Structure-based design of pan-coronavirus inhibitors targeting host cathepsin L and calpain-1.

Respiratory disease caused by coronavirus infection remains a global health crisis. Although several SARS-CoV-2-specific vaccines and direct-acting antivirals are available, their efficacy on emerging coronaviruses in the future, including SARS-CoV-2 variants, might be compromised. Host-targeting antivirals provide preventive and therapeutic strategies to overcome resistance and manage future outbreak of emerging coronaviruses. Cathepsin L (CTSL) and calpain-1 (CAPN1) are host cysteine proteases which play crucial roles in coronaviral entrance into cells and infection-related immune response. Here, two peptidomimetic α-ketoamide compounds, 14a and 14b, were identified as potent dual target inhibitors against CTSL and CAPN1. The X-ray crystal structures of human CTSL and CAPN1 in complex with 14a and 14b revealed the covalent binding of α-ketoamide groups of 14a and 14b to C25 of CTSL and C115 of CAPN1. Both showed potent and broad-spectrum anticoronaviral activities in vitro, and it is worth noting that they exhibited low nanomolar potency against SARS-CoV-2 and its variants of concern (VOCs) with EC50 values ranging from 0.80 to 161.7 nM in various cells. Preliminary mechanistic exploration indicated that they exhibited anticoronaviral activity through blocking viral entrance. Moreover, 14a and 14b exhibited good oral pharmacokinetic properties in mice, rats and dogs, and favorable safety in mice. In addition, both 14a and 14b treatments demonstrated potent antiviral potency against SARS-CoV-2 XBB 1.16 variant infection in a K18-hACE2 transgenic mouse model. And 14b also showed effective antiviral activity against HCoV-OC43 infection in a mouse model with a final survival rate of 60%. Further evaluation showed that 14a and 14b exhibited excellent anti-inflammatory effects in Raw 264.7 mouse macrophages and in mice with acute pneumonia. Taken together, these results suggested that 14a and 14b are promising drug candidates, providing novel insight into developing pan-coronavirus inhibitors with antiviral and anti-inflammatory properties.

Immunosensor for Rapid and Sensitive Detection of Digoxin.

Digoxin is a cardiac glycosylated steroid-like drug with a positive inotropic effect and has been widely used in treating congestive heart failure, atrial fibrillation, atrial flutter, and other heart diseases. Digoxin is also a dangerous drug, which can cause drug poisoning at a low blood drug concentration (2.73-3.9 nmol/L, i.e., 2.14-3.05 ng/mL). Therefore, the timely detection of a patient's blood drug concentration plays a significant role in controlling blood drug concentration, reducing the occurrence of drug poisoning events, and maximizing the role of drug therapy. In this study, a DNA vector for the expression of the antidigoxin antibody Fab fragment was constructed. With the vector, Fab was expressed in E. coli and purified, and 1.2 mg of antibodies was obtained from 100 mL of culture. An immunofluorescent sensor based on the mechanism of photoinduced electron transfer was constructed by labeling additional cysteines in the heavy chain variable region and light chain variable region of the antibody Fab fragment with fluorescent dyes. The assay for digoxin with the immunosensor could be finished within 5 min with a limit of detection of 0.023 ng/mL, a detectable range of 0.023 ng/mL to 100 µg/mL, and an EC50 of 0.256 ng/mL. A new approach for the rapid detection of digoxin was developed and will contribulte to therapeutic drug monitoring.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Synthesis of maleimide-braced peptide macrocycles and their potential anti-SARS-CoV-2 mechanisms.

Macrocycles often exhibit good biological properties and potential druggability, which lead to versatile applications in the pharmaceutical industry. Herein, we report a highly efficient and practical methodology for the functionalization and macrocyclization of Trp and Trp-containing peptides via Pd(II)-catalyzed C-H alkenylation at the Trp C4 position. This method provides direct access to C4 maleimide-decorated Trp-containing peptidomimetics and maleimide-braced 17- to 30-membered peptide macrocycles. In particular, these unique macrocycles revealed low micro- to sub-micromolar EC50 values with promising anti-SARS-CoV-2 activities. Further explorations with computational methodologies and experimental validations indicated that these macrocycles exert antiviral effects through binding with the N protein of SARS-CoV-2.

Investigation of binding mechanism for human plasminogen Kringle 5 with its potential receptor vWA1 domain in Cochlin by bio-specific technologies and molecular dynamic simulation.

Given the significant clinical potential of human plasminogen Kringle 5 on tumours, it is crucial to seek its receptors for a thorough comprehension of its physiological functions and mechanism. Eleven candidates have been screened out in our previous works. In the present work, we further inquired whether the candidate, von Willebrand factor type A domain 1 in coagulation factor C homology protein (abbr. vWA1), was a potential receptor of Kringle 5, and investigated their binding mechanism by bio-specific experiments, frontal affinity analysis (FA), and molecular dynamic simulation (MDS). After the potential was validated by bio-specific experiments, the FA results stated that vWA1 exhibited a strong interaction towards Kringle 5 in the proportion of 1:1 with the binding constant of 4.18 × 104 L/mol. The MDS results showed that the binding was mainly driven by electrostatic and Van der Waals forces and occurred spontaneously, during which vWA1 and Kringle 5 mutually fit each other by conformational changing into more flexible and suitable structures including fluctuations for five loops and partial transformation into a random coil for α6-helix in vWA1. Moreover, lysine binding site Leu71-Tyr74 was speculated responsible for Kringle 5 in binding and Tyr72 to be the key amino acid residue. In short, this work not only confirmed vWA1 as a potential Kringle 5 receptor but also provided valuable information on the detailed binding, facilitating the application development of Kringle 5 in regulating immune or inhibiting tumour migration through vWA1.

