Binding Of Glycoprotein Research Articles

SP-A binding to the SARS-CoV-2 spike protein using hybrid quantum and classical in silico modeling and molecular pruning by Quantum Approximate Optimization Algorithm (QAOA) Based MaxCut with ZDOCK.

The pulmonary surfactant protein A (SP-A) is a constitutively expressed immune-protective collagenous lectin (collectin) in the lung. It binds to the cell membrane of immune cells and opsonizes infectious agents such as bacteria, fungi, and viruses through glycoprotein binding. SARS-CoV-2 enters airway epithelial cells by ligating the Angiotensin Converting Enzyme 2 (ACE2) receptor on the cell surface using its Spike glycoprotein (S protein). We hypothesized that SP-A binds to the SARS-CoV-2 S protein and this binding interferes with ACE2 ligation. To study this hypothesis, we used a hybrid quantum and classical in silico modeling technique that utilized protein graph pruning. This graph pruning technique determines the best binding sites between amino acid chains by utilizing the Quantum Approximate Optimization Algorithm (QAOA)-based MaxCut (QAOA-MaxCut) program on a Near Intermediate Scale Quantum (NISQ) device. In this, the angles between every neighboring three atoms were Fourier-transformed into microwave frequencies and sent to a quantum chip that identified the chemically irrelevant atoms to eliminate based on their chemical topology. We confirmed that the remaining residues contained all the potential binding sites in the molecules by the Universal Protein Resource (UniProt) database. QAOA-MaxCut was compared with GROMACS with T-REMD using AMBER, OPLS, and CHARMM force fields to determine the differences in preparing a protein structure docking, as well as with Goemans-Williamson, the best classical algorithm for MaxCut. The relative binding affinity of potential interactions between the pruned protein chain residues of SP-A and SARS-CoV-2 S proteins was assessed by the ZDOCK program. Our data indicate that SP-A could ligate the S protein with a similar affinity to the ACE2-Spike binding. Interestingly, however, the results suggest that the most tightly-bound SP-A binding site is localized to the S2 chain, in the fusion region of the SARS-CoV-2 S protein, that is responsible for cell entry Based on these findings we speculate that SP-A may not directly compete with ACE2 for the binding site on the S protein, but interferes with viral entry to the cell by hindering necessary conformational changes or the fusion process.

Natural furin inhibitor(s) as potent therapeutic molecule to mitigate SARS-CoV-2 infection

The COVID 2019 has had been going pandemic as per WHO situation reports. The major differentiating point in this virus is the presence of a unique furin cleavage site. Our insilico study points out to the effectiveness of a potent plant origin furin inhibitor. We exploited the aspect of the cleavage machinery of furin subunits which is critical and indispensible to the entry of SARS-CoV-2 in human cells and subsequent massive contagion. In-silico analysis was done to observe the interactions of proposed analogs of protease inhibitor of plant origin against furin protein as well as the furin spike glycoprotein (SGP) binding machinery. This was done by docking protocols using Hex 6.0 software followed by molecular Dynamic simulation (MDS) analysis in 100 ns scale using Amber94. Further, the images were analysed with PyMol software. The analogs I, II and III included in our study showed strong interactivity against furin individually, as well as the furin-Spike Glycoprotein 1 binding machinery. The findings were confirmed using molecular dynamic simulation analysis which indicated good structural stability and ability to neutralise furin and furin-spike glycoprotein 1. Analog II was found to be the best interactive molecule against furin, showing the least deviation (1.484 ± 0.0064). Also, it was the most effective against furin + Spike glycoprotein I machinery [1.575 ± 0.01]. We report the first of its kind of natural furin inhibitor(s) which would disrupt the furin machinery of SARS-CoV-2 and help in controlling the COVID-19 contagion. Communicated by Ramaswamy H. Sarma

Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells.

The plasma protein histidine-rich glycoprotein (HRG) is implicated in the polarization of macrophages to an M1 antitumoral phenotype. The broadly expressed secreted protein stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. With the aim to biochemically characterize the HRG and STC2 complex, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using the quartz crystal microbalance (QCM) methodology. The functionality of recombinant proteins was tested in a phagocytosis assay, where HRG increased phagocytosis by monocytic U937 cells while STC2 suppressed HRG-induced phagocytosis. The binding of HRG to STC2, measured using QCM, showed an affinity between the proteins in the nanomolar range, and both HRG and STC2 bound individually and in combination to vitamin D3-treated, differentiated U937 monocytes. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of their differentiation stage in part through the interaction with heparan sulfate. These data show that HRG and STC2 bind to each other as well as to U937 monocytes with high affinity, supporting the relevance of these interactions in monocyte/macrophage polarity.

Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins

CD163, a receptor for porcine reproductive and respiratory syndrome virus (PRRSV), possesses nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. To identify CD163 regions involved in PRRSV infection, CD163 mutants were generated. Infection experiments showed resistance to infection following deletion of the SRCR4/5 interdomain or the Exon 13 that encodes a portion of PSTII. The mutation of a pentapeptide domain in SRCR5 and SRCR7 also conferred resistance. Mutant CD163 proteins that resisted infection retained the ability to interact with GP2, GP3, GP4 and GP5 viral glycoproteins. The contribution of multiple domains to infection but not to the binding of viral glycoproteins suggests that the envelope proteins may form multiple interactions with CD163, or that receptor regions important for infection have other cellular binding partners required for PRRSV infection. Finally, we mapped the localization the anti-CD163 2A10 antibody epitope.

Does the imbalance in the apolipoprotein E isoforms underlie the pathophysiological process of sporadic Alzheimer's disease?

Human apolipoprotein E (apoE) is a 299-amino acid secreted glycoprotein binding cholesterol and phospholipids, and with three common isoforms (APOE ε2, APOE ε3, and APOE ε4). The exact mechanism by which APOE gene variants increase/decrease Alzheimer's disease (AD) risk is not fully understood, but APOE isoforms differently affect brain homeostasis and neuroinflammation, blood-brain barrier (BBB) permeability, glial function, synaptogenesis, oral/gut microbiota, neural networks, amyloid beta (Aβ) deposition, and tau-mediated neurodegeneration. In this perspective, we propose a comprehensive interpretation of APOE-mediated effects within AD pathophysiology, describing some specific cellular, biochemical, and epigenetic mechanisms and updating the different APOE-targeting approaches being developed as potential AD therapies. Intracisternal adeno-associated viral-mediated delivery of APOE ε2 is being tested in AD APOE ε4/ε4 carriers, while APOE mimetics are being used in subjects with perioperative neurocognitive disorders. Other approaches including APOE ε4 antisense oligonucleotides, anti-APOE ε4 monoclonal antibodies, APOE ε4 structure correctors, and APOE-Aβ interaction inhibitors produced positive results in transgenic AD mouse models.

Bioactive components of different nasal spray solutions may defeat SARS-Cov2: repurposing and in silico studies.

The recent outbreak "Coronavirus Disease 2019 (COVID-19)" is caused by fast-spreading and highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). This virus enters into the human respiratory system by binding of the viral surface spike glycoprotein (S-protein) to an angiotensin-converting enzyme2 (ACE2) receptor that is found in the nasal passage and oral cavity of a human. Both spike protein and the ACE2 receptor have been identified as promising therapeutic targets to develop anti-SARS-CoV2 drugs. No therapeutic drugs have been developed as of today except for some vaccines. Therefore, potent therapeutic agents are urgently needed to combat the COVID-19 infections. This goal would be achieved only by applying drug repurposing and computational approaches. Thus, based on drug repurposing approach, we have investigated 16 bioactive components (1-16) from different nasal spray solutions to check their efficacies against human ACE2 and SARS-CoV2 spike proteins by performing molecular docking and molecular dynamic (MD) simulation studies. In this study, three bioactive components namely ciclesonide (8), levocabastine (13), and triamcinolone acetonide (16) have been found as promising inhibitory agents against SARS-CoV2 spike and human ACE2 receptor proteins with excellent binding affinities, comparing to reference drugs such as nafamostat, arbidol, losartan, and benazepril. Furthermore, MD simulations were performed (triplicate) for 100ns to confirm the stability of 8, 13, and 16 with said protein targets and to compute MM-PBSA-based binding-free energy calculations. Thus, bioactive components 8, 13, and 16 open the door for researchers and scientist globally to investigate them against SARS-CoV2 through in vitro and in vivo analysis.

HSMotifDiscover: identification of motifs in sequences composed of non-single-letter elements.

The functional sub-string(s) of a biopolymer sequence defines the specificity of its interaction with other biomolecules and is often referred to as motifs. Computational algorithms and software have been broadly developed for finding such motifs in sequences in which the individual elements are single characters, such as those in DNA and protein sequences. However, there are more complex scenarios where the motifs exist in non-single-letter contexts, e.g. preferred patterns of chemical modifications on proteins, DNAs, RNAs or polysaccharides. To search for those motifs, we describe a new method that converts the modified sequence elements to representative single-letter codes and then uses a modified Gibbs-sampling algorithm to define the position specific scoring matrix representing the motif(s). As a proof of principle, we describe the implementation and application of an R package for discovering heparan sulfate (HS) motifs in glycan sequences, which are important in regulating protein-protein interactions. This software can be valuable for analyzing high-throughput glycoprotein binding data using microarrays with HS oligosaccharides or other biological polymers. HSMotifDiscover is freely available as an open source R package released under an MIT license at https://github.com/bioinfoDZ/HSMotifDiscover and also available in the form of an app at https://hsmotifdiscover.shinyapps.io/HSMotifDiscover_ShinyApp/. Supplementary data are available at Bioinformatics online.

