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Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors., (© 2024 The Protein Society.)

Background: Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CL pro ) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico ., Objective: We aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CL pro and ACE2 neutralization were major targets and explored whether any additional targets existed., Methods: We tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CL pro and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, in vitro 3CL pro activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA)., Results: AM and GM reduced SARS-CoV-2 3CL pro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC 50 of 0.70-3.05 μM, in which wild-type B was the most susceptible strain (EC 50 0.70-0.79 μM). AM was slightly more efficient in the variants (EC 50 0.88-2.41 μM), resulting in higher selectivity indices (SI 3.65-10.05), compared to the GM (EC 50 0.94-3.05 μM, SI 1.66-5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CL pro activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM., Conclusion: AM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC 50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

Controlled release of proteins, such as growth factors, from biocompatible silk fibroin (SF) hydrogel is valuable for its use in tissue engineering, drug delivery, and other biological systems. To achieve this, we introduced silk fibroin-mimetic peptides (SFMPs) with the repeating unit (GAGAGS) n . Using green fluorescent protein (GFP) as a model protein, our results showed that SFMPs did not affect the GFP function when conjugated to it. The SFMP-GFP conjugates incorporated into SF hydrogel did not change the gelation time and allowed for controlled release of the GFP. By varying the length of SFMPs, we were able to modulate the release rate, with longer SFMPs resulting in a slower release, both in water at room temperature and PBS at 37 °C. Furthermore, the SF hydrogel with the SFMPs showed greater strength and stiffness. The increased β-sheet fraction of the SF hydrogel, as revealed by FTIR analysis, explained the gel properties and protein release behavior. Our results suggest that the SFMPs effectively control protein release from SF hydrogel, with the potential to enhance its mechanical stability. The ability to modulate release rates by varying the SFMP length will benefit personalized and controlled protein delivery in various systems., (© 2024. The Author(s).)

The COVID-19 pandemic has created an urgent need for effective therapeutic and diagnostic strategies to manage the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the emergence of numerous variants of concern (VOCs) has made it challenging to develop targeted therapies that are broadly specific in neutralizing the virus. In this study, we aimed to develop neutralizing nanobodies (Nbs) using computational techniques that can effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2 VOCs. We evaluated the performance of different protein-protein docking programs and identified HDOCK as the most suitable program for Nb/RBD docking with high accuracy. Using this approach, we designed 14 novel Nbs with high binding affinity to the VOC RBDs. The Nbs were engineered with mutated amino acids that interacted with key amino acids of the RBDs, resulting in higher binding affinity than human angiotensin-converting enzyme 2 (ACE2) and other viral RBDs or haemagglutinins (HAs). The successful development of these Nbs demonstrates the potential of molecular modeling as a low-cost and time-efficient method for engineering effective Nbs against SARS-CoV-2. The engineered Nbs have the potential to be employed in RBD-neutralizing assays, facilitating the identification of novel treatment, prevention, and diagnostic strategies against SARS-CoV-2., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Longsompurana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drug development requires significant time and resources, and computer-aided drug discovery techniques that integrate chemical and biological spaces offer valuable tools for the process. This study focused on the field of COVID-19 therapeutics and aimed to identify new active non-covalent inhibitors for 3CL pro , a key protein target. By combining in silico and in vitro approaches, an in-house database was utilized to identify potential inhibitors. The drug-likeness criteria were considered to pre-filter 553 compounds from 12 groups of natural products. Using structure-based virtual screening, 296 compounds were identified that matched the chemical features of SARS-CoV-2 3CL pro peptidomimetic inhibitor pharmacophore models. Subsequent molecular docking resulted in 43 hits with high binding affinities. Among the hits, caffeic acid analogs showed significant interactions with the 3CL pro active site, indicating their potential as promising candidates. To further evaluate their efficacy, enzyme-based assays were conducted, revealing that two ester derivatives of caffeic acid (4k and 4l) exhibited more than a 30% reduction in 3CL pro activity. Overall, these findings suggest that the screening approach employed in this study holds promise for the discovery of novel anti-SARS-CoV-2 therapeutics. Furthermore, the methodology could be extended for optimization or retrospective evaluation to enhance molecular targeting and antiviral efficacy of potential drug candidates., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

3CL pro is a viable target for developing antiviral therapies against the coronavirus. With the urgent need to find new possible inhibitors, a structure-based virtual screening approach was developed. This study recognized 75 pharmacologically bioactive compounds from our in-house library of 1052 natural product-based compounds that satisfied drug-likeness criteria and exhibited good bioavailability and membrane permeability. Among these compounds, three promising sulfonamide chalcones were identified by combined theoretical and experimental approaches, with SWC423 being the most suitable representative compound due to its competitive inhibition and low cytotoxicity in Vero E6 cells (EC 50 = 0.89 ± 0.32 μM; CC 50 = 25.54 ± 1.38 μM; SI = 28.70). The binding and stability of SWC423 in the 3CL pro active site were investigated through all-atom molecular dynamics simulation and fragment molecular orbital calculation, indicating its potential as a 3CL pro inhibitor for further SARS-CoV-2 therapeutic research. These findings suggested that inhibiting 3CL pro with a sulfonamide chalcone such as SWC423 may pave the effective way for developing COVID-19 treatments.

New N -containing xanthone analogs of α-mangostin were synthesized via one-pot Smiles rearrangement. Using cesium carbonate in the presence of 2-chloroacetamide and catalytic potassium iodide, α-mangostin ( 1 ) was subsequently transformed in three steps to provide ether 2 , amide 3 , and amine 4 in good yields at an optimum ratio of 1:3:3, respectively. The evaluation of the biological activities of α-mangostin and analogs 2 - 4 was described. Amine 4 showed promising cytotoxicity against the non-small-cell lung cancer H460 cell line fourfold more potent than that of cisplatin. Both compounds 3 and 4 possessed antitrypanosomal properties against Trypanosoma brucei rhodesiense at a potency threefold stronger than that of α-mangostin. Furthermore, ether 2 gave potent SARS-CoV-2 main protease inhibition by suppressing 3-chymotrypsinlike protease (3CL pro ) activity approximately threefold better than that of 1 . Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CL pro active site regarding an additional ether moiety. Thus, the series of N -containing α-mangostin analogs prospectively enhance druglike properties based on isosteric replacement and would be further studied as potential biotically active chemical entries, particularly for anti-lung-cancer, antitrypanosomal, and anti-SARS-CoV-2 main protease applications.

Bone morphogenetic protein-2 (BMP-2) is a promising osteogenic agent in tissue engineering. BMP-2 is usually expressed in Escherichia coli owing to the high yield and low cost, but the protein is expressed as inclusion bodies. Thus, the bottleneck for BMP-2 production in E. coli is the refolding process. Here, we explored the effects of the refolding buffer composition on BMP-2 refolding. The BMP-2 inclusion body was solubilized in urea and subjected to refolding by the dilution method. Various additives were investigated to improve the BMP-2 refolding yield. Nonreducing SDS-PAGE showed that BMP-2 dimers, the presumably biologically active form, were detected at approximately 25 kDa. The highest yield of the BMP-2 dimers was observed in the refolding buffer that contained ionic detergents (sarkosyl and cetylpyridinium chloride) followed by zwitterionic and nonionic detergents (NDSB-195, NP-40, and Tween 80). In addition, sugars (glucose, sorbitol, and sucrose) in combination with anionic detergents (sodium dodecyl sulfate and sarkosyl) reduced BMP-2 oligomers and increased the BMP-2 dimer yield. Subsequently, the refolded BMP-2s were tested for their bioactivity using the alkaline phosphatase assay in osteogenic cells (SaOS-2), as well as the luciferase reporter assay and the calcium assays. The refolded BMP-2 showed the activities in the calcium deposition assay and the luciferase reporter assay but not in the alkaline phosphatase activity assay or the intracellular calcium assay even though the dimers were clearly detected. Therefore, the detection of the disulfide-linked dimeric BMP-2 in nonreducing SDS-PAGE is an inadequate proxy for the bioactivity of BMP-2., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Parallel cascade selection molecular dynamics-based ligand binding-path sampling (LB-PaCS-MD) was combined with fragment molecular orbital (FMO) calculations to reveal the ligand path from an aqueous solution to the SARS-CoV-2 main protease (M pro ) active site and to customise a ligand-binding pocket suitable for delivering a potent inhibitor. Rubraxanthone exhibited mixed-inhibition antiviral activity against SARS-CoV-2 M pro , relatively low cytotoxicity, and high cellular inhibition. However, the atomic inhibition mechanism remains ambiguous. LB-PaCS-MD/FMO is a hybrid ligand-binding evaluation method elucidating how rubraxanthone interacts with SARS-CoV-2 M pro . In the first step, LB-PaCS-MD, which is regarded as a flexible docking, efficiently samples a set of ligand-binding pathways. After that, a reasonable docking pose of LB-PaCS-MD is evaluated by the FMO calculation to elucidate a set of protein-ligand interactions, enabling one to know the binding affinity of a specified ligand with respect to a target protein. A possible conformation was proposed for rubraxanthone binding to the SARS-CoV-2 M pro active site, and allosteric inhibition was elucidated by combining blind docking with k-means clustering. The interaction profile, key binding residues, and considerable interaction were elucidated for rubraxanthone binding to both M pro sites. Integrated LB-PaCS-MD/FMO provided a more reasonable complex structure for ligand binding at the SARS-CoV-2 M pro active site, which is vital for discovering and designing antiviral drugs., (© 2022. The Author(s).)

Intelectins are putative innate immune lectins that are found throughout chordates. The first intelectin reported was Xenopus laevis cortical granule lectin-1 (XCGL-1 or XL-35). XCGL-1 is critical in fertilization membrane development in Xenopus . Here, we explored the biochemical properties of XCGL-1. The cysteines responsible for forming intermolecular disulfide bonds were identified. XCGL-1 adopted a four-lobed structure as observed by electron microscopy. The full-length XCGL-1 and the carbohydrate recognition domain (CRD) bind galactose-containing carbohydrates at nanomolar to micromolar affinities. Molecular modeling suggested that galactoside ligands coordinated the binding site calcium ion and interacted with residues around the groove made available by the non-conserved substitution compared to human intelectin-1. Folding conditions for production of recombinant XCGL-1 CRD were also investigated. Our results not only provide new biochemical insights into the function of XCGL-1, but may also provide foundation for further applications of XCGL-1 as glycobiology tools., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

SARS-CoV-2 causes the current global pandemic coronavirus disease 2019. Widely-available effective drugs could be a critical factor in halting the pandemic. The main protease (3CLpro) plays a vital role in viral replication; therefore, it is of great interest to find inhibitors for this enzyme. We applied the combination of virtual screening based on molecular docking derived from the crystal structure of the peptidomimetic inhibitors (N3, 13b, and 11a), and experimental verification revealed FDA-approved drugs that could inhibit the 3CLpro of SARS-CoV-2. Three drugs were selected using the binding energy criteria and subsequently performed the 3CLpro inhibition by enzyme-based assay. In addition, six common drugs were also chosen to study the 3CLpro inhibition. Among these compounds, lapatinib showed high efficiency of 3CLpro inhibition (IC50 value of 35 ± 1 μM and Ki of 23 ± 1 μM). The binding behavior of lapatinib against 3CLpro was elucidated by molecular dynamics simulations. This drug could well bind with 3CLpro residues in the five subsites S1', S1, S2, S3, and S4. Moreover, lapatinib's key chemical pharmacophore features toward SAR-CoV-2 3CLpro shared important HBD and HBA with potent peptidomimetic inhibitors. The rational design of lapatinib was subsequently carried out using the obtained results. Our discovery provides an effective repurposed drug and its newly designed analogs to inhibit SARS-CoV-2 3CLpro., Competing Interests: The authors have declared that no competing interests exist.

A global crisis of coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted millions of people's lives throughout the world. In parallel to vaccine development, identifying potential antiviral agents against SARS-CoV-2 has become an urgent need to combat COVID-19. One of the most attractive drug targets for discovering anti-SARS-CoV-2 agents is the main protease (M pro ), which plays a pivotal role in the viral life cycle. This study aimed to elucidate a series of twenty-one 12-dithiocarbamate-14-deoxyandrographolide analogues as SARS-CoV-2 M pro inhibitors using in vitro and in silico studies. These compounds were initially screened for the inhibitory activity toward SARS-CoV-2 M pro by in vitro enzyme-based assay. We found that compounds 3 k , 3 l , 3 m and 3 t showed promising inhibitory activity against SARS-CoV-2 M pro with >50% inhibition at 10 μM. Afterward, the binding mode of each compound in the active site of SARS-CoV-2 M pro was explored by molecular docking. The optimum docked complexes were then chosen and subjected to molecular dynamic (MD) simulations. The MD results suggested that all studied complexes were stable along the simulation time, and most of the compounds could fit well with the SARS-CoV-2 M pro active site, particularly at S1, S2 and S4 subsites. The per-residue decomposition free energy calculations indicated that the hot-spot residues essential for ligand binding were T25, H41, C44, S46, M49, C145, H163, M165, E166, L167, D187, R188, Q189 and T190. Therefore, the obtained information from the combined experimental and computational techniques could lead to further optimization of more specific and potent andrographolide analogues toward SARS-CoV-2 M pro ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Piper nigrum , or black pepper, produces piperine, an alkaloid that has diverse pharmacological activities. In this study, N -aryl amide piperine analogs were prepared by semi-synthesis involving the saponification of piperine ( 1 ) to yield piperic acid ( 2 ) followed by esterification to obtain compounds 3 , 4 , and 5 . The compounds were examined for their antitrypanosomal, antimalarial, and anti-SARS-CoV-2 main protease activities. The new 2,5-dimethoxy-substituted phenyl piperamide 5 exhibited the most robust biological activities with no cytotoxicity against mammalian cell lines, Vero and Vero E6, as compared to the other compounds in this series. Its half-maximal inhibitory concentration (IC 50 ) for antitrypanosomal activity against Trypanosoma brucei rhodesiense was 15.46 ± 3.09 μM, and its antimalarial activity against the 3D7 strain of Plasmodium falciparum was 24.55 ± 1.91 μM, which were fourfold and fivefold more potent, respectively, than the activities of piperine. Interestingly, compound 5 inhibited the activity of 3C-like main protease (3CL Pro ) toward anti-SARS-CoV-2 activity at the IC 50 of 106.9 ± 1.2 μM, which was threefold more potent than the activity of rutin. Docking and molecular dynamic simulation indicated that the potential binding of 5 in the 3CL pro active site had the improved binding interaction and stability. Therefore, new aryl amide analogs of piperine 5 should be investigated further as a promising anti-infective agent against human African trypanosomiasis, malaria, and COVID-19.

Intelectins are immune lectins expressed in chordates, including several fish species, in which intelectins are known to be upregulated upon infection. However, the basic biochemical properties and bacteria binding specificities of several fish intelectins are not well studied. We focus our investigation on zebrafish intelectin-2 (DrIntL-2) that is predominantly expressed in the gastrointestinal tract. The disulfide-linked oligomeric state and the cysteine responsible for intermolecular disulfide bonds are identified. DrIntL-2 is a globular particle of around 30 nm. In addition to the typical exocyclic 1,2-diol ligands, DrIntL-2 binds β-1,3-glucan and recognizes Salmonella typhimurium and Pseudomonas aeruginosa. This investigation not only shed light on the fish innate immunity that will be essential for the aquaculture industry, but will also provide a foundation for further application of DrIntL-2 in bacteria detection and identification., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The coronavirus disease pandemic is a constant reminder that global citizens are in imminent danger of exposure to emerging infectious diseases. Therefore, developing a technique for inhibitor discovery is essential for effective drug design. Herein, we proposed fragment molecular orbital (FMO)-based virtual screening to predict the molecular binding energy of potential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitors. The integration of quantum mechanical approaches and trajectory analysis from a microsecond molecular dynamics simulation was used to identify potential inhibitors. We identified brominated baicalein as a potent inhibitor of the SARS-CoV-2 main protease and confirmed its inhibitory activity in an in vitro assay. Brominated baicalein did not demonstrate significant toxicity in either in vitro or in vivo studies. The pair interaction energy from FMO-RIMP2/PCM and inhibitory constants based on the protease enzyme assay suggested that the brominated baicalein could be further developed into novel SARS-CoV-2 protease inhibitors.

Background: Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations., Methods: A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures., Results: The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection., Conclusions: The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing coronavirus disease (COVID-19) pandemic which is characterized by respiratory illness and severe pneumonia, and currently accounts for > 2.5 million deaths worldwide. Recently, diverse mutations in the spike protein of SARS-CoV-2 were reported in United Kingdom (Alpha) and South Africa (Beta) strains which raise concerns over the potential increase in binding affinity towards the host cell receptor and diminished host neutralization capabilities. In order to study the effect of mutation in the binding efficiency of SARS-CoV-2 receptor binding domain (RBD) with anti-SARS-CoV/CoV-2 monoclonal antibodies (mAbs), we have produced SARS-CoV-2 RBD and two variants SARS-CoV-2 RBD (Alpha RBD and Beta RBD) in Nicotiana benthamiana by transient expression. Plant-produced SARS-CoV-2 RBD-Fc, Alpha RBD-Fc and Beta RBD-Fc exhibited specific binding to human angiotensin converting enzyme 2 (ACE2) receptor determined by ELISA. Intriguingly, the binding of plant-produced SARS-CoV-2 RBD proteins to plant-produced mAbs CR3022, B38, and H4 was found to be different depending on the variant mutation. In contrary to the plant-produced SARS-CoV-2 RBD-Fc and Alpha RBD-Fc, Beta RBD-Fc variant showed weak binding affinity towards the mAbs. The result suggested that the Beta RBD variant might have acquired partial resistance to neutralizing antibodies compared to other variants. However, further studies with sera from convalescent or vaccinated individuals are required to confirm this finding., Competing Interests: This study has received funding from National Research Council of Thailand and commercial source Baiya Phytopharm Co., Ltd. WP from Chulalongkorn University is a co-founder/shareholder of Baiya Phytopharm Co., Ltd. Authors BS and KR have potential financial competing interest due to paid employment provided by Baiya Phytopharm Co., Ltd. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18-49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher k cat /K m values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca 2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

The 3C-like main protease of SARS-CoV-2 (3CL Pro ) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CL Pro is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CL Pro inhibitors rely on peptide-based colorimetric or fluorogenic substrates. However, the COVID-19 pandemic has limit or delay access to these substrates, especially for researchers in developing countries attempting to screen natural product libraries. We explored the use of the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) as an alternative assay for inhibitor identification. Fluorescence enhancement upon binding of ANS to 3CL Pro was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CL Pro inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CL Pro was explored with molecular modeling. Our results suggested that ANS could be employed in a competitive binding assay to facilitate the identification of novel SARS-CoV-2 antiviral compounds., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Lipopolysaccharide (LPS) is a crucial component in the outer membrane of Gram-negative bacteria that contributes to both pathogenicity as well as immunity against pathogenic bacteria. Typical LPS contains GlcN disaccharide as the core of lipid A. However, some bacteria such as Acidithiobacillus ferrooxidans and Leptospira interrogans contain GlcN3N in lipid A instead. This modification has been shown to dampen the host immune response and increase resistance to antimicrobial peptides. Therefore, investigation of the enzymes responsible for the biosynthesis of GlcN3N has promising applications in the development of vaccines, antibiotics, or usage of the enzymes in chemoenzymatic synthesis of modified LPS. Here, we describe biochemical and structural investigation of GnnA from A. ferrooxidans ( Af GnnA) that is responsible for oxidation of UDP-GlcNAc, which subsequently undergoes transamination to produce UDP-GlcNAc3N as a precursor for LPS biosynthesis. Af GnnA is specific for NAD + and UDP-GlcNAc. The crystal structures of Af GnnA in combination with molecular dynamics simulation and mutational analysis suggest the substrate recognition mode and the catalytic mechanism. K91 or H164 is a potential catalytic base in the oxidation reaction. The results will not only provide insights into the biosynthesis of unusual LPS but will also lay the foundation for development of more immunogenic vaccines, novel antibiotics, or utilization of GnnA in the synthesis of UDP-sugars or modified LPS.

Last decade witnessed the outbreak of many life-threatening human pathogens including Nipah, Ebola, Chikungunya, Zika, Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute respiratory syndrome coronavirus (SARS-CoV) and more recently novel coronavirus (2019-nCoV or SARS-CoV-2). The disease condition associated with novel coronavirus, referred to as Coronavirus disease (COVID-19). The emergence of novel coronavirus in 2019 in Wuhan, China marked the third highly pathogenic coronavirus infecting humans in the 21st century. The continuing emergence of coronaviruses at regular intervals poses a significant threat to human health and economy. Ironically, even after a decade of research on coronavirus, still there are no licensed vaccines or therapeutic agents to treat coronavirus infection which highlights an urgent need to develop effective vaccines or post-exposure prophylaxis to prevent future epidemics. Several clinical, genetic and epidemiological features of COVID-19 resemble SARS-CoV infection. Hence, the research advancements on SARS-CoV treatment might help scientific community in quick understanding of this virus pathogenesis and develop effective therapeutic/prophylactic agents to treat and prevent this infection. Monoclonal antibodies represent the major class of biotherapeutics for passive immunotherapy to fight against viral infection. The therapeutic potential of monoclonal antibodies has been well recognized in the treatment of many diseases. Here, we summarize the potential monoclonal antibody based therapeutic intervention for COVID-19 by considering the existing knowledge on the neutralizing monoclonal antibodies against similar coronaviruses SARS-CoV and MERS-CoV. Further research on COVID-19 pathogenesis could identify appropriate therapeutic targets to develop specific anti-virals against this newly emerging pathogen.

We extend our study of the structural stability of helical and nonhelical regions in chain A of human intelectin-1 to include a second human intelectin (4WMY) and the frog protein "Xenopus embryonic epidermal lectin" (XEEL). These unique lectins have been shown to recognize carbohydrate residues found exclusively in microbes, thus they could potentially be developed into novel microbe detection and sequestration tools. We believe that by studying the structural stability of these proteins we can provide insights on their biological role and activities. Using a geometrical model introduced previously, we perform computational analyses of protein crystal structures that quantify the resiliency of the native state to steric perturbations. Based on these analyses, we conclude that differences in the resiliency of the human and frog proteins can be attributed primarily to differences in non-helical regions and to residues near Ca ions. Since these differences are particularly pronounced in the vicinity of the ligand binding site, they provide an explanation for the finding that human intelectin-1 has a higher affinity for a ligand than XEEL. We also present data on conserved and position-equivalent pairs of residues in 4WMY and XEEL. We identify residue pairs as well as regions in which the influence of neighboring residues is nearly uniform as the parent protein denatures. Since the structural signatures are conserved, this identification provides a basis for understanding why both proteins exhibit trimeric structures despite poor sequence conservation at the interface., (Copyright © 2018 Elsevier Inc. All rights reserved.)

UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small-molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His 6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies for optimizing inhibitor potency by exploiting UGM conformational flexibility.

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEELCRD) forms a stable trimer in solution. The structure of XEELCRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The roles of the extracellular domain of type II TGF-β receptor (TBRII-ECD) in physiological processes ranging from development to cancer to wound healing render it an attractive target for exploration with chemical tools. For such applications, large amounts of active soluble protein are needed, but the yields of TBRII-ECD we obtained with current folding protocols were variable. To expedite the identification of alternative folding conditions, we developed an on-plate screen. This assay indicated that effective folding additives included the non-detergent sulfobetaine-201 (NDSB-201). Although NDSB-201 can facilitate protein folding, the mode by which it does so is poorly understood. We postulated that specific interactions between NDSB-201 and TBRII-ECD might be responsible. Analysis by X-ray crystallography indicates that the TBRII-ECD possesses a binding pocket for NDSB-201. The pyridinium group of the additive stacks with a phenylalanine side chain in the binding site. The ability of NDSB-201 to occupy a pocket on the protein provides a molecular mechanism for the additive's ability to minimize TBRII-ECD aggregation and stabilize the folded state. NDSB-201 also accelerates TBRII-ECD crystallization, suggesting it may serve as a useful crystallization additive for proteins refolded with it. Our results also suggest there is a site on TBRII-ECD that could be targeted by small-molecule modulators., (Copyright © 2015. Published by Elsevier Inc.)

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. We used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol-modified glycans, heptoses, D-glycero-D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D-manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in human glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. This ligand selectivity suggests that hIntL-1 functions in microbial surveillance.