is also rationalized on the base of ligand-target interactions. Finally, efforts are described to circumvent the effect of non-susceptible strains by the design of new drugs based on existing ones, like the case of diones that act through the same mechanism as quinolones or the newly released quinole-carbonitrile derivatives that inhibit type II topoisomerases through a new mechanism.

Molecular determinants of the bacterial resistance to fluoroquinolones: a computational study.

Quinolones constitute a large class of antibacterial agents whose action is mediated through the formation of a ternary complex with DNA and either, DNA Gyrase or topoisomerase IV, resulting in the inhibition of DNA replication. In order to get a deeper insight into the features of the complex formation, we carried out docking studies of fifteen diverse quinolones to the cleaved topoisomerase IV-DNA complex. Docking studies were performed using the crystal structures of the cleaved complex with levofloxacin and moxifloxacin (pdb entries 3K9F and 2XKK, respectively) using the GOLD software. Ligands dock in positions similar to those of the crystal structures. Analysis of the results reveals that bound quinolones appear intercalated between the two nucleotides that are involved in the DNA cleavage and exhibit hydrogen bonds with Arg(117) and, the latter mediated though a water molecule. Arg(117) has not been described to be involved in resistance, since it is putatively involved in the enzymatic reaction and its mutation would be lethal for the organism. Mutants of Ser(79) exhibit resistance to quinolones which can be explained by the loss of an important anchoring point. Interestingly, quinolone resistance observed in Asp(83) mutants cannot be explained directly on the basis of the loss of a direct interaction, but could be explained on the basis of its involvement at the entrance of the ligands to their binding pocket since the residue is located at the mouth of the pocket. The results of the present study suggest that the 4-keto and 3-carboxyl groups of the fluoroquinolones bind a Mg(2+) before binding to the cleaved topoisomarase IV-DNA complex and use Asp(83) for entry into the binding pocket. Accordingly, mutations that do not conserve the binding capacity for the quinolone-Mg(2+) complex will prevent the binding of this class of ligands. The results we present here are also compared with the structure of PD0305970 a 2,4-dione active against the Ser(79) and Asp(83) mutants.