The regulatory role of compound 4 against SRD5A1 involved both suppression of SRD5A1 expression and mixed mode SRD5A1 inhibition. The Ki value of compound 4 was 2.382 µM based on the whole-cell kinetic studies under specific conditions. Molecular docking and molecular dynamics simulations with AlphaFold generated the human SRD5A1 structure and confirmed the stability of compound 4 at the SRD5A1 catalytic site with greater interactions, including hydrogen bonding of the key M119 amino-acid residue than those of finasteride and dutasteride. Thus, compound 4 shows the potential for further development as an SRD5A1 suppressor for androgenic alopecia treatment.Caffeic acid derivatives containing amide moieties similar to those of finasteride and dutasteride were synthesized. An in vitro inhibitory activity evaluation of caffeic acid ( The isozymes are produced in various human tissues and have diverse functional roles in human health and diseases. Steroid 5α-reductase type 1 (SRD5A1) is mostly found in hair follicles, scalp, sebaceous glands, non-genital skin, and liver; type 2 (SRD5A2) is abundant in genital tissues and scalp2; and type 3 (SRD5A3) is detected in benign and malignant tissues3. Both SRD5A1 and SRD5A2 exhibit amino-acid sequence similarity ranging from 40 to 60%4. They significantly control the conversion of testosterone to dihydrotestosterone (DHT), a more potent androgen. Overexpression of the steroid 5α-reductase leads to DHT overproduction, causing androgen-dependent diseases, including benign prostatic hyperplasia, hirsutism, androgenic alopecia, and prostate cancer1. Both male and female patients with androgenic alopecia have high levels of SRD5A1 and SRD5A2 in frontal and occipital hair follicles. SRD5A1 was found as the major isozyme in scalp-hair follicles, in which SRD5A1 is approximately five times higher than SRD5A25. Therefore, SRD5A1 serves as a crucial target for the treatment of androgenic alopecia by using the orally administered drugs, finasteride and dutasteride10. However, basic research and clinical studies reported several adverse effects of the steroidal inhibitors, finasteride and dutasteride, including impotence, abnormal ejaculation, abnormal sexual function, gynecomastia, and severe myopathy6. Therefore, there is a need to identify other novel steroid 5α-reductase inhibitors. Steroidal and non-steroidal inhibitors have been continuously reported in the past few decades16. Notably, a group of non-steroidal inhibitors are highly selective and exhibit lower interactions with other enzymes and receptors than steroid 5α-reductases in the steroidal endocrine system.Steroid 5α-reductases, also known as 3-oxo-5α-steroid 4-dehydrogenases, are microsomal and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane-bound enzymes in the oxidoreductase family; they are involved in steroid metabolism. Currently, these enzymes are classified into types 1, 2, and 3Avicennia marina17; osthenol, a coumarin from Angelica koreana18; physcion, a dihydroxyanthraquinone from Polygonum multiflorum19; and piperine, an alkaloid from Piper nigrum20. Moreover, polyphenols, such as curcumin from Curcuma longa21 and theaflavin-3,3′-digallate from Camellia sinensis (green tea)22, and triterpenoids, such as soyasaponin I and kaikasaponin III from Puerariae thomsonii23, exhibit moderate steroid 5α-reductase inhibition.Some natural compounds isolated from medicinal plants are promising inhibitors of steroid 5α-reductase with few side effects. These compounds include avicequinone C, a furanonaphthoquinone from 25 or human keratinocytes (HaCaTs)17 using non-radioactive and direct DHT detection by high-performance thin-layer chromatography (HPTLC) was developed. A HaCaT cell-based assay showed that avicequinone C, a natural furanonaphthoquinone isolated from Avicennia marina, was discovered as a promising SRD5A1 inhibitor with a half-maximal inhibitory concentration (IC50) of 4.45 ± 0.42 µM17.Building on previous studies, a method to determine a compound’s inhibitory effect on SRD5A1 using a cell-based assay with human-hair dermal papilla cells (HHDPCs)30. Moreover, the three-dimensional (3D) pharmacophore model of caffeic acid amides displays unique hydrophobic- and hydrophilic-binding interactions regarding the 3,4-dihydroxycinnamoyl scaffold against nitric oxide (NO) targets31. Recently, caffeic acid is reported as an active substance in ethanol-extracted propolis that significantly stimulated keratinocyte proliferation and hair growth in mice via anagen induction and hair elongation32. Thus, the 3,4-dihydroxycinnamic motif could be studied for its structure–activity relationship against SRD5A1 inhibition to identify candidates for treatment of androgenic alopecia.Caffeic acid, also known as 3,4-dihydroxycinnamic acid, belongs to a natural class of hydroxycinnamic acids found in several medicinal plants, fruits, and vegetables. Interestingly, caffeic acid and its derivatives exhibit many biological activities, especially antioxidant and anti-inflammatory activities33. The lack of a crystal structure of the human SRD5A1 adversely affects our ability to understand the mechanism of the enzyme’s activity and delays the discovery and development of effective inhibitors. Study of homologous protein structures of both SRD5A1 and SRD5A2 revealed a conserved catalytic site in steroid 5α-reductases catalyzing NADPH-mediated steroids reduction involving their signature Q, E, and Y residues34. The crystal structure of human SRD5A2 in complex with finasteride and NADP (PDB: 7BW1) led to the discovery of the ligand-binding catalytic pocket35, in which the amino-acid residues E57 and Y91 are crucial hydrogen-bonding sites for reducing testosterone to DHT. Moreover, residue R114 forms a hydrogen bond with the tert-butylacetamide tail group of finasteride35. The findings of an in-silico study support the importance of a specific amide moiety that could selectively bind to catalytic pocket of steroid 5α-reductase.To date, the crystal structure of human SRD5A1 is unknowntert-butyl, and trifluoromethyl substituted phenyl amide , and the SRD5A1 inhibitory activity assay with HaCaTs and non-radioactive HPTLC17. The underlying molecular mechanism was examined via mRNA and protein expression. The kinetic inhibitory profiles were also investigated by employing whole-cell-based studies. Furthermore, the molecular docking and molecular dynamics AlphaFold-generated SRD5A1 structure predicted the possible binding sites and stability of the potent caffeic acid amides. These findings could lead to the discovery of a non-steroidal inhibitor for SRD5A1 for the use as a tropical treatment for androgenic alopecia.In this study, a series of caffeic acid derivatives were structurally designed, focusing on the amide structures similar to those in finasteride and dutasteride, including phenyl, ide Fig. . Caffeictert-butyl, and N- substituents corresponding to compounds 2, 3, and 4, respectively, were designed based on their distinct chemical structures. The phenyl group is an inductively withdrawing group, the tert-butyl is a bulky electron-donating group, and the N- is a sterically electron-withdrawing group. Caffeic acid (1) was employed as the precursor to react with amine reagents in the presence of amide coupling reagents under suitable conditions to generate the designed caffeic acid amide derivatives, compounds 2, 3, and 4, with high yield (71%–77%)  analysis. Figure 1) and its amide analogs (2 − 4) were tested for their in vitro cytotoxicity to human keratinocytes (HaCaTs) and dermal papilla (DP) cells to identify their respective subtoxic concentrations. Caffeic acid (1) and its amide analogs containing phenyl (2) and tert-butyl (3) substituents exhibited no toxicity against HaCaT or DP cells at 0.5–50 µM phenyl]-containing analog (4) exhibited cytotoxicity against HaCaTs at IC50 29.99 ± 8.69 µM and no toxicity to DP cells at 0.5–50 µM.Caffeic acid phenyl]-containing amide (4), with a similar amide moiety to dutasteride, significantly inhibited the production of DHT by up to 46% at 1 µM, with high cell viability (88%). In contrast, caffeic acid (1) at 20 µM displayed a 30% inhibition of DHT production, with 86% cell viability, indicating a weaker inhibitory activity than compound 4. In addition, the phenyl-containing amide 2 and the tert-butyl-containing amide 3 showed essentially no inhibition of the DHT production phenyl] amide (compound 4) was chosen for further study. Serial dilutions within the range of sub-cytotoxic concentrations were prepared for determining the compound’s effective inhibitory concentration. Its concentrations of 0.2, 0.5, 1, and 2.5 µM displayed no significant cytotoxicity between control and treatment groups. Compound 4 exhibited potent cell-based inhibition of DHT production with IC50 at 1.44 ± 0.13 µM phenyl] amide (4) would be a promising non-steroidal modulator for SRD5A1, which led to the reduction of DHT production. Furthermore, compound 4 composes of three scaffolds, including a 3,4-dihydroxycinnamoly group, amide core, and N- amide derivative and the binding residues including the catalytic triad  and the surrounding residues within 3.5 Å  (dL-binding site); (3) # atom contacts within 3.5 Å sphere of ligand; and (4) # H-bonds formed between the compound and target protein. The last one was calculated using two different conditions. The distance and angle between the hydrogen bond donor (HD) and hydrogen acceptor (HA) of ≤ 2.8 Å and ≥ 150° were used to determine strong H-bonds, while those of ≤ 3.5 Å and ≥ 120° were applied for counting all possible H-bonds. The obtained results from the last 100 ns were selected for discussion.SRD5A1 bound with the potential compound 4 and its adduct were stably within the active site  as seen by the snapshots per time in Fig. 4  showed a similar pattern GBSA methods in Table 4 complex provided the binding free energy of − 36.58 ± 0.14 kcal/mol for MM/PBSA and − 15.02 ± 0.14 for MM/GBSA, which was relatively lower than that of the complex of free compound 4 . Although the electrostatic interaction was found to be the main force for complexation, particularly the adduct  the root-mean-square fluctuation (RMSF), (2) B-factor and (3) secondary