A Review of Bioinformatics Studies on the Function of Structural and Nonstructural Proteins and the Level of Glycoprotein Inhibiting Heme Metabolism by SARS-CoV-2 Virus

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection. Its virus called SARS-COV-2 which is an RNA virus with high homology to the bat coronavirus. In this review study, first the molecular and cellular characteristics and the proliferation and replication of SARS-COV-2 are investigated. Then, by reviewing bioinformatics studies regarding protected domain analysis, homology modeling, and molecular docking, the biological role of some specific SARS-COV-2 proteins are examined. The results showed that the open reading frame 8 (ORF8) and surface glycoprotein could bind to porphyrin. At the same time, ORF1ab, ORF10, and ORF3a can attack the heme part of hemoglobin to dissociate iron and form porphyrin. This attack reduces hemoglobin ability to carry oxygen and carbon dioxide. As a result, lung cells become severely inflamed due to their inability to exchange carbon dioxide and oxygen, which leads to large ground-glass opacities on CT scan images. Based on the bioinformatics results, chloroquine can prevent ORF1ab, ORF3a, and ORF10 from attacking hemoglobin to form porphyrin and avoid the binding of ORF8 and surface glycoprotein to porphyrin, which effectively relieves the symptoms of acute respiratory syndrome. In the current pandemic, bioinformatics studies are of great importance for preventing the spread of COVID-19, developing drugs and vaccines, and clinical practice.

Mechanism of Multi-Organ Injury in Experimental COVID-19 and Its Inhibition by a Small Molecule Peptide.

Severe disease from SARS-CoV-2 infection often progresses to multi-organ failure and results in an increased mortality rate amongst these patients. However, underlying mechanisms of SARS- CoV-2-induced multi-organ failure and subsequent death are still largely unknown. Cytokine storm, increased levels of inflammatory mediators, endothelial dysfunction, coagulation abnormalities, and infiltration of inflammatory cells into the organs contribute to the pathogenesis of COVID-19. One potential consequence of immune/inflammatory events is the acute progression of generalized edema, which may lead to death. We, therefore, examined the involvement of water channels in the development of edema in multiple organs and their contribution to organ dysfunction in a Murine Hepatitis Virus-1 (MHV-1) mouse model of COVID-19. Using this model, we recently reported multi-organ pathological abnormalities and animal death similar to that reported in humans with SARS-CoV-2 infection. We now identified an alteration in protein levels of AQPs 1, 4, 5, and 8 and associated oxidative stress, along with various degrees of tissue edema in multiple organs, which correlate well with animal survival post-MHV-1 infection. Furthermore, our newly created drug (a 15 amino acid synthetic peptide, known as SPIKENET) that was designed to prevent the binding of spike glycoproteins with their receptor(s), angiotensin- converting enzyme 2 (ACE2), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (SARS-CoV-2 and MHV-1, respectively), ameliorated animal death and reversed altered levels of AQPs and oxidative stress post-MHV-1 infection. Collectively, our findings suggest the possible involvement of altered aquaporins and the subsequent edema, likely mediated by the virus-induced inflammatory and oxidative stress response, in the pathogenesis of COVID- 19 and the potential of SPIKENET as a therapeutic option.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/β) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats. Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism. Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain. The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/β receptors of lung and breast neoplasia.

