key: cord-0879947-xabkzjrp authors: Hou, Bo; Zhang, Yu-Min; Liao, Han-Yi; Fu, Li-Feng; Li, De-Dong; Zhao, Xin; Qi, Jian-Xun; Yang, Wei; Xiao, Geng-Fu; Yang, Lian; Zuo, Zheng-Yu; Wang, Lin; Zhang, Xiang-Lei; Bai, Fang; Yang, Liu; Gao, George F.; Song, Hao; Hu, Jiang-Miao; Shang, Wei-Juan; Zhou, Jun title: Target-Based Virtual Screening and LC/MS-Guided Isolation Procedure for Identifying Phloroglucinol-Terpenoid Inhibitors of SARS-CoV-2 date: 2022-01-27 journal: J Nat Prod DOI: 10.1021/acs.jnatprod.1c00805 sha: 1e05e91d50781e0ecf799a43a4fddcfaa0050588 doc_id: 879947 cord_uid: xabkzjrp [Image: see text] The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 5 million deaths worldwide to date. Due to the limited therapeutic options so far available, target-based virtual screening with LC/MS support was applied to identify the novel and high-content compounds 1–4 with inhibitory effects on SARS-CoV-2 in Vero E6 cells from the plant Dryopteris wallichiana. These compounds were also evaluated against SARS-CoV-2 in Calu-3 cells and showed unambiguous inhibitory activity. The inhibition assay of targets showed that compounds 3 and 4 mainly inhibited SARS-CoV-2 3CLpro, with effective K(d) values. Through docking and molecular dynamics modeling, the binding site is described, providing a comprehensive understanding of 3CLpro and interactions for 3, including hydrogen bonds, hydrophobic bonds, and the spatial occupation of the B ring. Compounds 3 and 4 represent new, potential lead compounds for the development of anti-SARS-CoV-2 drugs. This study has led to the development of a target-based virtual screening method for exploring the potency of natural products and for identifying natural bioactive compounds for possible COVID-19 treatment. Target-Based Virtual Screening Strategy A subset of natural compounds in Table S1 was used for the virtual screen. The chemical shifts OH-2 (16.49 ppm) and OH-6 (11.28 ppm) of the hydroxy protons of the butyrylphloroglucinol-type ring in the 1 H NMR spectrum of 3 indicated that the cyclization skeleton was related to the phenolic hydroxy moiety at C-4 instead of C-6. Furthermore, signals of the cyclization fragment were observed from the HMBC correlations of the OH-2 and OH-6 groups with C-1 ( C 106.1 ppm). The resonance at  H 3.52 (s) and C-7 at  H 17.2 suggested a methylene moiety situated between hexadioldienone (ring A) and butyrylphloroglucinol-type unit (ring B). Thus, the chemical structure of 3 was defined as shown in Figure 2B . Conformational search was initially performed using Discovery Studio 4.0 Client. The molecular conformations were fully optimized without imposing any symmetry constraints. The geometries and energies of the stationary points on the potential energy surface were calculated using the B3LYP method in conjunction with the 6-31G(d). The Boltzmann population of conformers was calculated using the free energy values. Then the selected stable conformers (> 5% distribution rate at 298.15K) were used for TD-DFT computation of the 50 excited stats at the same levels with methanol using PCM model and the gas phase. The calculated ECD spectra were obtained by weighing the Boltzmann distribution rate of each geometric conformation. The ECD spectra were produced by SpecDis 1.6 software and compared with the experimental values. 29, 30 The molecular orbital information was generated by Gaussion09. 31 The lowest-energy conformers were selected for key NTOs analysis. The fch file was used to generate corresponding cube file by Multiwfn 3.1. 32 Lastly, the isosurface of NTOs were created by VMD software. 33 Mass-Spectral Studies on Some Naturally Occurring Phloroglucinol Derivatives .3. Mass-Spectra Of Some Monocyclic and Bicyclic Phloroglucinol Derivatives from Rhizomes of Different Dryopteris Species Acylphloroglucinols and flavonoid aglycones produced by external glands on the leaves of two dryopteris ferns and currania robertiana Phloroglucinol Derivatives of Dryopteris abbreviata Phloroglucinol derivatives in Dryopteris parallelogramma and D. patula Phenolic Constituents from the Rhizomes of Dryopteris crassirhizoma Phloroglucinol Derivatives of Dryopteris abbreviata Inhibitory effects of phloroglucinols from the roots of Dryopteris crassirhizoma on melanogenesis Separation of Naturally occurring acylphloroglucinols by high-performance liquid chromatography Phloraspyron and Phloraspidinol, New Phloroglucinol Derivatives from Dryopteris Ferns A phloroglucinol derivative of Dryopteris abbreviata The Phloroglucinols of Dryopteris aitoniana PICHI SERM. (Dryopteridaceae, Pteridophyta) Anti-Influenza Virus (H5N1) Activity Screening on the Phloroglucinols from Rhizomes of Dryopteris crassirhizoma. Molecules Phloroglucinol Derivatives of Dryopteris-Dickinsii and Some Related Ferns Ichthyotoxic Phloroglucinol Derivatives from Dryopteris fragrans and Their Anti-tumor Promoting Activity A new acylphloroglucinol of Dryopteris gymnosora Piscicidal Components from Dryopteris Fragrans Two new acyl-phloroglucinols from Dryopteris atrata Phloroglucinol derivatives of Hagenia abyssinica Unusual terpenylated acylphloroglucinols from Dryopteris wallichiana Die Phloroglucide von drei Dryopteris-Arten von den Azoren sowie zwei Arten von Madeira und den Kanarischen Inseln zum Vergleich Phloroglucinol derivatives in Dryopteris sect. Fibrillosae and related taxa (Pteridophyta, Dryopteridaceae) New 3-Ring Phloroglucinol Derivatives from Dryopteris Austriaca Structure-Molluscicidal Activity Relationships of Acylphloroglucinols from Ferns Phloroglucinol Derivatives of Dryopteris-Crassirhizoma From Japan Study on the Chemical Components of Dryopteris Crassirhizoma (Ⅰ) Dryocrassin: A new acylphloroglucinol from Dryopteris crassirhizoma The Phloroglucinols of Dryopteris aitoniana PICHI SERM. (Dryopteridaceae, Pteridophyta) ECD cotton effect approximated by the Gaussian curve and other methods SpecDis: Quantifying the Comparison of Calculated and Experimental Electronic Circular Dichroism Spectra