structure analysis were investigated and presented in Fig. 4 could be stabilized within the pocket by interacting with R98, A120, F123, E202, F224, T225 and F228 residues. While NADP-dihydro-4 bind better to SRD5A1 with additional interactions to the 9 residues including V23, R40, K110, M112, L172, R176, N198, F224 and R232. Interestingly, the trifluoromethyl phenyl group of the amide side chain of NADP-dihydro-4 hydrophobically interact with the catalytic residue M119. The binding of the NADP-dihydro-4 to the V23, R40, A120, A120, F123, R176, N198, F224, F228 and R232 showed a similar result as the docking study. Besides, the hydroxyl group on the 3,4-dihydroxycinnamoyl moiety of compound 4 forms hydrogen bonds with E202 (100 ± 0% and 100 ± 0%) and W206 (69 ± 6%). Whereas hydrogen bond formation between NADP-dihydro-4 and SRD5A1 was increased in the (1) adenine ring with N198 (98%) and (2) phosphate group with R232 .Furthermore, the protein structural investigations of SRD5A1 in complex with compound 1H and13C nuclear magnetic resonance (NMR) spectra were obtained on a Bruker ADVANCE NEO 400 MHz NMR spectrometer, and deuterated methanol (CD3OD) served as the internal standard for both1H (3.31 and 4.78 ppm) and13C (49.15 ppm) spectra. Accurate mass spectra were obtained using an Agilent 6540 UHD Q-TOF LC/MS spectrometer. HPTLC was performed using a Merk silica gel 60 F254 glass HPTLC plate. All HPTLC mobile phases including cyclohexane , ethyl acetate  and triethylamine  were purchased. HPTLC instruments include a semi-automatic sampler Linomat 5, TLC Plate Heater, TLC Scanner 3, Visualizer 2 . Each chromatogram was captured and processed by HPTLC Software visionCATS 3.0 .Commercial caffeic acid (Sigma Life Science) was stored at 0 °C until use. All other commercial reagents were from TCI chemicals. Anhydrous solvents were dried over 4 Å molecular sieves. All reactions were performed in oven-dried glassware and magnetically stirred under an inert atmosphere at room temperature unless otherwise described. In addition, all reactions were monitored by thin-layered chromatography (TLC) using an aluminum silica gel 60 F254 (Merck). Flash column chromatography was carried out using silica gel as the stationary phase. Additionally, all solvents, such as methanol, ethyl acetate, dichloromethane, and hexane, were purified by distillation before use. Moreover, infrared (IR) spectra were measured on a Perkin Frontier Fourier Transform Infrared Spectrometer. Furthermore,l-glutamine (GlutaMAX), sodium pyruvate, trypsin-ethylene diamine tetra-acetic acid, phosphate-buffered saline, Dulbecco’s phosphate-buffered saline, and 3--2,5-diphenyl tetrazolium bromide (MTT)  were purchased. Dimethyl sulfoxide (DMSO) was procured from Merck Millipore .Human keratinocyte (HaCaT)  and DP cells  were obtained. In addition, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotic–antimycotic, 1) , 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide , and hydroxybenzotriazole  were weighed into an oven-dried round-bottomed flask and dissolved in dry tetrahydrofuran (5 mL). Then, an N2 gas balloon was used to create the inert atmosphere. The reaction mixture was mixed using a magnetic stir bar for 30 min at room temperature. Afterward, aniline  was added to the mixture, and the reaction was stirred at room temperature for 24 h and monitored by TLC using an ethyl acetate:hexane solution (7:3 v/v) mixture as the mobile phase. Then, the volatile solvent was removed under reduced pressure, and the residue was redissolved in ethyl acetate (20 mL). The resulting solution was transferred to a separatory funnel and washed with water to remove the water-soluble by-products. Next, the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to produce a crude product. The crude product was purified using flash column chromatography with silica gel as the stationary phase and the ethyl acetate:hexane mixture as the eluent. Caffeic acid phenyl amide, or 3--N-phenyl-2-propenamide (2), was obtained with an isolation yield of 71% (50.04 mg) as a yellow solid. IR  3368.9 (br), 1735.2, 1655.2, 1597.0, 1525.9, 1498.9, 1441.4, 1352.5, 1281.5, 1183.2, 1112.1, 973.3, 848.2, 810.7, 754.4, 691.2, 562.0, and 456.9;1H − NMR  7.64 , 7.52 , 7.31 , 7.10 , 7.06 , 6.95 , 6.79 , 6.56 ;13C − NMR  167.52, 149.15, 146.90, 143.55, 140.24, 129.96 (3C), 128.39, 125.27, 122.52, 121.35, 118.84, 116.63, 115.31; HRMS m/z of caffeic acid phenyl amide was observed at 256.0969 .Caffeic acid (1) , 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide , and hydroxybenzotriazole  were weighed into an oven-dried round-bottomed flask respectively and dissolved in dry tetrahydrofuran (5 mL). Then, an N2 gas balloon was used to create the inert atmosphere. The reaction mixture was mixed using a magnetic stir bar for 30 min at room temperature. Next, tert-butyl amine  was added, and the reaction was stirred at room temperature for 24 h and monitored by TLC using an ethyl acetate:hexane solution (7:3 v/v) as the mobile phase. Then, the volatile solvent was removed under reduced pressure, and the residue was redissolved in ethyl acetate (20 mL). Next, the resulting solution was transferred to a separatory funnel and washed with water to remove the water-soluble by-products. Afterward, the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to produce a crude product. The crude product was purified using flash column chromatography with silica gel as the stationary phase and the ethyl acetate:hexane solution as the eluent. The tert-butyl amide of caffeic acid (3), or 3--N--2-propenamide, was obtained with an isolation yield of 75% (48.67 mg) as a yellow solid. IR  3228.7 (br), 2923.3, 1753.9, 1648.6, 1596.2, 1514.0, 1443.2, 1375.5, 1357.7, 1275.7, 1221.7, 1179.5, 1163.1, 1111.1, 1034.5, 974.5, 909.8, 856.7, 795.1, 779.1, 738.2, 567.7;1H − NMR  7.54 , 7.03 , 6.94 , 6.77 , 6.24 , 1.29 ;13C − NMR  169.87, 149.73, 147.08, 146.96, 127.81, 123.06, 116.62, 115.24, 114.96, 52.13, 30.59 (3C); HRMS m/z of tert-butyl amide of caffeic acid was observed at 236.1281 .Caffeic acid (1) , N,N′-dicyclohexylcarbodiimide , and 3,5-bis(trifluoromethyl)aniline  were dissolved in dry tetrahydrofuran (5 mL) in an oven-dried round-bottomed flask. Then, the reaction mixture was refluxed for 3 h and monitored by TLC using an ethyl acetate: hexane solution (1:1 v/v) as the mobile phase. After TLC monitoring indicated completion, the reaction mixture was filtered to remove the solid by-products, concentrated, and purified by flash column chromatography using silica gel as the stationary phase and the ethyl acetate:hexane solution as the mobile phase. Caffeic acid N- amide (4), or 3--N--2-propenamide, was produced with the isolation yield of 77% (83.1 mg) as a pale-yellow powder. IR  3399.9 (br), 2929.5, 2856.8, 1667.6, 1600.7, 1565.1, 1472.8, 1440.2, 1382.4, 1278.2, 1174.5, 1135.4, 975.6, 937.0, 886.6, 850.9, 811.0, 701.1, 682.2, 561.0, 436.6;1H − NMR  8.28 , 7.60 , 7.58 , 7.06 , 6.95 , 6.78 , 6.52 ;13C − NMR  167.84, 149.56, 146.95, 145.02, 142.54, 133.84 & 133.51 & 133.18 & 132.85 , 128.92 & 126.22 & 123.52 &120.82 , 128.05, 122.90, 120.46 & 120.42 , 117.79, 117.54 & 117.51 & 117.47 & 117.43 & 117.39 , 116.64, 115.39; HRMS m/z of caffeic acid N- amide was observed at 392.0720 .Caffeic acid . The RNA was purified by DNase I treatment . cDNA was synthesized using RevertAid First Stand cDNA Synthesis Kit . The specific forward and reverse primers for synthesis of cDNA was presented as following:- Human Actin forward primer: 5′- ATGATGATATCGCCGCGCTC -3′- Human Actin reverses primer: 5′- GCGCTCGGTGAGGATCTTCA -3′- Human SRD5A1 forward primer: 5′- CTGATGCGAGGAGGAAAGCCTATGC-3′- Human SRD5A1 reverses primer: 5′- GTCCAGATGCCTTTGCCTCACCTTG -3′- Human SRD5A2 forward primer: 5′- TGCCTTCTGCACTGGAAATGGAGTC-3′- Human SRD5A2 reverses primer: 5′- GGAGTGGGTTTGCTCTGGGTCTTTG -3′4 cell/well in 96-well plates in a complete DMEM medium, incubated overnight, and treated with test compounds at 0–50 µM for 24 h. The stock solutions of all the test compounds were prepared in DMSO. Each compound was serially diluted in DMEM to achieve concentrations at 0.5–50 µM; DMEM was used as the negative control. After 24 h of treatment, the culture medium was removed, and cell viability was determined by adding 100 µL of 0.5 mg/mL MTT at 37 °C in darkness for 3 h. After removing the MTT solution, 100 µL of DMSO was added to each well to dissolve the purple formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 value was calculated. Each experiment was performed in triplicates.The cytotoxicity of caffeic acid and its amide derivatives were evaluated with two cell lines, human keratinocyte (HaCaT) and DP cells, using the common cell viability assay with MTT. HaCaT and DP cells were seeded at 1 × 105 cells/well in DMEM with 0.1% DMSO and allowed to attach for 24 h. Then, 1 mL of 20 µM testosterone (T), the enzyme substrate, in DMEM with 0.1% DMSO was added. The controls were 1 mL of DMEM with 0.1% DMSO and 1 mL of 5.0 µM dutasteride (DU), the enzyme inhibitor. For the preliminary screening, caffeic acid (1) and two amides 2 and 3 were prepared at 40 µM, while amide 4 was prepared at 2 µM. For the evaluation of SRD5A1 inhibitory concentration, the N- amide of caffeic acid (4) was serially diluted and prepared at 0.4, 1.0, 2.0, 5.0, and 10.0 µM. Each test compound was dissolved in DMEM containing 0.1% DMSO. The cells were treated with 1 mL of each test compound at a non-toxic concentration. The cells were incubated in a total of 2 mL of the medium at 37 °C under a 5% CO2 atmosphere for 24 h. Therefore, the final concentration of test compounds was reduced by half. Afterward, the cell culture medium was separated from the cells, extracted with 2 mL of ethyl acetate, and centrifuged at 10,000 rpm at 25 °C for 5 min before a layer of 1 mL of ethyl acetate was collected. The extracted process was repeated twice by adding 0.5 mL ethyl acetate each time and collecting the layer of 0.5 mL of ethyl acetate. Next, 2 mL of the combining ethyl acetate extract was air-dried and reconstituted with 20 µL methanol. Then, 8 µL of the methanol solution was spotted on a silica gel 60 F254 glass HPTLC plate using a HPTLC sample loader (CAMAG Linomat 5). A