SARS-CoV-2突变背景下的病毒入侵机制研究进展与干预策略

<p indent="0mm">Since coronavirus disease 2019 (COVID-19) was reported in late 2019, its causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading worldwide for more than two years, and the trend continues unabated. SARS-CoV-2 is a single positive-stranded RNA virus from the same β-coronavirus family as SARS-CoV, which caused a severe acute respiratory syndrome (SARS) epidemic in 2003. Entry into the cell is mediated by the binding of the spike glycoprotein on the envelope to angiotensin converting enzyme 2 (ACE2) on the host cell, possibly with the help of co-receptors, adhesion factors, or alternative receptors. During the process of virus assembly, release, cell adhesion, and entry into the host cell, the SARS-CoV-2 spike protein must undergo a series of structural changes, including cleavage and conformational changes, to achieve membrane fusion function. After binding to cell surface receptors, SARS-CoV-2 has two invasion pathways: (i) The direct fusion pathway at the cell membrane interface, and (ii) the clathrin-mediated endocytosis pathway. In both cases, the SARS-CoV-2 spike protein must be cleaved by furin-like proteins at the S1/S2 site. Host proteases, mainly TMPRSS-2 or cathepsin, are involved in cleavage at the S2′ site, corresponding to pathway (i) or (ii). The entire entry process ends when the viral genome enters the cytosol. During the replication cycle of SARS-CoV-2, genomic RNA can be mutated. Accumulation of mutations, particularly in the spike protein, may result in new variants that differ in infectivity, transmissibility, virulence, and/or immune escape capability. These changes can alter the prevailing lineage in circulation, trigger a new wave of public health events, and threaten scientific research, clinical prevention, and treatment. The World Health Organization (WHO) has identified five mutant strains as variants of concern (VOCs) by assessing their public health significance worldwide: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). The Omicron variant is currently in circulation. The entry-related characteristics of VOCs may indicate global epidemiological trends and shed light on future pandemic dynamics. The entry mechanism of SARS-CoV-2, the first and crucial step in the establishment of a viral infection, has been extensively studied over the past two years. As the molecular mechanisms involved in entry are gradually being elucidated, interventions in key events may prevent viral entry and provide information for drug development and clinical treatment. Because the cellular entry mechanism of coronaviruses is relatively conserved, the first step in interrupting viral infection—blocking the entry process—is a promising target for the development of new preventive and therapeutic drugs against COVID-19 and even pan-coronavirus inhibitors, including small molecules, peptides, and monoclonal antibodies. Researchers have identified potential drug targets involved in the SARS-CoV-2 entry process, such as spike proteins, ACE2, and host proteases. Inhibition of the viral entry process is expected to reduce viral load in patients, thereby alleviating symptoms and improving prognosis. Recently, computer-aided drug design has emerged as a powerful and productive tool for drug discovery, in addition to experimental methods in the wet lab. In future anti-SARS-CoV-2 drug discovery, the flexible combination and optimized design of drug discovery strategies to improve the efficiency of drug development will be the trend. In this review, we outline the mechanism of SARS-CoV-2 entry into cells, describe the entry properties of VOCs currently defined by the WHO, and present the progress in the discovery of SARS-CoV-2 entry inhibitors.

Human variation in the protein receptor ACE2 affects its binding affinity to SARS-CoV-2 in a variant-dependent manner

SARS-CoV-2 infection depend on the binding of the viral Spike glycoprotein (S) to the human receptor Angiotensin Converting Enzyme 2 (ACE2) to induce virus–cell membrane fusion. S protein evolved diverse amino acid changes that are possibly linked to more efficient binding to human ACE2, which might explain part of the increase in frequency of SARS-CoV-2 Variants Of Concern (VOCs). In this work, we investigated the role of ACE2 protein variations that are naturally found in human populations and its binding affinity with S protein from SARS-CoV-2 representative genotypes, based on a series of in silico approaches involving molecular modelling, docking and molecular dynamics simulations. Our results indicate that SARS-CoV-2 VOCs bind more efficiently to the human receptor ACE2 than the ancestral Wuhan genotype. Additionally, variations in the ACE2 protein can affect SARS-CoV-2 binding and protein-protein stability, mostly making the interaction weaker and unstable in some cases. We show that some VOCs, such as B.1.1.7 and P.1 are much less sensitive to ACE2 variants, while others like B.1.351 appear to be specifically optimized to bind to the widespread wild-type ACE2 protein. Communicated by Ramaswamy H. Sarma

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle.

ABSTRACTThe COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections.IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.

Impact of SARS-CoV-2 spike protein on α7 nicotinic acetylcholine receptor in cells

SARS-CoV-2 infection relies on its Spike glycoprotein (S) binding to ACE2 in the host cells. S may also interact with other receptors to cause cellular damage. The α7 nicotinic acetylcholine receptor (α7nAChR), a primary component in the cholinergic anti-inflammatory pathway and a modulator of coagulation, has been proposed as a target of S. However, experimental evidence for full-length S binding to α7nAChR remains to be substantiated. Here we report our study of S/α7nAChR interactions using fluorescence imaging of cells.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Biological effect of black phosphorus nanosheets on the interaction between SARS-CoV-2 S protein and ACE2.

