New Sorbicillinoids from the Mangrove Endophytic Fungus Trichoderma reesei SCNU-F0042

Three new dimeric sorbicillinoids (1–3) and one new 3,4,6-trisubstituted α-pyrone (5), along with seven analogues (4 and 6–11), were isolated from the mangrove endophytic fungus Trichoderma reesei SCNU-F0042 under the guidance of molecular networking approach. Their chemical structures were established by 1D and 2D NMR HR-ESI-MS and ECD analysis. In a bioassay, compound 2 exhibited moderate SARS-CoV-2 inhibitory activity with an EC50 value of 29.0 μM.

Molecular networking, a strategy that organizes and analyses MS/MS data based on chemical similarity, can be used for dereplication in natural products discovery [17].After extraction and concentration, the EtOAc extract was subjected to LC-MS/MS analysis, then the data were uploaded to the Global Natural Product Social Molecular Networking (GNPS; www.gnps.ucsd.edu)platform, followed by MN analysis using the online workflow.A comprehensive examination of the MS 2 spectra libraries allowed the annotation of the node at m/z 497.217 (C 28 H 33 O 8 , [M + H] + ) as the bislongiquinolide (4) based on its fragmentation pattern [18].By the guidance of bislongiquinolide node, more new bioactive sorbicillinoids remain to be discovered in the metabolites from the fungus Trichoderma reesei SCNU-F0042 (Figure 1 and Figure S28).Eventually, under the guidance of molecular networking, three new dimeric sorbicillinoids (1-3) and one new 3,4,6-trisubstituted α-pyrone (5), as well as seven analogues (4 and 6-11) were isolated from the mangrove-derived fungus Trichoderma reesei SCNU-F0042 (Figure 2).Details of the isolation, structure elucidation, and bioactivities of these compounds are reported herein.

Fungal Material
The fungal strain Trichoderma reesei SCNU-F0042 was isolated from the fresh bark of the mangrove plant Bruguiera gymnorhiza collected from Qi'ao Island Mangrove Nature Reserve, Zhuhai City, Guangdong Province, China.The fungus was obtained using the standard protocol for isolation.The sequence data of the fungal strain have been deposited at Gen Bank with accession no.OP978317.A BLAST search result showed that the sequence was the most similar (99%) to the sequence of Trichoderma reesei (compared to OK445677.1).
A voucher strain was deposited in School of Chemistry, South China Normal University, Guangzhou, China, with the access code Trichoderma reesei SCNU-F0042.

General Experimental Procedures
Spores of the fungal strain were inoculated into solid autoclaved rice medium in 400 1L Erlenmeyer flasks, each of which contained 50 g rice and 50 mL 0.3% sea salt, culturing in room temperature under static condition for 30 days.The mycelia and solid rice medium were soaked with MeOH and extracted with EtOAc three times.The organic solvents were evaporated under 48 • C with reduced pressure and obtained 158.6 g of organic crude extract.The extract was isolated by column chromatography over silica gel eluting with a gradient of PE/EA (1:0−0:1) to yield 5 fractions (Frs.1−5).Fr. 3

LC-MS/MS and Molecular Networking Analysis
The EtOAc extract of Trichoderma reesei SCNU-F0042 was analyzed by LC−MS/MS.In positive-ionization conditions (m/z 200−800), the mobile phase consisted of 1‰ HCOOH formic acid in H 2 O and CH 3 CN.The elution gradient conditions for the LC mobile phase were as follows, based on times (t): t = 0−1 min, hold at 90% H The molecular networking was performed using the GNPS data analysis workflow and the spectral clustering algorithm.Parameters for molecular network generation were set as follows: precursor mass tolerance m/z 2.0 Da, fragment ion tolerance m/z 0.5 Da, cosine score above 0.7, minimum matched fragment ions 6, minimum cluster size 2, network TopK10.Data were visualized using Cytoscape 3.8.2software.

Plasmids
ACE2 packaging construct (GeneCopoeia, EX-U1285-Lv105) uses a cytomegalovirus (CMV) promoter to express ACE2 and bears a puromycin selection marker in the integrating cassette.

RT-qPCR Analysis
HEK 293T-hACE2 cells were seeded in 12-well flat-bottom plate at a density of 1.2 × 10 4 cells/well.After 24 h, cells were incubated in media consisting of BA.2 (MOI of 0.01) and different concentrations of each compound for 1 h at 37 • C.After the incubation, cells were washed with sterile phosphate-buffered saline (PBS) once and incubated with media mixed with different concentrations of each compound, respectively, for further 48 h.Total RNA of each well was extracted from the cell culture supernatant using LogPure Viral DNA/RNA Kit (Magen, Guangzhou, China).Reverse transcription and qPCR were performed with Detection Kit for Novel Coronavirus (2019-nCoV) RNA (DA0932, DAAN GENE, Guangzhou, China).Samples were read on the QuantStudio7 Flex real-time PCR detection system (Thermo Fisher Scientific, Shanghai, China).The qPCR was performed in duplicates for each sample, and results were calculated using 2 -∆CT , where CT is threshold cycle [29].

Cell Viability Assay
The test compounds at a serial final concentration of 50 to 1 µM were evaluated against HEK 293T-hACE2 using the MTT method.Tested cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 mg/mL streptomycin, and 100 units/mL penicillin (Invitrogen).The cultures were maintained at 37 • C in a humidified atmosphere of 5% CO 2 .

Table 3 .a
Results of anti-SARS-CoV-2 activity and cytotoxicity for 293T cell.Positive control; b Data are shown as mean from three parallel experiments; c NS means not sensitive at 80 µM.