mixture of cyclohexane: ethyl acetate: triethylamine solution (1.5:1:0.1 v/v) was used as the mobile phase. The HPTLC plate was developed twice, stained with 42.5% phosphoric acid in ethanol, and heated at 120 °C for 20 min. The product produced by 5α-reductase, dihydrotestosterone (DHT), was detected under UV light at 366 nm and captured by a TLC scanner CAMAG TLC Scanner 3. The obtaining chromatogram was visualized and analyzed by CAMAG HPTLC Software visionCATS. The inhibition of SRD5A1 was calculated according to the intensity of the DHT produced compared to that produced in the presence of the positive control, dutasteride (DU). In parallel, the viability of the attached treated cells was verified immediately after removing the cell culture medium for the MTT assay. Cell viability should be higher than 80% at each concentration of the test compound to avoid false-positive results. Each experiment was repeated in triplicates.The HaCaT-based SRD5A1 inhibitory assay involves culturing the cells for 48 h, treating the cells with a compound for 24 h, and evaluating cell viability in parallel with the MTT assay and steroid 5α-reductase inhibitory activity assay using HPTLC. HaCaTs were seeded in 6-well plates at 2 × 105 cells/well in 6 well-plates) were treated with compound 4  for 12 and 24 h. Then, total RNA was extracted by using Purelink RNA mini purification kit. The isolated RNA was purified by DNase I and further used as a template for synthesis of cDNA using RevertAid First Stand cDNA Synthesis Kit. Quantitative real-time PCR (RT-qPCR) was performed with a C100 Thermal Cycler  using Luna Universal qPCR Master Mix (M3003). Specific forward and reverse primers of the interested gene was indicated as following.HaCaT cells  of target genes were normalized with GAPDH (Ct) values.5 cells/well in 6-well plates) were cultured in DMEM with or without compound 4 (0–2.5 µM) for 12 and 24 h. The cells were lysed with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor on ice for 45 min. Then, the total protein from each sample was determined by bicinchoninic acid (BCA) protein assay kit . The equal amount (40 µg) of protein sample was mixed with loading buffer and heated at 95 °C for 5 min before loaded to 10% sodium dodecyl sulfate–polyacrylamide gels (SDS-PAGE). The proteins were transferred onto PVDF membrane, which was further blocked with 5% BSA in TBST  at room temperature for 1 h. The primary antibody specific to SRD5A1  in dilution 1:1000 was added onto the membrane and incubated overnight at 4 °C. Then the membrane was washed 3 times × 7 min with TBST following with the immersion in the specific secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. The signal of target protein was determined by using chemiluminescence HRP substrate  and the intensity of target protein was analyzed by analyst/PC densitometric software .HaCaT cells  and DMEM with 0.1% DMSO were added. For the assessment of kinetic inhibition of compound 4 against HaCaT-produced SRD5A1, the cells were treated with various concentration of testosterone (0 − 50 μM) and compound 4 . DMEM with 0.1% DMSO was used as negative control. Then, the cells were incubated for 12 h. The collection of cell culture medium, extraction process, sample preparation and HPTLC detection were prepared by the method mentioned above (3.8). The obtaining chromatogram was visualized by CAMAG HPTLC Software vision CATS and analyzed by Image J software. The percentage of DHT intensity was calculated based on the intensity of DHT normalized by the standard of DHT at 100 μM. At the same time, the cell viability of the treated cells was assessed by the MTT assay. Each experiment was repeated in triplicate.The in vitrop-value < 0.05 was considered statistically significant.All experiments were performed independently three times and in triplicates; the data were presented as mean ± SD. Statistical analyses were carried out using GraphPad Prism 9.3.1 . The differences between the means were analyzed using a one-way analysis of variance. A 44. The structure of compound 4, finasteride, dutasteride, and the adducts of these compounds and NADP were built using Accelrys Discovery Studio 2.5 49. The protonation state of 5α-reductase and the compounds was predicted by PDB2PQR50 and ChemAxon51, respectively. The binding mode of compound 4 and NADP-dihydro-4 adduct inhibition compared with known drugs were studied using molecular docking with the GOLD program based on a genetic algorithm (GA)52.The 3D structure of human SRD5A1 (P18405) was retrieved from the UniProtKB database which was derived from AlphaFold protein structure prediction35 was downloaded from Protein Data Bank. The 3D structure of both human SRD5A1 and SRD5A2 was superimposed. The NADP-dihydrofinasteride adduct was defined as the center of the active site for docking study. The 5α-reductase system was set as 6.5 Å for sphere docking and GOLD score for the scoring function. All systems were used as 100 independent docking poses and the structure with the highest GOLD score was selected to study. The GoldScore function combines force field terms with empirical terms to account for some of the deficiencies in pure force-field-based scoring functions53. It is optimized to predict the binding sites of the ligand considering factors such as the energy of hydrogen bonds, van der Waals (vdW) energy, metal interactions and torsion deformations, which is a molecular mechanics–like function with four terms as follows:Shb(ext) is protein–ligand hydrogen-bond energy  and Svdw(ext) is protein–ligand vdW energy , Shb(int) is the contribution of intramolecular hydrogen bonds in the ligand to Fitness and Svdw(int) is the contribution due to intramolecular in the ligand. After the GOLD score and binding patterns were obtained, the mode of binding between the compounds and 5α-reductase was elucidated using Accelrys Discovery Studio 2.5 49 and ChimeraX 1.154.The crystal structure of human SRD5A2 complexed with an NADP-dihydrofinasteride adduct (7BW1.pdb)4 and NADP-dihydro-4 in complex with SRD5A1 were embedded in 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer followed by TIP3P water solvation and neutralization, using sodium and chloride ions at 150 mM as previously reported35. The geometries of the two ligands were optimized by HF/6-31(d) level of theory as per previously studies56 using the Gaussian09 program. The electrostatic potential (ESP) charges and restrained ESP (RESP) charges of compounds were obtained. The FF19SB and GAFF2 force fields were applied for protein and ligands, respectively. Any steric hindrance or improper geometry was removed by minimization using 5000 iterations of steepest descent (SD) followed by 10,000 iterations of conjugated gradient (CG). Then heated gradually to 310 K, using a Langevin thermostat was applied, while the Berendsen algorithm was used to regulate the pressure at 1 atm. The nonbonded interactions were considered using the short-range cutoff of 12 Å, whereas long-range electrostatic interactions were treated using Ewald’s method. Covalent bonds containing hydrogen atoms were constrained with the SHAKE algorithm. Finally, 1 μs NPT simulations at 310 K and 1 atom were performed for the production runs. Every 500 steps, the MD trajectories were collected for analysis. The RMSD, distance between the center of mass of actives site residues and compound, the number of contacts, intermolecular HB occupation, RMSF and B-factor were evaluated using the CPPTRAJ module. While the MM/(PB)GBSA and decomposition free energy were calculated by the MM/PBSA.py module. Noted that the distance and angle between the hydrogen bond donor (HD) and hydrogen acceptor (HA) were employed as a criterion for hydrogen bond calculations, with the distance and angle of ≤ 3.5 Å and ≥ 120°, respectively.All-atom MD simulations of the SRD5A1 complexes were carried out using the AMBER20 under periodic boundary conditions. Both compound N- amide derivative (compound 4) as a potential non-steroidal SRD5A1 suppressor, with an inhibitory activity at IC50 1.44 ± 0.13 µM and a low cytotoxicity at IC50 29.99 ± 8.69 µM. Compound 4 displayed three-fold more potent inhibitory activity against SRD5A1 than avicequinone C, a previously reported natural non-steroidal inhibitor. Importantly, compound 4 at a concentration of 2.5 µM regulated the activity of SRD5A1 via suppression of SRD5A1 protein expression and potentially directed SRD5A1 inhibition via a mixed mode of inhibition. The underlying inhibitory mechanism of compound 4 was also investigated by conducting a whole-cell kinetic study, which gave a Ki value of 2.382 µM under specific conditions. The stability of SRD5A1 is crucial to limit the in vitro binding study. Thus, the direct SRD5A1 inhibition behavior of compound 4 was predicted by MD simulations. Furthermore, the binding interactions of compound 4 with human SRD5A1 generated by AlphaFold were studied via molecular docking and molecular dynamics simulations. The NADP-dihydro-4 adduct stabilized within the catalytic pocket site of SRD5A1 by hydrophobically interacting with the key M119 residue along with the increased hydrogen bond interactions that were stronger than those of finasteride and dutasteride. The in vitro and in silico results indicate that compound 4 modulates SRD5A1 function, leading to a lower level of DHT production and SRD5A1 protein suppression. Therefore, compound 4 is a promising non-steroidal SRD5A1 suppressor and should be further investigated in the clinical development stage for treating androgenic alopecia.In this study, a series of caffeic acid amide derivatives were designed and synthesized, with a focus on the addition of amide substituents that were similar to finasteride and dutasteride. HaCaTs produced SRD5A1 as a major isozyme, which facilitates the use of a HaCaT cell-based assay for the screening of SRD5A1 inhibitor for androgenic alopecia. Evaluation of the inhibitory activity against SRD5A1 using a HaCaT-based assay with direct DHT detection by HPTLC analysis revealed a caffeic acid Below is the link to the electronic supplementary material.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Video 1.Supplementary Video 2.Supplementary Information 6.


