A series of branched and unbranched homologs of the lead-compound 2, and vinyl, ethinyl and benzyl analogues, were designed and synthesized. A comparison between the cytotoxic effect of these homologs and 2 on different human tumor cell lines was performed from a cell viability study using MTT with 48 h and 72 h incubations. In general, the compounds were less potent than CA-4, showing CC50 values ranging from 0.030 μM to 7.53 μM (MTT at 72 h) and 0.096 μM to 8.768 μM (MTT at 48 h). The antimitotic effect of the target compounds was demonstrated by cell cycle analysis through flow cytometry, and the cellular mechanism of cytotoxicity was determined by immunofluorescence. While the benzyl homolog 10 (LASSBio-2070) was shown to be a microtubule stabilizer, the lead-compound 2 (LASSBio-1586) and the methylated homolog 3 (LASSBio-1735) had microtubule destabilizing behavior. Molecular docking studies were performed on tubulin protein to investigate their binding mode on colchicine and taxane domain. Surprisingly, the benzyl homolog 10 was able to modulate EGFR phosphorylate activity in a phenotypic model. These data suggest LASSBio-2070 (10) as a putative dual inhibitor of tubulin and EGFR. Its binding mode with EGFR was determined by molecular docking and may be useful in lead-optimization initiatives.Combretastatin A-4 (CA-4,  Antimicrotubule agents are often used in antimicrobial therapy because their modularity and microtubule dynamics agents interfere with spindle capacity, pausing the cell cycle at the G2/M checkpoint. Therefore, the cell cycle comes to arrest in rapidly proliferating cancer cells leading to apoptosis by stabilizing or destabilizing the microtubule polymer ,2. Antim1) have been developed, aiming to maintain the broad-spectrum cytotoxicity profile of the natural compound and to circumvent its low water solubility and chemical instability, due to the inter-conversion of the olefinic double bond from the more active cis to less active trans-conformation [2) has been described as a stable CA-4 analogue. This acylhydrazone derivative displayed excellent results in tumor cell lines and in vivo cytotoxic activity, being able to decrease proliferation of the SF-295 and HCT-116 cell lines in the Hollow Fiber Assay. The ability of LASSBio-1586 to inhibit β-tubulin was confirmed, revealing this compound as a new anti-tubulin agent structurally correlated to CA-4. In attempt to establish the structural activity relationship of LASSBio-1586, Amaral and coworkers demonstrated the crucial role of the acylhydrazone framework and the trimethoxyphenyl subunit for cytotoxic activity [N-methylation of the sp3 nitrogen proved to be tolerated, resulting in compound LASSBio-1735 (3) with cytotoxic potency superior to LASSBio-1586 (2) against HL-60 cell lines [Several synthetic analogues of CA-4  cytotoxic activity, this paper describes the design and synthesis of new homologs (4–12) of compounds 2 and 3, their comparative cytotoxic profile, cellular mechanism of action and their binding mode with target proteins by molecular docking.Having in mind the role of homologation as an important strategy for molecular modification , and ass3–6) and branched (7) homologs, as well as the vinyl (8), ethinyl (9) and phenyl (10) homologs, are shown in 11) and ethyl-morpholine (12) subunits were also designed and are displayed in The design concept of the linear (2) and LASSBio-1735 (3) were synthetized using the previous methodology described by Amaral and coworkers [N-alkylation of the sp3 nitrogen of the intermediate 2 (LASSBio-1586). All the compounds were characterized by 1H NMR, 13C NMR, 13C DEPT-135 NMR, IR and mass spectroscopy, and their purity was determined by HPLC with a reverse-phase column (C-18) with an isocratic mobile phase in the same system of CH3CN: H2O (60:40 v/v).The homologs of LASSBio-1586 (CH) in the 13C NMR spectra of compounds 3–12, in addition to the presence of the characteristic band of the carbonyl group (ArCONRN=CHR) in the infrared spectra, allowed confirmation of selective N-alkylation, ruling out the hypothesis of C- or O-alkylated products (3). The physicochemical properties of the compounds reported in these studies are summarized in The presence of the characteristic signal of the imine carbon , LASSBio-1735 (3), CA-4 (1) and pelitinib as standards (The cytotoxic activity of compounds TT assay  against tandards . To detetandards . 