The binding of the spike glycoprotein (S protein) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to angiotensin-converting enzyme 2 (ACE2) is the main pathway that leads to serious coronavirus disease 2019 (COVID-19) infection. In the biomedical applications of various nanomaterials, black phosphorus nanosheets (BP) have been receiving increasing attention owing to their excellent characteristics. In this study, the biological effect of BP on the interaction between the S protein and ACE2 was investigated by molecular dynamics simulations. The results indicated that the ACE2 could be quickly and stably adsorbed on the BP surface by non-specific binding and retain its structural integrity. Compared with the case without BP, the interaction of the S protein bound to ACE2 adsorbed on the BP surface was greatly weakened, including hydrogen bonds, salt bridges, and van der Waals forces. This study not only reveals that BP could effectively obstruct the binding of S protein and ACE2, which may provide a potential and reasonable drug carrier to further enhance the curative effect of inhibitors against SARS-CoV-2 infection, but also presents a novel interference mechanism for protein-protein interactions caused by nanomaterials.

Nanodispersions of Polyelectrolytes Based on Humic Substances: Isolation, Physico-Chemical Characterization and Evaluation of Biological Activity.

Natural polyelectrolytes, including in the form of complexes with colloidal particles, are increasingly used in pharmacy due to the possibility of regulated attachment of medicinal substances and their targeted delivery to the target organ. However, the formation, stability, and molecular-mass characteristics of polyelectrolyte nanodispersions (ND) vary depending on the nature and composition of the medium of their origin. This is due to the lack of standardized approaches to quality control and regulatory documentation for most natural ND. In this paper, we first introduced the isolation, followed by investigations into their physico-chemical properties and bioactivity. Using the dried droplet method, we were able to detect the “coffee ring effect”. Fractographic studies of the surface structure of EHA and FA dried samples using SEM showed its heterogeneity and the presence of submicron particles encapsulated in the internal molecular cavities of polyelectrolyte. FTIR spectroscopy revealed the ND chemical structure of benzo-α-pyron and benzo-γ-pyron, consisting of nanoparticles and a branched frame part. The main elements detected by X-ray fluorescence in humic substance extract and fulvic acid include Si, P, S, K, Ca, Mn, Fe, Cu, Zn, whereas Fe is in high concentrations. The UV-spectra and fluorescent radiation demonstrated the possibility of studying the effect of the fulvate chromone structure on its optical properties. It is shown that dilution of the initial solutions of polyelectrolytes 1:10 contributes to the detection of smaller nanoparticles and an increase in the absolute value of the negative ζ-potential as a factor of ND stability. A study of the EHS effect on the SARS-CoV-2 virus infectious titer in the Vero E6 cell showed the effective against virus both in the virucidal scheme (the SI is 11.90–22.43) and treatment/prevention scheme (the SI is 34.85–57.33). We assume that polyelectrolyte ND prevent the binding of the coronavirus spike glycoprotein to the receptor. Taking into account the results obtained, we expect that the developed approach can become unified for the standardization of the ND natural polyelectrolytes complex, which has great prospects for use in pharmacy and medicine as a drug with antiviral activity.

The use of biomimetic chromatography to predict acute aquatic toxicity of pharmaceutical compounds

The potential of biomimetic chromatography to predict ecotoxicological endpoints of pharmaceutical compounds was investigated. For this purpose, a data set of previously and newly measured chromatographic retention data for 36 structurally diverse drugs was used. Standardized retention times were measured on the immobilized artificial membrane, human serum albumin, and alpha-1-acid glycoprotein stationary phases. As ecotoxicological endpoints, half-maximal lethal concentration values of fish and half-maximal effective concentration (immobilization) values of a water flea (Daphnia magna spp.) determined with a two-day static method were considered. Ecotoxicity values correlated with octanol-water partitioning and the positive charge of compounds contributed even more to the toxicity. Models based on membrane partition exhibited the best statistics and predictive performance, attributed to lipophilicity and membrane electrostatic interactions. Alpha-1-acid glycoprotein binding led to satisfactory models, owing to its function as a binder of neutral and basic lipophilic compounds. Albumin binding, however, did not result in sound models, as it is governed by lipophilicity and the negative charge of compounds, contrary to the mechanism of toxicity. Both membrane and alpha-1-acid glycoprotein models were superior statistically from those derived from the octanol-water system. Overall, membrane and alpha-1-acid glycoprotein retention can be suggested as promising indices to assess the ecotoxicological risk of drugs.

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2–UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

CAR-NK Cells Targeting Sars-Cov-2 Glycosites As COVID-19 Treatment

Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2.Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA.Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6; Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6; Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3; Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3; Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK; 65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05; 78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03; n=6; Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation.Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. [Display omitted] DisclosuresMarkovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding; BMS: Research Funding; Kiadis Pharma: Research Funding.