Acetobacterium wieringae strain Y, which hydrogenated isoprene to a mixture of methyl-1-butenes at an overall rate of 288.8 ± 20.9 μM day−1 with concomitant acetate production at a rate of 478.4 ± 5.6 μM day−1. Physiological characterization demonstrated that isoprene was not utilized in a respiratory process; rather, isoprene promoted acetogenesis kinetically. Bioinformatic analysis and proteomics experiments revealed the expression of candidate ene-reductases responsible for isoprene biohydrogenation. Notably, the addition of isoprene to strain Y cultures stimulated the expression of proteins associated with the Wood-Ljungdahl pathway, indicating unresolved impacts of isoprene on carbon cycling and microbial ecology in anoxic environments . Our new findings advance understanding of microbial transformation of isoprene under anoxic conditions and suggest that anoxic environments are isoprene sinks.Isoprene is a ubiquitously distributed, biogenic, and climate-active organic compound. Microbial isoprene degradation in oxic environments is fairly well understood; however, studies exploring anaerobic isoprene metabolism remain scarce, with no isolates for study available. Here, we obtained an acetogenic isolate, designated  Isoprene is a biogenically produced, volatile organic compound, and increased emissions progressively impact global climate –4. Isopr–5–Actinobacteriota and Proteobacteriota, can utilize isoprene as a carbon and energy source under oxic conditions  responsible for the stepwise conversion of isoprene via 1,2-epoxy-isoprene and a glutathione conjugate 1-hydroxy-2-glutathionyl-2-methyl-3-butene  contribute to the production and utilization of isoprene , 8. The 10–3-butene , 13–16. Methanospirillum hungatei and Methanothrix soehngenii converted unsaturated hydrocarbons, including squalene and 1-hexadecene, to methane and carbon dioxide, but isoprene was not transformed  in the presence of hydrogen and bicarbonate was reported  responsible for isoprene biohydrogenation. Notably, isoprene stimulated the expression of proteins associated with the Wood-Ljungdahl pathway (WLP) in strain Y, indicating unresolved functions and impacts of isoprene on gene regulation and microbial ecology in anoxic environments.To advance the understanding of isoprene metabolism under anoxic conditions, sediment collected from a historically contaminated river was used as the source material to enrich anaerobes able to transform isoprene. Repeated transfers combined with dilution-to-extinction procedures in completely synthetic basal salt medium  amended −1 in the eighth transfer enrichment cultures was about 2.8-fold and 3.8-fold faster than that observed in the microcosms (94.2 ± 2.0 μM day−1) and the first transfer cultures (69.2 ± 9.9 μM day−1), respectively  μmol of isoprene completely disappeared with concomitant formation of 56.6 ± 2.7 μmol of 2-methyl-1-butene (~97%) and 2.0 ± 0.2 μmol of 3-methyl-1-butene (~3%) over a 13-day incubation period, indicating preferably biohydrogenation of the second C=C bond  in isoprene . Isoprenectively  and C. Iectively . The sub10.1128/mbio.02086-22.1FIG S12 or without CO2/HCO3−, respectively. Download FIG S1, TIF file, 0.3 MB.(A) Biotransformation of isoprene in the ninth transfer of the enrichment cultures amended with acetate as carbon source. (B) Isoprene, methyl-1-butenes, and acetate in the HEPES-buffered strain Y cultures incubated for a week without HCopyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria, Bacteroidota, Chloroflexi, Firmicutes, and Spirochaetota, with relative abundances ranging from 4.7% to 36.6% , Paludibacter (15.4%), Bacteroides (13.7%), Lentimicrobium (10.0%), and Acetobacterium (3.2%). By comparison, the most abundant genera in the 11th transfer acetate-fed cultures were Youngiibacter (79.3%), Acetobacterium (14.8%), Christensenellaceae R-7 group (2.7%), and Sporobacter (2.6%)  responsible for anaerobic isoprene hydrogenation. Most of the sequences  obtained from the sediment microbiome were classified into five phyla, to 36.6% . Time-ser (2.6%) . The incs report .Acetobacterium  and Acetobacterium sp. strain SVCO-15, with 99.3% and 99.7% 16S rRNA gene sequence identities, respectively (A. wieringae strain DSM 1911 is unable to reduce isoprene (A. wieringae strain. The purity of strain Y culture was further supported by the uniform cellular morphology observed in scanning electron microscopy (SEM) imaging. Strain Y cells were straight rods approximately 1 to 2 μm in length and 0.3 to 0.5 μm in diameter  , and 16S rRNA gene amplicon sequencing revealed identical sequences belonging to acterium . The cloectively . Notablyisoprene , 20, sugdiameter  and C, iOT-3T) 2022. Spore10.1128/mbio.02086-22.2FIG S2Acetobacterium. Download FIG S2, TIF file, 0.2 MB.Examination of contamination in strain Y culture based on 16S rRNA gene amplicon sequencing. Isolation-1 and Isolation-6 indicate the first-round and sixth-round dilution-to-extinction isolation, respectively. All qualified 16S rRNA gene amplicon sequences derived from the sixth-round isolation were identical and belong to Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Acetobacterium, despite its indispensable roles for understanding the biochemistry and energy conservation in homoacetogens and potential applications for production of chemicals and fuels from carbon dioxide  values for the genome pairwise comparison of strain Y and A. wieringae strain DSM 1911 were all above the 95% species delineation threshold  protein-coding genes on the forward strand  interspersed with rRNA (red) and tRNA (blue) genes; (2) protein-coding genes on the reversed strand  interspersed with rRNA (red) and tRNA (blue) genes; (3) GC content (black); (4) GC skew ; (5) size markers for the circular genome. Download FIG S3, TIF file, 0.1 MB.Circular genome map of Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02086-22.8TABLE S1Acetobacterium strains. Download Table S1, DOCX file, 0.04 MB.Genome statistics of 15 Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 (ISO-1), 101.1 ± 7.3 μmol of isoprene was completely hydrogenated to 87.3 ± 1.3 μmol of 2-methyl-1-butene and 5.0 ± 0.6 μmol of 3-methyl-1-butene within 4 days, with concomitant production of 160.2 ± 6.2 μmol of acetate  (5 to (4.05 ± 0.22) × 107 cells mL−1 (97.6-fold increase) and (4.32 ± 0.36) × 107 cells mL−1 (104.1-fold increase), respectively , indicating that isoprene impacted growth and acetate production from H2 and CO2/HCO3−  expressed during H2 plus CO2 reductive acetogenesis. When CO2/HCO3− was not limiting reductive acetogenesis, a mass balance calculation indicated that ~2.4 mL of H2 (~107.1 μmol) was consumed to hydrogenate 10 μL of isoprene (~100.0 μmol) according to 2  was used for acetogenesis via the Wood-Ljungdahl pathway, based on Experiments were conducted to determine whether strain Y was able to conserve energy from isoprene reduction and to investigate whether other unsaturated hydrocarbons could be transformed by strain Y in bicarbonate-buffered medium. In strain Y cultures amended with isoprene and 20 mL Hrbonate)  and B. Arbonate) . Acetateectively . During O2/HCO3−  and C. NO2/HCO3− . These o7 versus [3.66 ± 0.96] × 107 cells mL−1) and acetate production (172.8 ± 0.3 μmol versus 173.7 ± 2.8 μmol) were measured in the isoprene-amended (≈100.0 μmol) cultures with 20.0 + 2.4 mL H2 and the isoprene-free cultures with 20.0 mL H2  in the isoprene-amended cultures was used to hydrogenate 99.4 ± 4.9 μmol of isoprene in a cometabolic manner.Additional experiments were performed to confirm cometabolic transformation of isoprene. Similar cell densities  , and we 10.1128/mbio.02086-22.4FIG S4−10. Different colors represent distinct ene-reductase clusters with similar biochemical activity. The putative ene-reductases found in A. wieringae strain Y are colored blue and designated as IsoR. OYE, Old Yellow Enzyme (purple); MDR, medium-chain dehydrogenase/reductase (green); EnoR, enoate reductase (light blue); SDR, short-chain dehydrogenase/reductase (pink); QnoR, quinone reductase-like ene-reductase (orange). Download FIG S4, TIF file, 0.8 MB.The putative and characterized ene-reductases SSN generated with an alignment score threshold of 10Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. wieringae strain Y, proteomic analysis was performed with strain Y cultures grown in the H2-amended, bicarbonate-buffered mineral salt medium with or without isoprene. A total of 2,198 proteins were detected via 21,421 unique peptides. A total of 1,015 differentially expressed proteins were identified under the two different growth conditions, of which 540 were upregulated and 475 were downregulated in the isoprene-fed cultures when we used a fold change (FC) value of ≥1.2 in protein expression as a screening threshold for a physiologically significant change (Data Set S3). Seventeen putative ERs were upregulated, and the FAD-dependent oxidoreductase with protein ID LNN31_08025  and the NAD(P)H-dependent oxidoreductase with protein ID LNN31_03100  were abundantly expressed (8 (8) (To identify putative ER(s) catalyzing isoprene biohydrogenation in xpressed . These txpressed  28, 29), 29A. wiessed (8 . The putd (8 (8) .A. wieringae strain ISORED-2. Genes coding for the homologous proteins similar to the ER-like enzyme LNN31_08025 were also found in other sequenced Acetobacterium genomes. Another ER-like protein (LNN31_03100) exhibited 92 to 100% amino acid identities to the NAD(P)H-dependent oxidoreductases of A. wieringae strain ISORED-2, Firmicutes bacterium HGW-Firmicutes-17, Acetobacterium sp. strain KB-1, and A. woodii strain DSM 1030 (A. woodii strain DSM 1030 (The ER-like enzyme LNN31_08025 shared 100% amino acid identity to the FAD-dependent oxidoreductase (GenBank accession number WP_228882120.1) of the DSM 1030 . Since tDSM 1030 , the FAD10.1128/mbio.02086-22.5FIG S5FIG S5, TIF file, 0.5 MB.Phylogenetic tree of the putative ene-reductases, including LNN31_08025 (A) and LNN31_03100 (B). Trees were constructed with the neighbor-joining method using bootstrap resampling with 1,000 replications. Download Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Acetobacterium spp. and has been extensively investigated  and accessory proteins. These genes were markedly divergent from those in the A. woodii strain DSM 1030 genome with low sequence identities ranging between 30.3 and 44.4%. Cluster II  consists of genes encoding formate tetrahydrofolate ligases (Fhs1 and Fhs2), cyclodeaminase/cyclohydrolase family protein (FchA), 5,10-methenyltetrahydrofolate cyclohydrolase (FolD), electron transport complex subunit RsxC (RnfC), methylenetetrahydrofolate reductase C-terminal domain-containing protein (MetV), and methylenetetrahydrofolate reductase (MetF) (Acetobacterium genomes (Data Set S4). Interestingly, proteomic analysis revealed that WLP proteins, with the exception of Fdh, were expressed in higher abundances in the presence of isoprene. Twelve proteins encoded in cluster II and cluster III were differentially expressed when we compared the isoprene-amended and isoprene-free cultures. Specifically, 11 of these differentially expressed proteins  were significantly upregulated, but only one  was slightly downregulated in the presence