Design and Synthesis of Novel Phthalide Derivatives containing 1,3,4-Oxadiazole/1,2,4-Triazole Units as Potential Antifungal Agents.

Four series of novel 1,3,4-oxadiazole/1,2,4-triazole hybrids of phthalide derivatives were designed and synthesized to search for novel potential antifungal agents. Preliminary antifungal activity assay results showed that compounds 4 a, 4 b, 4 m, 5 b, 5 f, 5 h, and 7 h exhibited moderate to excellent inhibitory activity against some phytopathogenic fungi. Among them, compound 5 b displayed the most outstanding antifungal effects against V. mali and S. sclerotiorum, with the EC50 mean of 3.96 µg/mL and 5.60 µg/mL, respectively, which was superior to those of commercial fungicides hymexazol and chlorothalonil. Furthermore, compound 5 b could completely suppress the spore germination of V. mali at a concentration of 10 µg/mL. Finally, molecular docking revealed that the potential target for the antifungal activity of compound 5 b was succinate dehydrogenase (SDH). This research provides novel candidate compounds for the prevention of phytopathogenic fungi.

Proton- versus Cation-Selective Transport of Saccharide Rim-Appended Pillar[5]arene Artificial Water Channels.

Transport of water across cell membranes is a fundamental process for important biological functions. Herein, we focused our research on a new type of symmetrical saccharide rim-functionalized pillar[5]arene (PA-S) artificial water channels with variable pore structures. To point out the versatility of PA-S channels, we systematically varied the nature of anchoring/gate keepers d-mannoside, d-mannuronic acid, or sialic acid H-bonding groups on lateral pillar[5]arene (PA) arms, known as good membrane adhesives, to best describe the influence of the chemical structure on their transport activity. The control of hydrophobic membrane binding-hydrophilic water binding balance is an important feature influencing the channels' structuration and efficiency for a proper insertion into bilayer membranes. The glycosylated PA channels' transport performances were assessed in lipid bilayer membranes, and the channels were able to transport water at high rates (∼106-107 waters/s/channel within 1 order of magnitude as for aquaporins), serving as selective proton railways with total Na+ and K+ rejection. Molecular simulation substantiates the idea that the PAs can generate supramolecular pores, featuring hydrophilic carbohydrate gate-keepers that serve as water-sponge relays at the channel entrance, effectively absorbing and redirecting water within the channel. The present channels may be regarded as a rare biomimetic example of artificial channels presenting proton vs cation transport selectivity performances.

Development of pH-responsive active film materials based on purple corncob and its application in meat freshness monitoring.

The use of petroleum-based food packaging materials is causing environmental damage and increasing greenhouse gas production. Consequently, there is a great interest in developing smart and sustainable alternative materials. In this study, an agricultural waste product (purple corncob extract, PCCE) was used as a raw material to prepare environmentally friendly pH-sensitive packaging materials. Natural pH-sensitive pigments (anthocyanins) and lignin-containing cellulose nanocrystals (LCNC) were extracted from the purple corncobs. A cationic biopolymer (chitosan) was used as a scaffolding material to assemble the film matrix. Composite film (LCNC-PCCE-chitosan) was produced using a simple solvent casting method. Fourier transform infrared spectroscopy and scanning electron microscopy analyses showed that the PCCE and LCNC were well dispersed within the chitosan matrix and they interacted with the matrix through hydrogen bonding and electrostatic interactions. The addition of LCNC improved the hydrophobicity and mechanical properties of the film and imparted antioxidant activity and UV-blocking properties. The presence of anthocyanins in the PCCE endowed the film with a sensitive and reversible pH response, which could be well used to monitor changes in the freshness of pork and shrimp products.

Enzymatic synthesis of sodium caseinate-EGCG-carboxymethyl chitosan ternary film: Structure, physical properties, antioxidant and antibacterial properties.

Proteins and polysaccharides have been frequently used in recent years to prepare environment-friendly packaging materials. However, films based on proteins or polysaccharides alone often have poor performance as packaging, so they need to be combined to improve properties. In this work, we applied enzyme technology to prepare sodium caseinate (SC)-carboxymethyl chitosan (CMC) films, incorporating epigallocatechin gallate (EGCG) as bridging molecules and antibacterial agents. SC-EGCG-CMC ternary conjugate was firstly synthesized by tyrosinase (Tyr), and the composite films were then prepared with the aid of glycerol. Under tyrosinase catalytic conditions, EGCG could cross-link with SC and CMC covalently. The effects of different concentrations of EGCG and tyrosinase on mechanical properties, water vapor permeability, antibacterial properties and free radical scavenging ability were studied. The crosslinking degree and mechanical properties were improved with the increase of EGCG and tyrosinase content. The film showed good antibacterial activity against Gram-positive bacteria. In addition, the antibacterial activity and free radical scavenging ability increased with the increase of EGCG concentration. This work provides an efficient enzymatic method to prepare films with good strength and antibacterial properties, which can be used to improve the storage quality of foods.