Phytochemical and biological studies of plants from the genus Meconopsis.

In this review, the literature data on the phytochemical and biological investigations on the genus of Meconopsis are summarized from 49 references. Up to now, more than 95 compounds were isolated from 19 Meconopsis plant species. The chemical constituents are mostly alkaloids, flavonoids, phenols, steroids, and terpenes, together with minor constituents of essential oil, and others. The crude extracts and metabolites have been found to possess various bioactivities including antitumor activity, central action, cardiovascular system effects, antibiosis, antiviral activity, anti-inflammatory effects, and other biological activities.

The genus Scutellaria an ethnopharmacological and phytochemical review.

Scutellaria (HUANG QIN) (Lamiaceae), which includes about 350 species commonly known as skullcaps, is widespread in Europe, the United States and East Asia. Some species are taken to clear away the heat-evil and expel superficial evils in traditional Chinese medicine (TCM). The present paper reviews the ethnopharmacology, the biological activities and the correlated chemical compounds of Scutellaria species. More than 295 compounds have been isolated, among them flavonoids and diterpenes. Studies show that Scutellaria and its active principles possess wide pharmacological actions, such as antitumor, anti-angiogenesis, hepatoprotective, antioxidant, anticonvulsant, antibacterial and antiviral activities. Currently, effective monomeric compounds or active parts have been screened for pharmacological activity from Scutellaria in vivo and in vitro. Increasing data supports application and exploitation for new drug development.

[Study on the effect of Part III from Cynomorium songaricum on immunosuppressive mice induced by cyclophosphamide].

OBJECTIVE: To study the immunomodulatory effect of Part III of Cynomorium songaricum Rupr. on the immunosuppressive mice. METHODS: The immunity-deficiency model was induced by intraperitoneal injection of cyclophosphamide (CTX) at the dose of 100 mg/kg in mice; all the animals were divided into normal control group, immunity-deficiency model group, Part III treated group (300 mg/kg) and positive control group (TSPG, 300 mg/kg). The hemogram of peripheral blood, the index of immune organs, the phagocytosis activity of macrophage, the content of serum hemolysin were measured. RESULTS: The index of organs, the phagocytosis activity of macrophage and the content of serum hemolysin in the model group increased after administrated of Part III. CONCLUSION: Part III from Cynomorium songaricum Rupr. has protective effect on the immunosuppressive mice, which may be related to the increasing of humoral immunity and nonspecific immunity.

[Establishing and identifying insulin resistance 3T3-L1 adipocytes cell model].

OBJECTIVE: To establish insulin-resistant 3T3-L1 adipocytes cell model. METHODS: 3T3-L1 preadipocytes were cultured and induced to differentation. We established insulin resistant 3T3-L1 induced by dexamethasone and determined the change of glucose concentration in cell culture. RESULTS: In high glucose DMEM culture media, the glusose concentration significantly decreased. Achieved the peak of insulin-resisitant at the 96th hour induced by dexamethasone, and at this time compared with the blank the mRNA expression of Resistin of the model step up about three times. CONCLUSION: 3T3-L1 adipocyte exposed to 1 micromol/L dexamethasone for 24 hours are able to induce a state of insulin resistance which can be maintained for 216 hours.

[Effects of Rehmannia glutinosa oligosaccharides on proliferation of HepG2 and insulin resistance].

OBJECTIVE: To investigate the effects of Rehmannia glutinosa oligosaccharides (ROS) on the proliferation of HepG2 and insulin resistance. METHOD: The HepG2 cells were divided into control group, rosiglitazone (3.4 mg x L(-1)) treated group and ROS (0.1-30 mg x L(-1)) treated group. The proliferation of HepG2 was detected by MTT method. Insulin resistant HepG2 cells model was induced by high concentration of insulin, then the effects of ROS on glucose consumption in insulin resistant HepG2 cells were investigated. RESULT: In the middle glucose culture medium, the absorbance at 570 nm of HepG2 was increased by high concentration of ROS, and decreased by low concentration of ROS by using MTT method, a concentration-dependent manner. ROS increased glucose consumption in HepG2 cells, and showed a better effect at the dose of 10 mg x L(-1). ROS promoted the glucose consumption in insulin resistance of HepG2 cells, improved the sensitivity of insulin resistance of HepG2 cells to insulin. CONCLUSION: High concentration of ROS can promote the proliferation of HepG2, and however low concentration of ROS inhibits the proliferation of HepG2. ROS can significantly improve insulin resistance of HepG2 cells induced by high insulin.

[The antitumor activities of the extracts from Stellera chamaejasmel L. in vitro by means of systematic solvent extraction].

OBJECTIVE: To screen the antitumor extract from Stellera chamaejasmel L.. METHODS: The means of systematic solvent extraction was used to separate the 95% ethanol extract. The antitumor activities of the different extracts in vitro were analyzed with MTT assay. RESULTS: The petroleum ether extract and the chloroform extract strongly inhibited cell proliferation of human cancer cell lines such as leukemia HL-60, stomach cancer SGC-7901 and hepatocarcinoma BEL-7402, and showed significant dose-dependent response while the n-butanol extract and the methanol extract exhibited low antitumor activities in vitro. The petroleum ether extract was most active. CONCLUSION: The petroleum ether extract is the principal antitumor extract from Stellera chamaejasmel L..