Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB) showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator

A Closer Look at Dexamethasone and the SARS-CoV-2-Induced Cytokine Storm: In Silico Insights of the First Life-Saving COVID-19 Drug

The term cytokine storm refers to an uncontrolled overproduction of soluble inflammatory markers known as cytokines and chemokines. Autoimmune destruction of the lungs triggered by the release of these inflammatory markers often induces acute respiratory distress syndrome (ARDS). ARDS is an emergency condition with a high mortality rate in COVID-19 patients. Dexamethasone is the first repurposed corticosteroid with life-saving efficacy in patients with severe SARS-CoV-2 infection. Dexamethasone has traditionally been known to suppress the production of inflammatory markers at the transcriptional level, but its role as a direct therapeutic to neutralize cytokines, chemokines, their receptors, and functionally critical SARS-CoV-2 proteins has not yet been explored. Herein, we demonstrated that dexamethasone binds with high affinity to interlukin-1 (IL-1), IL-6, IL-8, IL-12, IL-21, INF2, TGFβ-1, INF-γ, CXCL8, some of the receptors, IL-1R, IL-21R, IFNGR, INFAR, IL-6αR-gp130, ST2 and the SARS-CoV-2 protein NSP macro X, and 3CLpro, forming stable drug–protein complexes. Our work implied that dexamethasone has the potential to directly neutralize inflammatory markers, further supporting its life-saving potential in patients with severe manifestations of COVID-19

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background: Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results: An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 103 U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63–75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine–HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion: Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease

Discovery of a potent cyclooxygenase-2 inhibitor, S4, through docking-based pharmacophore screening, in vivo and in vitro estimations

Cyclooxygenase (COX; EC: 1.14.99.1), the key enzyme in prostaglandin production in the human body, is a major pharmacological target for developing anti-inflammatory agents. Nonsteroidal anti-inflammatory drugs exhibit anti-inflammatory and analgesic activities when inhibiting COX-2 but cause gastrointestinal toxicity and other side effects because of concurrent inhibition of COX-1. Thus, potent and safe inhibitors against COX-2 are urgently required. We constructed a novel docking-based pharmacophore model for screening selective COX-2 inhibitors and discovered compounds S1, S2, S3, and S4, which apparently inhibit COX-2. Particularly, S4 inhibits COX-2 in vitro and shows a potent anti-inflammatory effect in vivo without cytotoxicity. Molecular docking analyses revealed that S4 interacted satisfactorily with the active site of COX-2 but not with that of COX-1. This reveals that S4 more specifically inhibits COX-2 and has potential for application in developing anti-inflammatory and anticancer agents

Design of Novel FLT-3 Inhibitors Based on Dual-Layer 3D-QSAR Model and Fragment-Based Compounds <i>in Silico</i>

FMS-like tyrosine kinase 3 (FLT-3) is strongly correlated with acute myeloid leukemia, but no FLT-3–inhibitor cocomplex structure is available to assist the design of therapeutic inhibitors. Hence, we propose a dual-layer 3D-QSAR model for FLT-3 that integrates the pharmacophore, CoMFA, and CoMSIA. We then coupled the model with the fragment-based design strategy to identify novel FLT-3 inhibitors. In the first layer, the previously established model, Hypo02, was evaluated in terms of its correlation coefficient (<i>r</i>), RMS, cost difference, and configuration cost, with values of 0.930, 1.24, 106.45, and 16.44, respectively. Moreover, Fischer’s cross-validation test of data generated by Hypo02 yielded a 98% confidence level, and the validation of the testing set yielded a best <i>r</i> value of 0.87. The features of Hypo02 were separated into two parts and then used to screen the MiniMaybridge fragment compound database. Nine novel FLT-3 inhibitors were generated in this layer. In the second layer, Hypo02 was subjected to an alignment rule to generate CoMFA- and CoMSIA-based models, for which the partial least-squares validation method was utilized. The values of <i>q</i>², <i>r</i>², and predictive <i>r</i>² were 0.58, 0.98, and 0.76, respectively, derived from the CoMFA model with steric and electrostatic fields. The CoMSIA model with five different fields yielded values of 0.54, 0.97, and 0.76 for <i>q</i>², <i>r</i>², and predictive <i>r</i>², respectively. The CoMFA and CoMSIA models were used to constrain 3D structures of the nine novel FLT-3 inhibitors. This dual-layer 3D-QSAR model constitutes a valuable tool to easily and quickly screen and optimize novel potential FLT-3 inhibitors for the treatment of acute myeloid leukemia