8), all compounds exhibited moderate to high cytotoxic potency with CC50 (concentration that reduced the proliferation of cells by 50%) values of 39 nM to 8.5 μM. As expected, CA-4 (1) exhibited cytotoxic potency in the nanomolar range for all cell lines studied (except for MCF-7). While pelitinib, a non-selective irreversible EGFR  inhibitor, showed potency ranging in the nanomolar to low micromolar range, with emphasis on its effect on lung tumor lines overexpressing EGFR .As shown in 2) analogues can be seen by the comparison between the potency of compounds 3–12. As demonstrated in 4–6) were active, none of them were better than the N-methylated compound (3), suggesting that increasing the carbon chain does not result in optimizing the cytotoxic effect. The homologation strategy by adding fragments with 2sp or sp hybridization resulted in compounds 8–10. In general, the vinyl (8), ethinyl (9) and phenyl (10) homologs displayed similar cytotoxic potency, but still being less active than LASSBio-1735 (3). The impact of homologation strategy to cytotoxic activity of LASSBio-1586 (N-methylated homolog 3 (LASSBio-1735) had a high cytotoxic potency against acute myeloid leukemia (HL-60 cell lines), being 53 times more potent (CC50 39 nM) than LASSBio-1586 (CC50 2.08 µM), corroborating our previous report [3) also showed great cytotoxic potency in breast adenocarcinoma (MCF-7) with CC50 of 0.28 µM. These data reveal that this homologous was 27 and 31 times more potent on MCF-7 than LASSBio-1586 (CC50 7.53 µM) and CA-4 (1) (CC50 8.58 µM), respectively  on the MCF-7 cell line, emerged as quite promising result (1).MCF-7 is a cell line known as a multidrug-resistant breast cancer cell line. Its resistant phenotype is attributed to the overexpression of the multiple drug carrier P glycoprotein (Pgp) ,13. Pgp g result , althoug50 on tumor and non-tumor cell lines [To establish the cytotoxic selective index (SI) of LASSBio-1586 homologs, they were studied on human lymphocyte cell lines (GM16000), using the MTT assay. The SI was calculated by the ratio between the CC1), most of the tested compounds showed low SI (SI< 10). For the three tumor lines studied, pelitinib exhibited the best SI (1), LASSBio-1586 (2) and their homologs (3–12) may be associated with their nonspecific cytotoxic mechanism of action, acting as antimicrotubule or anti-tubulin agents.As depicted in  best SI , probabl2–12 revealed that two homologs (4 and 11) stood out by showing SI higher than 10. LASSBio-2071 (4) exhibited SI of 22.2, 17.0 and 13.3 to HL60, LoVo and PC9 cell lines, respectively, while LASSBio-2074 (11) displayed an SI of 33.3, 39.5 and 20.8. against HL60, LoVo and PC9 tumor cell lines (2 showed a favorable cytotoxic selectivity index only for the LoVo cell line (SI = 39).The calculated SI for compounds ll lines . The lea3–12 were also studied in an MTT assay of 48 h, in a comparative manner to LASSBio-1586 (2), CA-4 (1) and pelitinib standards. All compounds tested were less potent in the 48 h MTT (3 (LASSBio-1735) and 4 (LASSBio-2071) showed more promise than the others. The former showed CC50 values of 0.096 µM, 0.152 µM and 0.479 against the H1975, H292 and LoVo cell lines, respectively. The latter presented a CC50 of 0.903 µM, 0.147 µM and 0.244 µM on H1975, H292 and PC9.The cytotoxic potencies of compounds 48 h MTT  when com48 h MTT . Compoun10) showed favorable SI, although only for two tumor cell lines (H292 and PC9). All others, including the standard drugs, exhibited SI values less than 