of isoprene  is the hallmark feature of stigated , 31. Sime (MetF) . Clusterisoprene . These reriments  and C.Acetobacterium in isoprene-reducing mixed cultures suggested a role of Acetobacterium in the biotransformation of isoprene under anoxic conditions. Here, we isolated a novel Acetobacterium wieringae strain, designated as strain Y, and demonstrated that strain Y was able to reduce isoprene to 2-methyl-1-butene and 3-methyl-1-butene during growth with H2 and CO2/HCO3−  play a role in the global cycling of isoprene  were provided, less acetate was produced in the cultures amended with isoprene than in those without isoprene addition. No differences in acetate production and growth yields were observed between strain Y cultivated with isoprene plus 22.4 mL hydrogen and strain Y grown only with 20 mL hydrogen without isoprene in the bicarbonate-buffered mineral salt medium. Although isoprene reduction did not exhibit energetic advantage, the presence of isoprene affects the kinetics of reductive acetogenesis and growth rates of Acetobacterium wieringae strain Y. Strain Y expressed WLP enzymes  in relatively higher abundances when exposed to isoprene, suggesting that the biohydrogenation of isoprene promotes the expression of enzymes involved in CO2 fixation. Isoprene, as a widely distributed and abundant natural product, has ecological implications and effects on anaerobic microbial processes. For instance, isoprene was found to moderately inhibit methanogenesis  was purchased from Sigma-Aldrich . All other chemicals were obtained from Macklin Co., Ltd.  or Sigma-Aldrich and were of analytical or higher grade.Isoprene and 2-methyl-1-butene (both ≥99%) were purchased from Aladdin-Reagents Co., Ltd. . 3-Methyl-1-butene (≥95%) was purchased from Shanghai Macklin Biochemical Co., Ltd. . Ethene, propene, 1,3-butadiene, and 1-butene  were purchased from Dalian Special Gases Co., Ltd. . Low-melting-temperature agarose  (2 (~414 μmol), Wolin vitamins . Unless otherwise specified, 160-mL glass serum bottles containing 100 mL of 30 mM bicarbonate-buffered (pH 7.2) mineral salt medium and a Nol/vol])  were usevitamins , and 7 μ2, and 5 mM acetate were added to 20-mL glass vials containing 9 mL of growth medium, 1% (wt/vol) low-melting-temperature agarose, and a N2/CO2 (80/20 [vol/vol]) headspace. A 1-mL aliquot of the ninth transfer of the isoprene-transforming enrichment culture served as the inoculum, which was serially diluted from 10−1 to 10−12. A white colony was picked up from the highest dilution tube showing complete conversion of isoprene to 2-methyl-1-butene and transferred to fresh liquid medium. This dilution-to-extinction procedure was repeated six times before culture purity was evaluated by SEM imaging, 16S rRNA gene amplicon sequencing, and Sanger sequencing.For isolation, 2 μL of neat isoprene, Wolin vitamins, 1 mL of H2/CO2 headspace (80/20 [vol/vol]) and 100 mL bicarbonate-buffered basal salt medium amended with 10 μL isoprene (~100.0 μmol), 20 mL H2, and Wolin vitamins. For energy metabolism analysis, strain Y cultures were grown under three different conditions: (i) isoprene-amended cultures with 20.0 mL H2 (ISO-1), (ii) cultures with 20.0 mL H2 without isoprene (H2 plus CO2), and (iii) cultures with isoprene and 22.4 mL H2 (ISO-2). Samples collected from the first two growth conditions were subjected to proteomic analysis. To investigate if isoprene transformation was HCO3−/CO2 and/or H2 dependent, the buffer system was replaced with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , and 100% N2 was provided in the headspace. To test hydrogenation of other compounds with at least one C=C bond, isoprene was replaced with 2 mL ethene (~0.14 mM in the liquid phase), 2 mL propene (~0.14 mM), 10 mM fumarate, or 2.5 mL 1,3-butadiene (~0.35 mM).Unless otherwise specified, the isolate, designated as strain Y, was routinely maintained in 160-mL serum bottles containing a Nwww.mothur.org) following MiSeq standard operating procedures  following the manufacturer’s instructions. For amplicon sequencing, the hypervariable V3-V4 region of the bacterial 16S rRNA gene was amplified using the primers V3-V4-F and V3-V4-R  39). Se. Se39). ocedures . Qualityocedures . Sequencocedures  using a ocedures . PCR pro10.1128/mbio.02086-22.9TABLE S2Table S2, DOCX file, 0.03 MB.Nucleotide sequences of the primers and probe used in the PCR and qPCR assays targeting bacterial 16S rRNA genes. Download Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the GGTAGTCCACGCCGTAAACG-3′) and Aceto-866R (5′-CAGGCGGAGTGCTTATTGC-3′), and probe Aceto-829 probe (5′–6-carboxyfluorescein–CTCAGTGCCGCAGCT–MGB–3′) targeting the 16S rRNA gene of strain Y were designed using Primer3Plus   primer set, Aceto-786F  . Primer ge, USA) . Each qPge, USA) . The 440reaction .10.1128/mbio.02086-22.7FIG S7A. wieringae strain Y. Plasmid DNA containing a 16S rRNA gene fragment of strain Y was diluted to obtain gene copy numbers ranging from 2.61 × 103 to 2.61 × 1010. All qPCR reactions were performed in triplicate. Download FIG S7, TIF file, 1.4 MB.Amplification plot (A) and standard curve (B) of the qPCR assay targeting the 16S rRNA gene of Copyright © 2022 Jin et al.2022Jin et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 30 min at 4°C. Genomic DNA was extracted using the sodium dodecyl sulfate (SDS) method  method . Whole-g) method . The com) method .g for 15 min. The treated pellets were rinsed gently with 100 mM phosphate-buffered saline (PBS) solution at pH 7.3 three times and then incubated with 1% (wt/vol) osmic acid for 2 h at room temperature. Cells were washed again with PBS and then dehydrated through a series of 30%, 50%, 70%, 85%, 95%, and 100% ethanol for 10 min each. Cell samples were immersed with tert-butanol and transferred into a glass flat plate coated with aluminum film. After overnight lyophilization in a freeze dryer , the samples were scanned and imaged using an FEI Inspect F50 field emission electron microscope  under high-performance conditions with accelerating voltages reaching 20 kV.For immersion fixation, freshly harvested strain Y cell pellets were suspended in 2.5% glutaraldehyde buffer at 4°C for 4 h, followed by centrifuging the suspension at 14,000 × g for 15 min. The supernatant was quantified with a bicinchoninic acid protein assay kit  according to the manufacturer’s instructions. SDS-PAGE (8 to 16%) for total crude proteins was used to perform a preliminary difference assessment. Protein digestion was performed following the previously described filter-aided sample preparation method . The six samples were ultrasonicated in a boiling water bath for 10 min. Cellular debris was removed by centrifugation at 13,000 × n method . Peptide18 trap precolumn  and separated using a C18 analytical column  at a flow rate of 300 nL min−1. The mobile phases consisted of deionized water with 0.1% formic acid (solution A) and 95% acetonitrile with 0.1% formic acid (solution B). The elution procedure was as follows: the fraction of solution B increased from 2% to 8% during the initial 2 min, increased to 30% over the next 40 min, further to 45% over 8 min, rapidly to 100% over an additional 1-min time period, and then held at 100% for 10 min. MS1 and MS2 spectra based on the higher-energy collisional dissociation (HCD) method were acquired in the Orbitrap in positive ionization mode at resolutions of 60,000 and 1,500, respectively. MS spectra of the full scan were acquired over a range of 350 to 2,000 m/z. The top 20 abundant precursor ions were selected for HCD fragmentation. The normalized collision energy was 32 eV. Raw data were retrieved using a search engine (Sequest HT) with default parameters in Proteome Discoverer 2.4 software (version 1.6.0.16) for protein identification against strain Y genome. The false-discovery rate (FDR) was set to 1%, and TMT reporter ion intensity was used for quantification. Expression data were grouped together by hierarchical clustering according to the protein level. Protein functional annotation was conducted using the Universal Protein (UniProt) database . Samples were injected into a Cdatabase .2SO4. Undiluted or diluted samples were separated at a flow rate of 0.6 mL min−1 using 4 mM H2SO4 as the mobile phase.Unsaturated hydrocarbons were measured by injecting 100 μL headspace gas into an Agilent 7890A gas chromatograph equipped with a flame ionization detector and an Agilent DB-624 column as described elsewhere . The hydhttps://itol.embl.de)  . The maxembl.de)  and Megaembl.de)  with defCP087994. The BioSample and BioProject accession numbers are SAMN23007773 and PRJNA778957, respectively. Raw sequencing reads have been deposited in the Sequence Read Archive under the accession numbers SRR18087747 (Illumina) and SRR18091988 (PacBio) for the strain Y genome and accession number PRJNA808885 for the 16S rRNA gene amplicon sequencing reads. Data Sets S1 to S4 are available through the Figshare platform and can be accessed via https://doi.org/10.6084/m9.figshare.20349294.v2.The complete genome sequence of strain Y has been deposited in GenBank under accession number


Saccharomyces cerevisiae strain for development as a vaccine candidate. To rapidly express the recombinant protein and avoid the need for expensive galactose as an inducer, the gal80 gene of S. cerevisiae was knocked out, and the conventional 72-h culture period was thus successfully shortened to 24 h. Mice vaccinated against variant B.1.617.1 showed robust humoral and cellular immune responses. Moreover, the antiserum in the B.1.671.1 group had neutralizing activity against wild-type RBD and high binding titers against RBD mutants of variants B.1.351 and B.1.1.7. Double deglycosylation at N331Q and N343Q resulted in marked reduction of the affinity of RBD binding to angiotensin converting enzyme 2 (ACE2) and escaped antibody neutralization. This study demonstrates that yeast surface display technology can provide an alternative approach to rapid large-scale preparation of promising SARS-CoV-2 vaccine candidates at low cost.Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a significant global human health threat. The most effective way to end the pandemic is through timely vaccination. In this study, the receptor-binding domains (RBDs) of Spike protein of the initial strain of SARS-CoV-2 and its variants, B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.1 (Kappa), were successfully displayed on the surface of a  Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused a global pandemic that, up to early July 2022, has infected more than 544 million people and has caused more than 6.3 million deaths around the world. This virus poses a serious global public health emergency. Developing vaccines against COVID-19 is considered the most promising approach for curbing the pandemic. Many teams around the world have already been engaged in SARS-CoV-2 vaccine development . RecentlSaccharomyces cerevisiae is the best-understood eukaryote at the molecular level, and technologies for its large-scale fermentation are well established  and mannan, can be used as natural adjuvants with the ability to stimulate or modulate the immune response  of wild-type (Wuhan-Hu-1 strain) SARS-CoV-2 Spike protein and the B.1.1.7, B.1.351, and B.1.671.1 variant mutant RBDs (mRBDs) were fused to the C-terminus of the S. cerevisiae Aga2 protein (one of the subunits of the a-agglutinin receptor). The gal80 gene of the yeast strain EBY100 was knocked out to achieve rapid expression of the recombinant protein using glucose as a carbon source instead of the more expensive galactose. Western blotting analysis and immunofluorescence microscopy confirmed the display of RBD fusion protein on the S. cerevisiae surface after 24 h in culture. Immunization experiments in mice indicated that variant B.1.671.1 triggered host humoral and cellular immune responses. In addition, by construction of deglycosylated mimetic N331Q and N343Q variants of wild-type RBD, we confirmed that glycosylation of the RBD is essential for its binding affinity to the receptor, ACE2.Here, we report the display of SARS-CoV-2 recombinant protein antigen and variants on the cell surface of Escherichia coli Top10 was the host strain used for the construction of expression vector and propagated in Luria–Bertani (LB) broth  at 37°C. S. cerevisiae EBY100  served as a host cell for RBD and its variants’ surface display and grown in YNB-CAA-Glucose medium  at 30°C. Minimal dextrose medium  was used to select yeast transformants.pYD1 plasmid  was used to construct SARS-CoV-2 RBD surface display vector. BamHI endonuclease sites of pYD1 plasmid. The variants B.1.1.7 (N501Y), B.1.351 (N501Y/E484K/K417N), and B.1.617.1 (L452R/E484Q) were constructed by a site-directed mutagenesis kit (see Section 2.7) with temple of wild-type RBD. The S. cerevisiae EBY200 strain was constructed by deleting the gal80 gene of S. cerevisiae EBY100 strain. The resultant shuttle plasmid pYD1-RBD was transformed into S. cerevisiae EBY200. Recombinant yeast transformants were plated on selective minimal dextrose plates at 30°C for 3 days. The positive clones were confirmed by DNA sequencing and then induced in YNB-CAA-Glucose medium  at 30°C for 24 h for RBD expression. Meanwhile, S. cerevisiae EBY200 containing empty pYD1 plasmid was used as a negative control for the following tests.The SARS-CoV-2 Spike RBD  encoding gene was synthesized by GenScript  and subcloned into S. cerevisiae PYD1-RBD/EBY200 and variant cells were harvested after 24 h induction in YNB-CAA-Glucose medium and washed three times in PBS. Collected cells were inactivated at 60°C for 1 h. After treatment, cells were washed three times with PBS and resuspended in 10 ml of sterile 10% skimmed milk and freeze-dried. The freeze-dried cells were stored at 4°C until use. Before immunization, 5 OD 600 nm freeze-dried cell powder was washed three times with 1 ml of PBS and resuspended in 100 μl of PBS.Two hundred microliters of recombinant S. cerevisiae pYD1-RBD/EBY 200 and variant cell pellets were harvested and washed twice with PBS buffer. Remove the PBS and resuspend the cell pellets in 250 µl of PBS, 1 mg/ml BSA, and 1 µg of rabbit anti-RBD (wild-type) antibody  on ice for 30 min with occasional mixing. Centrifuge the cells at 5,000 × g for 5 min at 4°C and washed twice with PBS. After the cells were washed, the second antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG , was reacted with them on ice for 30 min in the dark and the tube was occasionally inverted. After washing, the cell pellets were resuspended in 40 µl of PBS and spot 10 µl on a slide for immunofluorescence assay . The S. cerevisiae EBY200 strain with pYD1 plasmid was used as a negative control.For immunofluorescence assay and microscopy, 1 OD 600 nm of For Western blot analysis, 1 OD 600 nm of pYD1-RBD/EBY 200 and variants were harvested at 24 h post-induction. The pellets were washed twice with 500 µl of sterile distilled water and re-suspended with 10 µl of 4× SDS loading buffer and 30 µl of sterile distilled water. The suspension was boiled for 15 min. The treated samples were resolved by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane . Membranes were blocked in TBST buffer  containing 10% skimmed milk for 1 h at room temperature. After blocking, membrane was incubated with rabbit anti-RBD antibody  for 1 h at room temperature followed by three 10-min washes in TBST buffer. The membrane was followed incubated with an HRP-conjugated goat anti-rabbit IgG  with 2,500 dilution at room temperature for 1 h. After washing three times with TBST, Tanon High-sig ECL Substrate  was added to visualize the bands and imaged by Tanon Imager System .S. cerevisiae pYD1-RBD/EBY200 cells was weighed in 1.5-ml microtubes and washed twice with PBS. Collected cell pellets were resuspended in 100 µl of a series of diluted Anti-Spike-RBD human IgG  solutions [in PBS containing 1% (w/v) BSA] and incubated at room temperature for 1 h. Cell pellets were washed three times in PBS containing 0.05% Tween-20. After washing, cells were resuspended in 100 µl of PBS containing 0.1 µg of rabbit anti-human IgG-HRP antibody  and incubated at room temperature for 1 h. After washing in the same way, cells were developed with the addition of 100 µl of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB)  at room temperature in darkness for 30 min and stopped reactions by addition of 100 μl of 2 mol/L H2SO4. Finally, absorbance of cell supernatant was measured at 450 nm using a microplate reader .The quantity of RBD expressed in EBY200 surface was determined by quantitative ELISA, as described previously . In brieDpnI at 37°C for 1.5 h. Then, the mixtures were transformed to E. coli Top10 for screening.In order to construct RBD deglycosylated expression plasmid, site-directed mutagenesis was performed following the instruction of a site-directed mutagenesis kit . Briefly, the plasmid pYD1-RBD was used as the template to generate the mutagenesis in RBD gene of N331 and N343. Following site-directed mutagenesis PCR, the amplification fragments were digested using S. cerevisiae pYD1-RBD/EBY200 cell pellets was centrifuged in 1.5-ml microtubes and washed twice with PBS. Subsequently, cells were resuspended in 100 µl of a series of diluted biotinylated Human ACE2 protein  solutions in PBS containing 1% (w/v) BSA and incubated at room temperature for 1 h. Cell pellets were washed three times in PBS containing 0.05% Tween-20. After washing, cells were resuspended in 100 µl of PBS containing 0.1 µg of Streptavidin-HRP  and incubated at 37°C for 1 h to avoid light. After washing in the same way, cells were developed with the addition of 100 µl of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB)  at room temperature in darkness for 30 min and reactions were stopped by the addition of 100 μl of 2 mol/L H2SO4. Finally, absorbance of cell supernatant was measured at 450 nm using a microplate reader.To determine the affinity of RBD and its deglycosylated mutants to ACE2, 0.5 OD 600 nm of Female SPF BALB/c mice at 6–7 weeks of age were purchased from Shanghai Laboratory Animal Co. Ltd, China. Mice were housed under a 12:12h light/dark cycle at controlled temperature. Mice were allowed for a week adaptation period upon arrival. All animal experimental protocols were approved by the Animal Ethics Committee of the School of Basic Medical Sciences, Fudan University .S. cerevisiae pYD1/EBY200), inactivation treated surface-displayed RBD (wild) group [vaccinated with inactivated pYD1-RBD (wild)/EBY200], surface-displayed RBD (B.1.351) group [vaccinated with inactivated pYD1-RBD (B.1.351)/EBY200], surface-displayed RBD (B.1.1.7) group [vaccinated with inactivated pYD1-RBD (B.1.1.7)/EBY200], and surface-displayed RBD (B.1.617.1) group [vaccinated with inactivated pYD1-RBD (B.1.617.1)/EBY200]. Each group consisted of six mice. Yeast cells or PBS were injected subcutaneously in the mouse back skin for prime immunization and boosted on day 15, day 30, day 47, and day 69. Blood was collected on day 28, day 44, and day 64 after the initial immunization. Plasma was used for RBD-specific IgG antibody titer detection and neutralization titer detection. Mice were sacrificed 5 weeks after the fifth immunization (D120), and splenocytes were isolated and prepared to detect IFNγ levels (ELISPOT) and multifunctional T-cell response (flow cytometry).Mice were divided into six groups: PBS control group, vector-only control group . Briefly, a 96-well plate was pre-coated with human ACE2 protein and 50 μl of serially diluted sera samples ; positive control and negative control were added to each well followed by adding 50 μl of HRP-SARS-CoV-2 Spike RBD (wild) working solution (0.3 μg/ml). The plate was sealed with microplate sealing film and incubated at 37°C for 1 h to avoid light. After washing three times, 100 μl of substrate solution was added to each well and the plate was incubated at 37°C for 20 min. Then, 50 μl of stop solution was added to each well. After gently shaking the plate for 3 min, the absorbance at 450 nm was read using a microplate spectrophotometer. Results were judged according to the following criteria: (1) Percent inhibition = (1 − OD 450 of sample/OD 450 of negative control) × 100%; (2) Cutoff value = 20% signal inhibition; (3) Positive: Percent inhibition of sample ≥ Cutoff value; (4) Negative: Percent inhibition of sample < Cutoff value.The spleen was collected aseptically and was placed in a cell filter  with a diameter of 70 μM. Then, the spleen was ground with a disposable 1-ml syringe plug and rinsed with RPMI 1640 medium . Splenocytes were isolated by lymphoprep  according to the instructions.5 or 4 × 104) were co-cultured with wild-type RBD (40 µg/ml) or concanavalin A (ConA)  (5 µg/ml) in 100 µl of RPMI 1640 containing 10% FBS, 1% P/S, 1% HEPES buffer , 0.1% 2-mercaptoethanol , 1% L-glutamine, and 1% Mem NEAA  for 20 h in a humidified 37°C CO2 incubator, and because there is no stimuli control, splenocytes were left untreated. Spots were revealed by AEC coloring system  according to the kit manual and determined using an automatic ELISpot reader  and image analysis software (ImmunoSpot 5.1.36). The number of IFN-γ cells was adjusted by subtracting the background number in the no stimuli controls and normalized by the ConA controls based on the following formula: Nadjusted = (Ntest − Nblank)×F, F = 2000/[(NConA − Nblank)×5].IFN-γ ELISpot assays were performed using the Ms IFN-Gma ELISPOT Set  referring to the manufacturer’s instructions and reference . Freshly6) were inoculated in a 96-well cell culture plate , co-cultured with 40 μg/ml wild-type RBD for 29 h or positive stimulant PMA (0.1 μg/ml)  + Ion (1 μg/ml)  for 6 h. After stimulation, cells were stained referring to the instruction of Cytofix/Cytoperm W/Golgi Stop Kit . Briefly, the 96-well plate was centrifugated  and washed twice with PBS. Then, the cells were blocked on ice in 50 μl of CD16/CD32 antibody (1:100 in 2% FASC Buffer)  for 20 min, and stained with fluorescent-labeled anti-CD3, anti-CD4, and anti-CD8 monoclonal antibody   at 4°C for 40 min. After washing, cells were treated with the fixation and permeabilization solution . Then, cells were washed twice with PBS, and treated with 50 μl/well fluorescent conjugated IL-2/TNF-α/IFN-γ antibody   at 4°C for 40 min. After washing, cells were suspended with 150 μl of 2% FASC Buffer and analyzed by flow cytometry .Splenocytes (1.5×10S. cerevisiae EBY100 as a surface-expressing host. The DNA fragments encoding the RBD or mRBDs were cloned into the plasmid pYD1 to express the fusion proteins of RBD and the second subunit of the A-agglutinin receptor (C-terminus of Aga2)  through two disulfide bonds while Aga1 was secreted from the yeast cells and covalently attached to β-glucan in the extracellular matrix of the yeast cell wall . However, mice vaccinated with variant B.1.617.1 showed a significant increase in serum RBD-specific IgG level after the third immunization (day 44) and a further increase after the 4th immunization (day 64). Mice vaccinated with wild-type RBD and mRBDs from variants B.1.1.7 and B.1.351.1 showed no significant differences after fourth immunization except for individual mice in which RBD-specific IgG antibodies could be detected.As shown in Subsequently, we coated the mutant RBDs of variants B.1.1.7, B.1.617.1, and B.1.351 to detect the cross-reactivity of antiserum 2 weeks after the fourth immunization (day 64). As shown in The neutralization titer of mRBD of B.1.617.1 antiserum against wild-type RBD was determined by competitive ELISA. Serum samples were collected 2 weeks after the fourth immunization (day 64), and detected after mixing the samples from the same group in equal volume. As shown in + T cells in the spleen after RBD restimulation was much higher in the B.1.617.1 mRBD group than the empty vector control group   protein to bind to ACE2 to mediate host cell entry . The RBDblotting Figure 7S. cerevisiae surface. The B.1.617.1 mRBD vaccine induced high levels of RBD-specific IgG antibodies, and had cross-reactivity against wild-type RBD as well as mRBDs of B.1.1.7 and B.1.351.1 variants  should be maintained at each step. Maintaining a continuous cold chain may bring the cost increase of vaccines by about 80% . For exanditions . ELISA qnditions , E. OvePrevention of infection is related to the induction of functional specific antibodies, especially antibodies neutralizing the RBD : ACE2 interaction. However, the T-cell response also plays an important role in COVID-19 mitigation, especially in the patients without measurable humoral responses . In our study, SARS-CoV-2-specific CD4+ T-cell responses were more dominant than CD8+ T-cell responses after vaccination mice using yeast with the surface-displayed B.1.617.1 mRBD . Additio+ CD4+ T cells dominated the SARS-CoV-2 specific T-cell response at later time points in patients, and were associated with persistent antibodies .ZZ, GZ, XY, and HZ initiated and coordinated the project. HX completed plasmid construction, strain culture, protein expression, biochemical analysis, and vaccine preparation. LZ and HZ completed animal immunity experiment and data analysis. HX and LZ wrote the manuscript with contributions from all authors. PW, XY, YY, and ZZ completed the revision of the manuscript. All authors contributed to the article and approved the submitted version.This work was financially supported by the National Key Research and Development Program of China (Grant No. 2018YFA0900700), the National Natural Science Foundation of China (No. 32071425), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB27020206), the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences , and the Shanghai Municipal Science and Technology Major Project on Infectious Diseases.We thank Prof. Sibao Wang  for equipment support in immunofluorescence microscopy. We thank Dr. YingYing Chen  and Dr. XiaoYong Fan  for insightful suggestions on techniques and design of immunization experiments. We thank Qinglian Xie , Xiaoting Zhu , and Chenchen Feng  for technical assistance.Author LZ was employed by Zhejiang Hongguan Bio-pharma Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The CCK8 and EdU assay demonstrated the anti-tumor effect of the four derivatives, and IC50 for U87 was 3.99, 3.28, 7.60, and 11.84 μM, respectively. In U251, IC50 was 7.00, 5.43, 8.64, and 18.05 μM, respectively. D2 and D3 were further selected, and flow cytometry showed that apoptosis rates were increased after D2 or D3 treatment via ROS generation. Potential targets were predicted by network pharmacology analysis, most of which were associated with apoptosis, cell cycle, and metabolism pathway. CDC25B and DUSP1 were two of the most likely candidates for targets. The orthotopic glioblastoma model was established to evaluate the anti-glioma effect and side-effect of juglone derivatives, and the in vivo experiments confirmed the anti-glioma effects of juglone derivatives. In conclusion, new derivatives of juglone were created via chemical group substitution and could inhibit glioma cell viability and proliferation and induce apoptosis rate via ROS generation.Juglone has been extensively reported as a natural antitumor pigment. However, it is easy to be oxidized due to active hydroxy in the quinone. Here, we designed some new juglone derivatives, as the hydroxy was replaced by methyl (D1), allyl (D2), butyl (D3), and benzyl (D4) groups. Nuclear magnetic resonance spectra and mass spectrometry were applied to confirm the derivatives and oxidative products of juglone. U87 and U251 cell lines were used for tests  Glioma is the most common primary malignant brain tumor, and glioblastoma (GBM) contributes 50–60% of them. Despite the advance in molecular research of GBM, the overall survival remains as poor as 14.6 months even after comprehensive management . Temozolin vitro and in vivo in human GBM cells  and alkyl halide (1.5 mmol) was added to the solution of juglone  in CH2Cl2 (5 mL). The reaction mixture was stirred at room temperature for 24 h. After filtration through celite and removal of the solvent in vacuo, the residue was subjected to flash column chromatography on silica gel (230–400 mesh) using n-hexane/ethyl acetate as eluent to give the product D1-D4.New juglone derivatives (D1-D4) were prepared according to literature procedures . Ag2O  spectra were recorded on a Varian Inova 400 spectrometer at 400 and 100 MHz, respectively. All signals were reported in ppm unit with references to the residual solvent resonances of the deuterated solvents for proton and carbon chemical shifts. Mass spectra were obtained on a Thermo Finnigan MAT 95 XL spectrometer, Shanghai Institute of Organic Chemistry, CAS.All reactions were carried out in oven-dried glassware under an atmosphere of dry Nin vivo experiment is IDH-wildtype, MGMT unmethylation, and TP53 mutation. U251 and U87 were purchased from China Academia Sinica Cell Repository . Primary GBM cells and glioma cell lines were cultured in Dulbecco’s modified Eagle’s Medium  supplemented with 10% fetal bovine serum . Cell cultures were maintained in a 5% CO2 humidified incubator at 37°C.GBM primary cells were isolated by specimens derived from patients in Huashan Hospital with full consent after approval from the local ethic committee. The molecular pathology of the specimen for GBM specimen was placed in ice and transferred to the lab within 1 h after surgical resection. The specimen was washed with PBS to remove the blood and necrotic tissue. Then the minced GBM tissue by a surgical knife blade was digested with 0.25% trypsin in the falcon tube at 37°C for 15 min and shaken every 5 min. The tissues were triturated into single cells by a 5 ml pipette and a 40 μm filter was used to remove tissue debris. Single cells were centrifuged and resuspended with 1 ml RBC lysis buffer at room temperature for 5 min. At last, GBM cells were resuspended and cultured in DMEM with 10% FBS. For subculture, cells were passaged once they reached 80–90% confluence. An intracranial implantation experiment was performed on cells within passages two and five to minimize genetic mutation.4 cells/100ul/well and incubated overnight. Tumor cells were pretreated with and without NAC  for 1 h. After treatment of juglone or its derivatives in different concentrations for 48 h, cells were incubated for 1 h with 10 μl CCK-8 per well. The optical density (OD) was measured at 450 nm with a microplate spectrophotometer . Proliferation was examined using the EdU incorporation  assay, which was performed according to the manufacturer’s protocol, and the cells were examined under a fluorescence microscope. The experiment was triplicated, and each contained six replicates.Juglone  and derivatives were dissolved in dimethyl sulfoxide (DMSO) and diluted in DMEM. Cell viability was assessed by the Cell Counting Kit-8 assay . Briefly, tumor cells that were cultured in DMEM with 10% FBS were seeded in 96-well plates at a density of 1×105 cells were harvested, resuspended in 100 µl binding buffer, and incubated with 5 µl Annexin V-FITC, 10 µl of PI  in darkness at room temperature for 15 min. After that, 400 µl of binding buffer was added before being tested on a FACS Calibur cytometer . FlowJo software  was used to analyze the data. The experiments were triplicated.For apoptosis assay, glioma cells were treated with two kinds of juglone derivatives for 48 h, 1×105 cells were pretreated with different concentrations of juglone derivatives for 6 h and then loaded with DCFDA (10 μM) probe. After incubation at 37°C for 30 min, cells were harvested, washed, resuspended with PBS, and fluorescence intensity was measured by flow cytometry. FlowJo software was used to analyze the mean fluorescence intensity (MFI). The experiments were repeated three times.DCFDA fluorescent assay  was used to label intracellular reactive oxygen species (ROS) and then detected with flow cytometry. Briefly, 5×10http://swisstargetprediction.ch/). The top 100 genes were selected for the construction of a protein-protein interaction (PPI) network (https://string-db.org/). The TSV format file was downloaded from string and imported into Cytoscape software (version 3.8.0) for visualization. Molecular docking was performed using UCSF Chimera  C=CC2 = O)C (=C1)O) were obtained from SwissTargetPrediction ( Chimera . The strld.com/) .After treating with different concentrations of juglone derivatives for 48 h, total protein of glioma cell lines of U87 and U251 were obtained from RIPA lysis buffer with 1% PMSF . The protein concentration was determined by BCA assay , and samples were separated on 10% SDS-PAGE, and then transferred onto NC membranes . The membranes were incubated with primary Abs against cleaved-PARP  and β-actin  overnight at 4°C, followed by HRP conjugated secondary Ab . The protein bands were visualized using enhanced chemiluminescence  and a detection system All animal procedures were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Fudan University.5 cells/μL. Mice were anesthetized intraperitoneally with 10% chloral hydrate and secured into a stereotaxic apparatus. GBM cells in 10 uL PBS via a Hamilton syringe were injected into the right forebrain . The mice were randomly divided into three groups . The number in each group was five. D2 and D3 were dissolved in DMSO and diluted in PBS; the final concentration of DMSO was 20 mg/ml. PBS containing the same concentration of DMSO was used as vehicle control. Juglone derivatives treatment group was injected intraperitoneally with D2 and D3 (1 mg/kg) every 2 days, which was the same as the previous dosage. Bioluminescent imaging was performed on the twenty-eighth day after transplantation with IVIS-200  to test the tumor volume. Before anesthesia, D-luciferin  was injected intraperitoneally at 150 mg/kg body weight. Images of different groups were captured with the same parameters. Bioluminescence values of intracranial tumors were quantitated using the Living Image software. Mice were euthanized when neurological symptoms appeared and perfused with 4% paraformaldehyde in PBS.Female BALB/c-nu mice (3–4 weeks old) provided by SLAC Laboratory Animal Company  were used as orthotopic xenograft recipients. Mice were housed in an environment with a 12-h light/dark cycle. At least 1 week time was provided for mice to acclimatize new environment before experimentation. Human primary GBM cells were labeled with lentivirus expressing luciferase. Before intracranial transplantation, cells were digested into single cells and suspended with PBS at a density of 1×10The whole brain, heart, liver, and kidney of each mouse were collected. Fixation with 4% paraformaldehyde in PBS, dehydration with gradient ethanol, embedded in paraffin, and section cut in 5 μm thickness were performed. The sections were stained with hematoxylin and eosin (H&E). Cell morphology of different tissue was observed under a light microscope.p values. A p value < 0.05 was considered statistically significant.All quantified data were presented as mean ± SEM. For comparison between the two groups, two-tailed student’s t-tests were used to calculate The phenomenon that juglone is easy to be oxidated has previously been observed. Juglone solution took on dark brown color changes in a time-dependent manner when preserved at 4°C in an EP tube . The max1H NMR : δ 7.70 , 7.31 , 6.86 , 4.00 . These data are identical with those reported in the literature : δ 7.73 , 7.65 , 7.29 , 6.87 , 6.10 , 5.66 , 5.37 , 4.73 . These data are identical with those reported in the literature : δ 7.70 , 7.65 , 7.29 , 6.85 , 4.14 , 1.89 , 1.60 , 1.01 . 13C{1H} NMR : δ 185.2, 184.0, 159.1, 140.7, 135.9, 134.7, 133.8, 119.6, 118.8, 118.7, 69.0, 31.0, 19.0, 13.7. HRMS (EI) Calcd for C14H15O3+ (M + H+): 231.1016. Found: 231.1019 : δ 7.74 , 7.65 , 7.58 , 7.42 , 7.34 , 6.89 , 5.30 . These data are identical with those reported , protease (15.0%), and kinase 13.0%, . KEGG anhobicity .To further validate the effect of juglone derivatives on apoptosis, U87 and U251 cells were stained with Annexin V/PI after treatment of D2  and D3 . Flow cytometry analysis showed that D2 and D3 could induce apoptosis and increase the percentage of Annexin V+/PI + cells both in U87 and U251 groups . To subsvia p38-MAPK pathway activation. In this study, ROS production was also measured with a ROS assay kit by flow cytometry. As demonstrated, D2 and D3 could significantly induce ROS generation in U87 and U251 cells  and then assigned to the following groups randomly: control (n = 5), D2 (n = 5), D3 (n = 5). Three days after brain implantation of human primary glioma cells infected with lentivirus expressing luciferase into nude mice, vehicle, D2 (1 mg/kg) or D3 (1 mg/kg) were administrated intraperitoneally every other day. Both D2 and D3 had an inhibitory effect on glioma growth confirmed with in vivo imaging systems 28 days later after tumor transplantation  generation mechanism  into brain parenchyma depends on the biological features and the physicochemical properties of the compound such as molecular weight, hydrogen bonding capacity, and lipophilicity. And the unstable property or the poor BBB penetrating power of juglone hinders its effective use in clinical brain tumor therapy. It is indispensable to block potential oxidation susceptibility to preserve bioactivities. In general, adding halogen or alkyl chemical group could increase the lipophilicity of molecules, facilitating drug active substances crossing the BBB, and entering the central nervous system.via the ROS-based pathway. Mice are given an intraperitoneal injection of D2 and D3 at a dose of 1 mg/kg. The dose selection of these two derivatives is based on juglone used in our previous research, as the intraperitoneal median lethal dose is 25 mg/kg according to the manufacturer’s instructions of juglone , 3.28 μM (D2), 7.60 μM (D3), and 11.84 μM (D4), compared with 40 μM of juglone in our previous study . EdU and juglone . In vivovia ROS generation is still involved after chemical group modification of juglone. And exogenous antioxidant NAC could diminish the amount of ROS generation of juglone derivatives.It is reported that ROS has a dual role in tumor cell progression, as excessive generation of ROS and imbalance of redox reaction results in cell death while moderate increase promotes cell proliferation . In geneThe future drug of these new juglone derivatives surely needs further clinical validation. These novel chemical reagents would be good candidates, especially for those MGMT unmethylated gliomas or recurrent gliomas.


ION) of 76.07 μA/μm and ON-state to OFF-state current ratio (ION/IOFF) of 7 × 105, and those for NMOS are 48.57 μA/μm and 1 × 106. The CMOS inverter shows a voltage gain of 18V/V, a noise margin for high (NMH) of 0.17 V and for low (NML) of 0.43 V, with power consumption less than 0.9 μW at VDD of 0.8 V. Full functionality of CMOS ring oscillators (RO) are further demonstrated.In this work, low-temperature Schottky source/drain (S/D) MOSFETs are investigated as the top-tier devices for 3D sequential integration. Complementary Schottky S/D FinFETs are successfully fabricated with a maximum processing temperature of 500 °C. Through source/drain extension (SDE) engineering, competitive driving capability and switching properties are achieved in comparison to the conventional devices fabricated with a standard high-temperature (≥1000 °C) process flow. Schottky S/D PMOS exhibits an ON-state current ( The technology of 2D planar scaling is now facing major limitations, and in order to extend the semiconductor roadmap, 3D sequential integration, which consists of stacking transistors on top of each other, has been envisioned ,2. As itVTH) variation. Hence, low-temperature devices based on Si technology may be further developed.However, source/drain (S/D) activation is typically performed by spike annealing at high temperature ≥1000 °C). Decreasing thermal budget will impair CMOS device performance. Much work has been undertaken to circumvent the thermal budget limitation. For instance, nanosecond laser annealing (NLA)  and soli00 °C. DeSchottky S/D MOSFETs, using metal silicide to replace doped silicon as S/D , hold an1st) and SDE last (SDElast). In the SDE1st scheme, SDE implantation was performed before the spacer and followed by silicidation annealing, and SDE was formed by dopant segregation at the silicide/Si interface during silicidation. In contrast, SDE implantation was performed after silicide formation in the SDElast scheme, and an additional rapid thermal annealing (RTA), also named drive-in annealing, was used to drive the dopant to segregate at the silicide/Si interface, forming SDE. An amount of 3 nm Ni was deposited by sputtering, and B 1.5 keV 2 × 1015 cm−2 was implemented for SDE implantation in both schemes. A split, shown in 2/TiN/TaN and HfO2/TiAl/TiN were separately applied to pFinFETs and nFinFETs for VTH adjustment. The fabrication was completed with tungsten contact plug and Al metallization. Conventional pFinFETs, with and without silicide, were also fabricated with a standard high-temperature (≥1000 °C) process flow (The process flow of Schottky S/D FinFETs is summarized in ess flow c for comIDS-VGS characteristics of the Schottky S/D pFinFETs with three SDE processes are noted in ION/IOFF) of 5 orders, and excellent swings (SSs) of around 61 mV/decade obtained for physical gate length (LG) of 500 nm. As compared in ION was achieved with 550 °C SDElast, followed by 310 °C SDE1st and then 450 °C SDElast, with the values of 55.49 μA/μm, 40.78 μA/μm and 25.23 μA/μm at VDS = −0.8 V; whereas larger IOFF of 0.39 nA/μm and 0.21 nA/μm were resolved for 310 °C SDE1st and 450 °C SDElast with respect to that of 0.08 nA/μm for 550 °C SDElast. Further, the VTH defined as VGS corresponding to IDS = 10−7 A/μm were −0.13 V, −0.16 V and −0.16 V for the devices with 310 °C SDE1st, 450 °C SDElast and 550 °C SDElast processes, respectively. A reduction of around 30 mV in VTH was demonstrated for 310 °C SDE1st.To develop the optimal SDE process, SDE engineering, shown in RS/D) extracted by fitting ON-state resistance (RON) vs. 1/(VGS-VTH) at high gate bias in linear mode [RS/D measurements at VGS = −0.8 V were about 6.31 kΩ/μm, 8.71 kΩ/μm and 2.46 kΩ/μm for the devices with 310 °C SDE1st, 450 °C SDElast and 550 °C SDElast processes, respectively. Such high RS/D values are ascribed to the nano-fins scheme and to insufficiently silicided S/D, as uncovered in ION dependence on the SDE process thermal budget, which is involved with the annealing steps in ION in the SDElast scheme; it seems that SDE1st prevails over SDElast in driving capability at a lower thermal budget level.ear mode . The RS/1st holds the advantage in ION at a lower thermal budget level, which results from the larger gate-to-SDE overlap , by performing SDE implantation before the spacer, as illustrated in VTH of −0.13 V, larger IOFF of 0.39 nA/μm and lower RS/D of 6.31 kΩ/μm for 310 °C SDE1st, with reference to 450 °C SDElast, justify this point, which was performed with the same silicidation annealing. Second, in the SDElast scheme, increasing the thermal budget will lower RS/D, and thus improve ION. The lowered RS/D is probably attributable to the reductions in silicide resistance and injection resistance , an improvement in ION was achieved for pFinFETs due to the reduced RS/D.In accordance with the findings in ION of conventional pFinFETs is significantly improved. Since no shift of VTH was evidenced with silicide  or without (w/o) silicide, owing to the lower RS/D  process flow c. As shosilicide c, the IO 2 kΩ/μm b. The lower RS/D b and VTHS/D  of 18 v/v was achieved at VDD in the range of 0.3 V ~ 0.8 V (NM), a piecewise approximation of the VTC was used here to determine the boundary of the transition zone. As illustrated in VOH, VIH) as well as for low  were defined by the position of points where dVOUT/dVIN = −1. NM for high input (NMH = VOH − VIH) of 0.17 V and NM for low input (NML = VIL − VOL) of 0.43 V at VDD = 0.8 V were obtained. VIN at VDD = 0.8 V. The maximum static power consumption for VIN sweeping from 0 V to 0.8 V at VDD = 0.8 V was less than 0.9 μW. In The CMOS inverter voltage transfer characteristics (VTC) at nFinFETs . The tra ~ 0.8 V b, suggesION values of 76.07 μA/μm and 48.57 μA/μm, ION/IOFF ratios of 7 × 105 and 1 × 106 at VDD = 0.8 V were obtained for pFinFETs and nFinFETs, respectively. Excellent CMOS inverter and functional CMOS RO are successfully explored, offering a new method of high-performance 3D VLSI CMOS integration.In conclusion, low-temperature complementary Schottky S/D FinFETs were proposed as the top-tier devices for 3D sequential integration and were experimentally demonstrated in this work. The thermal budget for fabrication was no more than 500 °C. and the entire process flow was fully compatible with current Si technology. With optimal SDE engineering and competitive