The clinical characteristics and therapeutic outcomes of cryptococcal meningitis in elderly patients: a hospital-based study

Abstract Background The elderly, and especially those with an immuno-compromised status, are vulnerable to infectious diseases. The purpose of this study was to examine the clinical characteristics and therapeutic outcomes of cryptococcal meningitis (CM) in elderly patients in Taiwan. Methods Ninety-nine adult patients with CM were identified during a 15-year study period (2002–2016), of whom 38 elderly (≥ 65 years) patients (16 men and 22 women, median age 72.9 years; range 65–86 years) were included for analysis. The clinical characteristics and therapeutic outcomes of these patients were analyzed and compared to non-elderly adult patients (< 65 years) with CM. Results Among the 38 patients, diabetes mellitus was the most common underlying condition (15), followed by adrenal insufficiency (7), malignancy (6), hematologic disorders (5), chronic obstructive pulmonary disease (5), autoimmune diseases (3), liver cirrhosis (3) and acquired immunodeficiency syndrome (1). Altered consciousness (29), fever (21) and headache (17) were the leading clinical manifestations. Positive cerebrospinal fluid and blood cultures for Cryptococcus (C.) neoformans were found in 26 and 9 patients, respectively. There were significant differences in gender, altered consciousness and recent cerebral infarction between the elderly and non-elderly groups. The elderly group had a high mortality rate (36.8%, 14/38), and the presence of cryptococcemia was the most significant prognostic factor. Conclusions This study offers a preliminary view of the clinical characteristics of CM in the elderly. The results suggest that elderly patients (≥ 65 years) are more vulnerable to CM than adults aged < 65 years. Compared to the non-elderly group, the elderly group had female predominance, higher rates of altered consciousness and recent cerebral infarction as the clinical presentation. The presence of cryptococcemia was a significant prognostic factor in the elderly group. This study is limited by the small number of patients, and further large-scale studies are needed to better delineate this specific infectious syndrome

Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

International audienceA neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity

Serendipitous Discovery of Short Peptides from Natural Products as Tyrosinase Inhibitors

Tyrosinase, which is the crucial copper-containing enzyme involved in melanin synthesis, is strongly associated with hyperpigmentation disorders, cancer, and neurodegenerative disease; thus, it has attracted considerable interest in the fields of medicine and cosmetics. The known tyrosinase inhibitors show numerous adverse side effects, and there is a lack of safety regulations governing their use. As a result, there is a need to develop novel inhibitors with no toxicity and long-term stability. In this study, we use molecular docking and pharmacophore modeling to construct a reasonable and reliable pharmacophore model, called Hypo 1, that could be used for identifying potent natural products with crucial complementary functional groups for mushroom tyrosinase inhibition. It was observed that, out of 47 263 natural compounds, <b>A5</b> structurally resembles a dipeptide (WY) and natural compound <b>B16</b> is the equivalent of a tripeptide (KFY), revealing that the C-terminus tyrosine residues play a key role in tyrosinase inhibition. Tripeptides RCY and CRY, which show high tyrosinase inhibitory potency, revealed a positional and functional preference for the cysteine residue at the N-terminus of the tripeptides, essentially determining the capacity of tyrosinase inhibition. CRY and RCY used the thiol group of cysteine residues to coordinate with the Cu ions in the active site of tyrosinase and showed reduced tyrosinase activity. We discovered the novel tripeptide CRY that shows the most striking inhibitory potency against mushroom tyrosinase (IC₅₀ = 6.16 μM); this tripeptide is more potent than the known oligopeptides and comparable with kojic acid-tripeptides. Our study provides an insight into the structural and functional roles of key amino acids of tripeptides derived from the natural compound <b>B16</b>, and the results are expected to be useful for the development of tyrosinase inhibitors