Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses.

The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.

Innovative method of alopecia treatment by autologous adipose-derived SVF.

BACKGROUND: Alopecia refers to a condition developed by gradual reduction of hair loss by various abnormal causes such as endocrine system, genetic factors, and stress. Stromal vascular fraction (SVF) isolated from the fat is one of the latest innovative solutions in the field of regeneration therapy. We focused on presenting effectiveness of clinical cases to improve AGA through transplantation of autologous SVF into the scalp. OBJECTIVE: To confirm the efficacy of the autologous SVF usage to the patients with AGA. METHODS: Nine patients (age range 43-64 years; 4 men, grade IV to V and 5 women, grade I to III), who are suffering from androgenic alopecia (AGA), were treated with single transplantation of autologous SVF in the upper scalp. Autologous SVF was isolated and characterized prior to the injection of live 7-9 × 106 cells into the patients' treatment site. The hair loss improvement effect was assessed by three test criteria: hair skin quality, hair thickness and hair density 3 and 6 months after post-injection compared to pre-injection status. RESULTS: Hair density of SVF-treated side was significantly increased after 3 and 6 months of transplantation compared to non-treated side (P = 0.01 and P = 0.009 per each). And significant improvement in the score of the keratin on the scalp was seen in the injected area as compared to the non-injected area 6 months after transplantation (P = 0.032). Although thickness increase was observed at 3 and 6 months after transplantation, there was no statistical significance (P = 0.142 and 0.155, respectively). CONCLUSIONS: One transplantation of autologous SVF for the AGA patients, hair density and score for the keratin were significantly increased within 6 months. This study shows that SVF is a very effective way to treat hair loss and most of subjects are satisfied with the result after treatment.

Absolute Configuration and Corrected NMR Assignment of 17-Hydroxycyclooctatin, a Fused 5-8-5 Tricyclic Diterpene.

The absolute configuration and corrected NMR assignment of 17-hydroxycyclooctatin isolated from Streptomyces sp. M56 recovered from a nest of South African Macrotermes natalensis termites are reported. 17-Hydroxycyclooctatin is a unique tricyclic diterpene (C20) consisting of a fused 5-8-5 ring system, and in this study, its structure was unambiguously determined by a combination of HR-ESIMS and 1D and 2D NMR spectroscopic experiments to produce corrected NMR assignments. The absolute configuration of 17-hydroxycyclooctatin is reported for the first time in the current study using chemical reactions and quantum chemical ECD calculations. The corrected NMR assignments were verified using a gauge-including atomic orbital NMR chemical shifts calculation, followed by DP4 probability. To understand the pharmacological properties of 17-hydroxycyclooctatin, a network pharmacological approach and molecular docking analyses were used, which also predicted its effects on human breast cancer cell lines. Cytotoxicity and antiestrogenic activity of 17-hydroxycyclooctatin were determined, and it was found this compound may be an ERα antagonist.

Lobatamunsolides A-C, Norlignans from the Roots of Pueraria lobata and their Nitric Oxide Inhibitory Activities in Macrophages.

Phytochemical investigation of the methanol (MeOH) extract of Pueraria lobata roots, known as "kudzu", combined with liquid chromatography/mass spectrometry (LC/MS)-based analysis, resulted in the identification of four norlignans (1-4), including three new norlignans, lobatamunsolides A-C (1-3), and five known isoflavonoids (5-9). The structures of the new compounds were elucidated by a combination of spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and high resolution (HR)-electrospray ionization mass spectrometry (ESIMS), and their absolute configurations were determined by chemical reaction and quantum chemical electronic circular dichroism (ECD) calculations. The isolated compounds (1-9) were evaluated for their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Compound 9 displayed the strongest NO inhibitory effect and compound 2 showed a weak effect. The potential mechanism of the effect of compound 9 was investigated by analysis of its molecular docking on the active site of inducible nitric oxide synthase (iNOS), which showed the potential interactions of compound 9 with key amino acid residues and the heme cofactor of iNOS. The mechanism as the inhibition of transcriptional iNOS protein expression was confirmed by western blotting experiments.

Bioactive compounds from the seeds of Amomum tsaoko Crevost et Lemaire, a Chinese spice as inhibitors of sphingosine kinases, SPHK1/2.

Amomum tsaoko Crevost et Lemaire (Zingiberaceae), a traditional Chinese spice also known as "Caoguo" or "tsao-ko," has been considered to have many health benefits. As part of our continuous efforts to screen natural resources exhibiting potential bioactivity, we examined the seeds of A. tsaoko and found that its EtOH extract inhibited sphingosine kinases 1 and 2 (SPHK1/2). Bioactivity-based analysis and chemical investigation of the EtOH extract led to the isolation and identification of four aliphatic alcohols (1-4), five fatty acids (5-9), 12 phenolics (10-21), and four terpenoids (22-25), including four new compounds, an acetylated aliphatic alcohol (2), a fatty acid (5), and two phenolics (10-11). In addition, compound 1 was isolated for the first time from natural sources in this study. The structures of all compounds were elucidated based on spectroscopic analysis, including 1D and/or 2D NMR and HR-ESIMS as well as LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for the assignment of absolute configuration of compound 5. The absolute configurations of compounds 10 and 11 were determined using ECD calculations. All of the compounds (1-25) isolated from the active fraction were evaluated for their SPHK1/2 inhibitory effects at the concentration of 10 µM. Aliphatic alcohols 2-4, fatty acids 7 and 9, and phenolic compounds 13-15 and 21 showed inhibition against the activity of SPHK1 up to 20% and aliphatic alcohols 2 and 4, fatty acid 8, and phenolic compounds 10, 11, 18, and 22 showed inhibition against the activity of SPHK2 up to 40% compared with the control. Compound 2 showed the highest potency to inhibit SPHK1 enzymatic activity, by 59.75%, and compound 22 showed the highest potency in inhibiting the activity of SPHK2, by 22.75%, in comparison with the control, where both exhibited higher inhibition compared to those of positive controls. Docking modeling studies were conducted to suggest the binding mode of 2 and 22 in the substrate-binding pocket of SPHK1 and SPHK2, respectively.

Isoamericanoic Acid B from Acer tegmentosum as a Potential Phytoestrogen.

Phytoestrogens derived from plants have attracted the attention of the general public and the medical community due to their potentially beneficial role in relieving menopausal symptoms. The deciduous tree Acer tegmentosum Maxim (Aceraceae) has long been utilized in Korean folk medicine to alleviate many physiological disorders, including abscesses, surgical bleeding, and liver diseases. In order to explore structurally and/or biologically new constituents from Korean medicinal plants, a comprehensive phytochemical study was carried out on the bark of A. tegmentosum. One new phenolic compound with a 1,4-benzodioxane scaffold, isoamericanoic acid B (1), as well as with nine known phenolic compounds (2⁻10), were successfully isolated from the aqueous extracts of the bark of A. tegmentosum. A detailed analysis using 1D and 2D NMR spectroscopy, electronic circular dichroism (ECD) spectral data, and LC/MS afforded the unambiguous structural determination of all isolated compounds, including the new compound 1. In addition, compounds 2, 4, 5, and 9 were isolated and identified from the bark of A. tegmentosum for the first time. All isolated compounds were tested for their estrogenic activities using an MCF-7 BUS cell proliferation assay, which revealed that compounds 1, 2, and 10 showed moderate estrogenic activity. To study the mechanism of this estrogenic effect, a docking simulation of compound 1, which showed the best estrogenic activity, was conducted with estrogen receptor (ER) -α and ER-ß, which revealed that it interacts with the key residues of ER-α and ER-ß. In addition, compound 1 had slightly higher affinity for ER-ß than ER-α in the calculated Gibbs free energy for 1:ER-α and 1:ER-ß. Thus, the present experimental evidence demonstrated that active compound 1 from A. tegmentosum could be a promising phytoestrogen for the development of natural estrogen supplements.

Bioactivity-Guided Isolation of Anti-Inflammatory Constituents of the Rare Mushroom Calvatia nipponica in LPS-Stimulated RAW264.7 Macrophages.

Calvatia species, generally known as puffball mushrooms, are used both as sources of food and as traditional medicine. Among the Calvatia genus, Calvatia nipponica (Agaricaceae) is one of the rarest species. Using bioassay-guided fractionation based on anti-inflammatory effects, five alkaloids (1 - 5), two phenolics (6 and 7), and a fatty acid methyl ester (8) were isolated from the fruiting bodies of C. nipponica. Compound 8 was identified from C. nipponica for the first time, and all isolates (1 - 8) were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compound 7 showed mild inhibition while compound 8 significantly inhibited NO production with an IC50 value of 27.50 ± 0.08 µm. The mechanism of NO inhibition of compound 7 was simulated by molecular docking analysis against nitric oxide synthase (iNOS), which revealed the interactions of 7 with the key amino acid residue and the heme in the active site. With the most potent inhibition against LPS-induced inflammation, compound 8 was further investigated with respect to its mechanism of action, and the activity was found to be mediated through the inhibition of iNOS and COX-2 expression.