10.SI values in an MTT assay of 48h were also determined  and only3) on lung adenocarcinoma cell lines, including H1975 (the T790M-positive cell line that harbors the EGFRL858R/T790M double mutation), both at 72 h and 48 h MTT, this compound was selected for further study. In parallel, we also chose LASSBio-2070 (10) that showed good cytotoxic potency at 48 h MTT, allowing us to compare the impact of homologation by a phenyl introduction on the cytotoxic cellular mechanism of LASSBio-1735 (3).The excellent cytotoxic effect on MCF-7 observed for the MTT assay at 72 h of incubation was not observed when the experiment was performed at 48 h. Considering the great cytotoxic potency of LASSBio-1735 (1) and LASSBio-1586 (2) are antimitotic agents, we decided to investigate the ability of the new homologs 3–12 to interrupt M phase of the cell cycle, promoting mitotic catastrophe and leading the cell to apoptosis. Flow cytometry was used for cell cycle analysis to estimate the percentages of a cell population in the different phases of the cell cycle. The analysis was performed using the lung cancer cell line H1975 treated with CA-4 , LASSBio-1586 , LASSBio-1735 , LASSBio-2070  and LASSBio-2074 . As depicted in 50 determined by measuring the cell viability using MTT assay after 72 h of incubation) revealed very similar behavior between the target compounds and the standard drugs. They decreased cells in phase G0/G1 and S and increased cells in phase G2/M , LASSBio-1735 , LASSBio-2070 , and LASSBio-2074 , first identified as antimitotic agents , vincristine (0.5 µM) and taxol (0.1 µM) were used as standard controls. Compounds were assayed at a concentration two times higher than their CC50 to demonstrate their effect on the cells.Antitubulin agents have been broadly classified into destabilizing or stabilizing agents, based on their interference with the dynamic balance of polymerization or depolymerization of microtubules . To elucc agents , we perf1), vincristine and LASSBio-2070 (10) cell morphology was changed, assuming a rounded shape. The treatment with taxol, LASSBio-1586 (2), LASSBio-1735 (3) and LASSBio-2074 (11) resulted in cells with a more spread-out morphology. It was possible to observe a decrease in the number of cells for all fields treated with the compounds and the positive standards, probably indicating cell death from the high concentration of the drug used. The immunofluorescence studies demonstrated that CA-4 (1), vincristine and LASSBio-2070 (10) exhibited a typical response of microtubule destabilizing agents, or microtubules polymerization inhibitors, by binding to tubulin protein [1), vincristine and LASSBio-2070 (10). As shown in  protein . As show2), LASSBio-1735 (3) and LASSBio-2074 (11) displayed a cell morphology similar to microtubule-stabilizing drugs such as taxol, with consequent depolymerization inhibition (2) that described this prototype as a structural analogue of CA-4 (1) capable of binding to the colchicine site of tubulin in silico studies [On the other hand, cells treated with LASSBio-1586 (hibition . In thes1) binds to tubulin at the domain known as the colchicine binding site. Binding to this site induces microtubule depolymerization in a mechanism typical of microtubule destabilizer agents. This mechanism were observed only for the benzyl homolog 10, while LASSBio-1586 (2), the N-methyl homolog 3 and even the polar derivative 11 exhibited a cellular mechanism like microtubule stabilizer agents such as taxol, inducing polymerization of tubulin, stabilizing the polymer and preventing depolymerization [2, 3 and 11 interact with the taxane domain, while compound 10 binds to the colchicine domain on tubulin, docking studies were performed and are discussed later.Taken together, the results suggest that homologation has a significant impact on the binding domain of the compound to tubulin. Microtubule dynamics is controlled mainly by three established drug domains on tubulin, known