Thiol-based mucolytics exhibit antiviral activity against SARS-CoV-2 through allosteric disulfide disruption in the spike glycoprotein

Small molecule therapeutics targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have lagged far behind the development of vaccines in the fight to control the COVID-19 pandemic. Here, we show that thiol-based mucolytic agents, P2119 and P2165, potently inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity of repurposed mucolytic agents to the reduction of key disulfides, specifically, by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif (RBM) in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol warhead pointed directly towards Cys432. These collective findings establish the vulnerability of human coronaviruses to repurposed thiol-based mucolytics and lay the groundwork for developing these compounds as a potential treatment, preventative and/or adjuvant against infection.

Taurine ameliorates thioacetamide induced liver fibrosis in rats via modulation of toll like receptor 4/nuclear factor kappa B signaling pathway

Liver fibrosis is a significant health problem that can cause serious illness and death. Unfortunately, a standard treatment for liver fibrosis has not been approved yet due to its complicated pathogenesis. The current study aimed at assessing the anti-fibrotic effect of taurine against thioacetamide induced liver fibrosis in rats through the modulation of toll like receptor 4/nuclear factor kappa B signaling pathway. Both concomitant and late taurine treatment (100 mg/kg, IP, daily) significantly reduced the rise in serum ALT and AST activities and significantly reversed the decrease in serum albumin and total protein. These results were confirmed by histopathological examinations and immunehistochemical inspection of α-SMA, caspase-3 and NF-κB. The antioxidant potential of taurine was verified by a marked increase of GSH content and a reduction of MDA level in liver tissue. The anti-fibrotic effects of taurine were evaluated by investigating the expression of TLR4, NF-κB. The protein levels of IL-6, LPS, MyD88, MD2, CD14, TGF-β1 and TNF-α were determined. Docking studies were carried out to understand how taurine interacts inside TLR4-MD2 complex and it showed good binding with the hydrophobic binding site of MD2. We concluded that the anti-fibrotic effect of taurine was attributable to the modulation of the TLR4/NF-κB signaling.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

Differential Regulation of Bilastine Affinity for Human Histamine H1 Receptors by Lys 179 and Lys 191 via Its Binding Enthalpy and Entropy

Bilastine, a zwitterionic second-generation antihistamine containing a carboxyl group, has higher selectivity for H1 receptors than first-generation antihistamines. Ligand-receptor docking simulations have suggested that the electrostatic interaction between the carboxyl group of second-generation antihistamines and the amino group of Lys179ECL2 and Lys1915.39 of human H1 receptors might contribute to increased affinity of these antihistamines to H1 receptors. In this study, we evaluated the roles of Lys179ECL2 and Lys1915.39 in regulating the electrostatic and hydrophobic binding of bilastine to H1 receptors by thermodynamic analyses. The binding enthalpy and entropy of bilastine were estimated from the van ’t Hoff equation using the dissociation constants. These constants were obtained from the displacement curves against the binding of [3H] mepyramine to membrane preparations of Chinese hamster ovary cells expressing wild-type human H1 receptors and their Lys179ECL2 or Lys1915.39 mutants to alanine at various temperatures. We found that the binding of bilastine to wild-type H1 receptors occurred by enthalpy-dependent binding forces and, more dominantly, entropy-dependent binding forces. The mutation of Lys179ECL2 and Lys1915.39 to alanine reduced the affinity of bilastine to H1 receptors by reducing enthalpy- and entropy-dependent binding forces, respectively. These results suggest that Lys179ECL2 and Lys1915.39 differentially contribute to the increased binding affinity to bilastine via electrostatic and hydrophobic binding forces.

Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13

Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.

Removal of methylene blue in water by electrospun PAN/β-CD nanofibre membrane

The polyacrylonitrile (PAN) nanofiber membrane prepared by the electrospinning technology was used as the matrix, and β-cyclodextrin (β-CD) was introduced into it to synthesize the composite nanofiber membrane. Taking methylene blue as the object, the adsorption performance of nanofiber membranes for dyes in the aqueous solution was studied. The structure, morphology, and specific surface area of the nanofiber membrane were characterized using FTIR, SEM, BET, XRD, and EDS. Meantime, the adsorption equilibrium was also explored. After being modified by cyclodextrin, a large number of cavity structures and hydrophobic binding sites were provided for the nanofiber membrane, and the adsorption performance was significantly improved. The results showed that the maximum adsorption capacity of the PAN/β-CD blend nanofiber membrane (at 25℃) for methylene blue was 108.66 mg g−1. The effects of pH, adsorption time, ion concentration, and adsorbent dosage on the adsorption capacity were also investigated. In addition, by fitting with the adsorption model, the adsorption process was more complex with the quasi-secondary adsorption kinetics and Langmuir isotherm adsorption model.

Utilization Phase Transition Component Method to Prepare Specially Functionalized Nanoemulsion by Adding Resveratrol/Phenethyl Resorcinol Mixed Active Components and Application in Free Radicals Removal

To explore the possibility of using a specially functionalized nanoemulsion for the removal of free radical, resveratrol and phenethyl resorcinol were employed to form resveratrol/phenethyl resorcinol mixed active components, and the removal efficiency of free radicals was evaluated. Original nanoemulsion and specially functionalized-nanoemulsion were characterized by DLS and EPR. The free radical scavenging ability of the mixed active components of resveratrol/phenethyl resorcinol was determined as a function of the mass ratio of resveratrol to phenethyl resorcinol, temperature, and ionic strength. In the results, the removal of free radicals by the resveratrol/phenethyl resorcinol nanoemulsion system was found to be more effective than the nanoemulsion alone. At a higher resveratrol ratio, more than 68% of free radicals could be removed. The efficiency was also found to increase with rising temperature. However, efficiency decreased with the increase in ionic strength. In conclusion, compared with conventional nanoemulsion, the combined utilization of nanoemulsion and the mixed active components of resveratrol/phenethyl resorcinol achieved better results for the removal of free radicals because of synergistic effect between nanoemulsion droplets and the mixed active components of resveratrol/phenethyl resorcinol, involving hydrophobic binding, hydrogen binding, and partitioning.