Multivalent 9-O-Acetylated-sialic acid glycoclusters as potent inhibitors for SARS-CoV-2 infection.

The recent emergence of highly transmissible SARS-CoV-2 variants illustrates the urgent need to better understand the molecular details of the virus binding to its host cell and to develop anti-viral strategies. While many studies focused on the role of the angiotensin-converting enzyme 2 receptor in the infection, others suggest the important role of cell attachment factors such as glycans. Here, we use atomic force microscopy to study these early binding events with the focus on the role of sialic acids (SA). We show that SARS-CoV-2 binds specifically to 9-O-acetylated-SA with a moderate affinity, supporting its role as an attachment factor during virus landing to cell host surfaces. For therapeutic purposes and based on this finding, we have designed novel blocking molecules with various topologies and carrying a controlled number of SA residues, enhancing affinity through a multivalent effect. Inhibition assays show that the AcSA-derived glycoclusters are potent inhibitors of cell binding and infectivity, offering new perspectives in the treatment of SARS-CoV-2 infection.

A review of multilayer and composite films and coatings for active biodegradable packaging.

Active biodegradable packaging are being developed from biodegradable biopolymers which may solve the environmental problems caused by petroleum-based materials (plastics), as well as improving the shelf life, quality, nutritional profile, and safety of packaged food. The functional performance of active ingredients in biodegradable packaging can be extended by controlling their release profiles. This can be achieved by incorporating active ingredients in sandwich-structured packaging including multilayer and composite packaging. In multilayer materials, the release profile can be controlled by altering the type, structure, and thickness of the different layers. In composite materials, the release profile can be manipulated by altering the interactions of active ingredients with the surrounding biopolymer matrix. This article reviews the preparation, properties, and applications of multilayer and composite packaging for controlling the release of active ingredients. Besides, the basic theory of controlled release is also elaborated, including diffusion, swelling, and biodegradation. Mathematical models are presented to describe and predict the controlled release of active ingredients from thin films, which may help researchers design packaging materials with improved functional performance.

Fortification of edible films with bioactive agents: a review of their formation, properties, and application in food preservation.

Biodegradable films constructed from food ingredients are being developed for food coating and packaging applications to create more sustainable and environmentally friendly alternatives to plastics and other synthetic film-forming materials. In particular, there is a focus on the creation of active packaging materials from natural ingredients, especially plant-based ones. The film matrix is typically constructed from film-forming food components, such as proteins, polysaccharides and lipids. These matrices can be fortified with active ingredients, such as antioxidants and antimicrobials, so as to enhance their functional properties. Edible active films must be carefully designed to have the required optical, mechanical, barrier, and preservative properties needed for commercial applications. This review focuses on the fabrication, properties, and functional performance of edible films constructed from natural active ingredients. It provides an overview of the type of active ingredients that can be used, how they interact with the film matrix, how they migrate through the films, and how they are released. It also discusses the potential application of these active films for food preservation. Finally, future trends are highlighted and areas where further research are required are discussed.

Discovery of potential small molecular SARS-CoV-2 entry blockers targeting the spike protein.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

Identification of Potential Binding Sites of Sialic Acids on the RBD Domain of SARS-CoV-2 Spike Protein.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still an emergent pandemic for humans. The virus infection is achieved by penetrating its spike protein to host cells via binding with ACE2. Moreover, recent studies show that SARS-CoV-2 may have multiple receptors that need to be further revealed. SARS-CoV-2 shares similar sequences of the spike protein with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which can invade host cells by binding to either DPP4 or sialic acids. Sialic acids can be linked to the terminal of glycoproteins and gangliosides are used as one of the receptors of many types of viruses. Therefore, it is very interesting to determine whether sialic acid is a potential receptor of SARS-CoV-2. To address this question, we took N-Acetylneuraminic acid (Neu5Ac), a type of predominant sialic acid found in human cells, as the molecular probe to computationally search the surface of the spike protein to locate the potential binding sites of Neu5Ac. SPR analysis and mass spectrum analysis confirmed the interaction between Neu5Ac and spike protein. This study shows that sialic acids can moderately interact with the spike protein of SARS-CoV-2 by binding between the two RBDs of the spike protein, indicating it could be a potential secondary or auxiliary receptor of SARS-CoV-2.

Accurate Retention Time Prediction Based on Monolinked Peptide Information to Confidently Identify Cross-Linked Peptides.

Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome-wide level mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the retention time as an extra filter, the false positive rate can be reduced by around 86% with a sensitivity loss of 10%. The method combined with DTSS (T-DTSS) not only benefits improving identification confidence but also leads to lower cutoff scores and facilitates substantially increasing inter-cross-link identification. T-DTSS was successfully applied to the identification of inter-cross-links obtained from Escherichia coli cell lysate cross-linked by a newly synthesized enrichable cross-linker, pDSBE. The approach can be applicable to both cleavable and noncleavable methods.

Avidity-Based Selection of Tissue-Specific CAR-T Cells from a Combinatorial Cellular Library of CARs.

Using T-cell chimeric antigen receptors (CAR-T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor-associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106-member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR-T's produce cytokines known to be associated with T cell activation in a CD38 expression-dependent manner. This avidity-based selection endows the engineered T cells with minimal off-tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR-T therapy to TAAs previously considered undruggable.

Synthesis of functionalized copillar[4+1]arenes and rotaxane as heteromultivalent scaffolds.

In this study, novel copillar[4+1]arenes were used as central heteromultivalent scaffolds via orthogonal couplings with a series of biologically relevant molecules such as carbohydrates, α-amino acids, biotin and phenylboronic acid. Further modifications by introducing maleimides or cyclooctyne groups provided molecular probes adapted to copper-free click chemistry. An octa-azidated fluorescent rotaxane bearing two distinct ligands was also generated in a fully controlled manner.

Decision Tree Searching Strategy to Boost the Identification of Cross-Linked Peptides.

We describe an efficient decision tree searching strategy (DTSS) to boost the identification of cross-linked peptides. The DTSS approach allows the identification of a wealth of complementary information to facilitate the construction of more protein-protein interaction networks for human cell lysate, which was tested by the use of a recently reported cross-linking data set (ACS Cent. Sci. 2019, 5, 1514-1522). A variant of the PhoX-linker, named pDSPE, was synthesized and applied to cross-link Escherichia coli cell lysate to demonstrate that the acquisition of doubly charged ions can significantly improve identification results. The method can be seamlessly integrated to other search engines to maximize the number of identified cross-links.

Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor.

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.

Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.

BACKGROUND: CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. RESULTS: We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. CONCLUSION: G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

Pharmacokinetics, Tissue Distribution, and Excretion Study of Cajanonic Acid A in Rats by UPLC-MS/MS.

Cajanonic acid A (CAA), a prenylated stilbene derivative extracted from the leaves of pigeon pea (Cajanus cajan), has been reported to possess inhibitory activity on the peroxisome proliferator-activated receptor gamma (PPARγ) and protein tyrosine phosphatase 1B (PTP1B). Its hypoglycemic activity in rats is comparable to that of the approved antidiabetic agent rosiglitazone. Therefore, CAA is a potential candidate for the treatment of type 2 diabetes and a lead compound for the discovery of novel hypoglycemic drugs. To achieve a thorough understanding of the biological behavior of CAA in vivo, our current study was designed to investigate the pharmacokinetics, bioavailability, distribution, and excretion of CAA in rats by UPLC-MS/MS. Chromatographic separation was performed on BEHC18 column (2.1 mm × 50 mm, 1.7 µm). Quantification was performed under the negative ion mode by using single reaction monitoring (SRM) of the transitions of m/z 353.14 → 309.11 for CAA and m/z 269.86 → 224.11 for genistein, respectively. Standard calibration curve showed excellent linearity (r2 > 0.99) within the range of 2 - 800 ng/mL. The accuracies and precisions were within the acceptance limits (all < 20%). CAA was quickly absorbed into bloodstream and distributed rapidly and widely to various tissues. The excretion ratio of CAA in the 3 main pathways via bile, feces, and urine was only 5.17%. These results indicate that CAA was quickly and thoroughly metabolized in vivo and excreted mainly as metabolites.

A novel mass spectrometry-cleavable, phosphate-based enrichable and multi-targeting protein cross-linker.

Chemical cross-linking mass spectrometry (XL-MS) is a powerful technology for obtaining protein structural information and studying protein-protein interactions. We report phospho-bisvinylsulfone (pBVS) as a novel water-soluble, MS-cleavable, phosphate-based enrichable and multi-targeting cross-linker. In this approach, the fragmentation of pBVS cross-linked peptides occurs in situ through retro-Michael addition. The phosphate group is successfully used as a small affinity tag to isolate cross-linked peptides from the highly abundant non-cross-linked peptides. In addition, the linker targets multiple types of amino acid residues, including cysteine, lysine and histidine. This method was applied to cross-link bovine serum albumin (BSA), myoglobin and Lbcpf1 demonstrating the ability to yield accurate and abundant information to facilitate protein structure elucidation.

Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation.

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for 'proteolysis-targeting chimeras'). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation.