Alpha and gamma mangostins inhibit wild-type B SARS-CoV-2 more effectively than the SARS-CoV-2 variants and the major target is unlikely the 3C-like protease.

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 (3CLpro) 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 3CLpro 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 3CLpro 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 3CLpro 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 3CLpro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC50 of 0.70-3.05 µM, in which wild-type B was the most susceptible strain (EC50 0.70-0.79 µM). AM was slightly more efficient in the variants (EC50 0.88-2.41 µM), resulting in higher selectivity indices (SI 3.65-10.05), compared to the GM (EC50 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. 3CLpro 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.

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

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 (IC50 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.

Optimizing protein delivery rate from silk fibroin hydrogel using silk fibroin-mimetic peptides conjugation.

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 ß-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.

Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2.

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.

Identification of Promising Sulfonamide Chalcones as Inhibitors of SARS-CoV-2 3CLpro through Structure-Based Virtual Screening and Experimental Approaches.

3CLpro 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 (EC50 = 0.89 ± 0.32 µM; CC50 = 25.54 ± 1.38 µM; SI = 28.70). The binding and stability of SWC423 in the 3CLpro active site were investigated through all-atom molecular dynamics simulation and fragment molecular orbital calculation, indicating its potential as a 3CLpro inhibitor for further SARS-CoV-2 therapeutic research. These findings suggested that inhibiting 3CLpro with a sulfonamide chalcone such as SWC423 may pave the effective way for developing COVID-19 treatments.

N-Containing α-Mangostin Analogs via Smiles Rearrangement as the Promising Cytotoxic, Antitrypanosomal, and SARS-CoV-2 Main Protease Inhibitory Agents.

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 (3CLpro) activity approximately threefold better than that of 1. Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CLpro 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.

Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts.

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.

Promising SARS-CoV-2 main protease inhibitor ligand-binding modes evaluated using LB-PaCS-MD/FMO.

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 (Mpro) 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 Mpro, 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 Mpro. 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 Mpro 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 Mpro sites. Integrated LB-PaCS-MD/FMO provided a more reasonable complex structure for ligand binding at the SARS-CoV-2 Mpro active site, which is vital for discovering and designing antiviral drugs.

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1.

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.

Discovery of C-12 dithiocarbamate andrographolide analogues as inhibitors of SARS-CoV-2 main protease: In vitro and in silico studies.

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 (Mpro), 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 Mpro inhibitors using in vitro and in silico studies. These compounds were initially screened for the inhibitory activity toward SARS-CoV-2 Mpro 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 Mpro with >50% inhibition at 10 µM. Afterward, the binding mode of each compound in the active site of SARS-CoV-2 Mpro 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 Mpro 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 Mpro.

Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening.

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.

Semi-Synthesis of N-Aryl Amide Analogs of Piperine from Piper nigrum and Evaluation of Their Antitrypanosomal, Antimalarial, and Anti-SARS-CoV-2 Main Protease Activities.

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 (IC50) 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 (3CLPro) toward anti-SARS-CoV-2 activity at the IC50 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 3CLpro 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.

Halogenated Baicalein as a Promising Antiviral Agent toward SARS-CoV-2 Main Protease.

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.

Molecular properties and ligand specificity of zebrafish intelectin-2.

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 ß-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.

Global diversity of the gene encoding the Pfs25 protein-a Plasmodium falciparum transmission-blocking vaccine candidate.

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.

Plant-produced SARS-CoV-2 receptor binding domain (RBD) variants showed differential binding efficiency with anti-spike specific monoclonal antibodies.

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.

Interaction of 8-anilinonaphthalene-1-sulfonate with SARS-CoV-2 main protease and its application as a fluorescent probe for inhibitor identification.

The 3C-like main protease of SARS-CoV-2 (3CLPro) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CLPro is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CLPro 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 3CLPro was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CLPro inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CLPro 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.

Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases.

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 kcat/Km 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.

Biochemical and structural characterization of a recombinant fibrinogen-related lectin from Penaeus monodon.

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 Ca2+ 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.

Biochemical and Structural Investigation of GnnA in the Lipopolysaccharide Biosynthesis Pathway of Acidithiobacillus ferrooxidans.

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 (AfGnnA) that is responsible for oxidation of UDP-GlcNAc, which subsequently undergoes transamination to produce UDP-GlcNAc3N as a precursor for LPS biosynthesis. AfGnnA is specific for NAD+ and UDP-GlcNAc. The crystal structures of AfGnnA 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.