as taxane, vinca alkaloid and colchicine sites. CA-4 , LASSBio-1735  and LASSBio-2070 , exhibited a comparable EGFR inactivation index, although only the result for the benzyl homolog 10 was statistically significant. Osimertinib, a third-generation EGFR inhibitor that has been reported as a potent and selective inhibitor of EGFR mutations forms  [10) as able to modulate EGFR activity in a phenotypic model by inhibiting the phosphorylation of EGFR on PC-9 cell line  , exhibitell line . Therefo2 and its methyl homolog 3 versus that found for the benzyl analogue 10. To understand the molecular reasons for LASSBio-1586 (2) and LASSBio-1735 (3) profiles similar to taxol, and the behavior of LASSBio-2070 (10) similar to CA4, we performed a molecular docking study using tubulin structure co-crystallized with colchicine and taxol retrieved from the Protein Data Bank server (PDB ID: 5XIW and 6WVR). As previously discussed, the experimental data obtained from the immunofluorescence studies  anticipa1) , and highly polarizable amino acids, such as Cys239 and Met257. A steric clash, promoted by colchicine A and ana1) B, with a1), where interactions with Cys239, Leu246, Leu253, Asn256 Met257, Ala314, Ile316, Lys350, Ala352, Ile368 amino acids are described as pharmacophoric [10) (10), in which the cells treated with this ligand displayed a cell morphology similar to that with microtubule-destabilizing drugs such as CA-4 (1), with polymerization inhibition  C at the hibition G.10) allowed a similar fitting at the colchicine binding site when compared with CA-4 (1). Additionally, LASSBio-2070 (10) promoted more interactions than CA-4 (1) due to the extra phenyl group that interacts with amino acids within the α-subunit . This probably justified the molecular reason for LASSBio-2070 (11) to act as a destabilizing agent, mimicking CA-4 (1) pharmacological behavior as demonstrated in the following docking study.It is also possible to visualize the increase in the number of hydrophobic interactions promoted by the benzyl group with the residues of Val181, Asn256, Met257, Ala314, Ala315, and Lys350. Other fragments of that structure interacted with additional residues important for selectivity of ligands at the colchicine site that act as tubulin destabilizers, such as Asn101, Leu240, Ile316 ,33. ThesAs shown in 2) . The imine phenyl group interacts with residues from the M-loop  with more intensity in terms of distance when compared with hydrophobic interactions performed by LASSBio-1735 (3) (2) on the M-loop. These results possibly contribute to understanding the mechanism by which this molecule acts as a MSA [LASSBio-1586 (2) E showed  subunit . The N-a1735 (3) F for theas a MSA ,37.3) adopts a different conformation (sin-periplanar) related to the amide group (O=C-N-C), its interactions between the trimethoxyphenyl ring and the H7-loop are similar, except for the loss of the hydrogen bond with Arg278, and van der Waals interaction with His229, this loss being counterbalanced by the hydrophobic interaction with Ala233. The main difference occurs when analyzing the interaction at the hydrophobic pocket on the M-loop, in which only the methyl group of the N-acylhydrazone subunit is facing towards the cavity, while the phenylmethylidene group is directed to the H7-loop. The change of conformation favors the imine phenyl group to make additional pi-stacking interaction with the Phe272 present on the S9-loop. Together, this result also revealed the capacity of the LASSBio-1735 (3) to act as a MSA like LASSBio-1586 (2).Although LASSBio-1735  . Docking2) (ChemPLP fitness: 58.3), LASSBio-1735 (3) (ChemPLP fitness: 51.7), and LASSBio-2070 (10) (ChemPLP fitness: 71.1) fit into the ATP binding site  (ChemPLP fitness: 58.3), the non-homologated compound, projects the phenyl group deeply into the hydrophobic pocket, making hydrophobic interactions with residues that interact with the aniline subunit of erlotinib, namely Leu764, Met742, Thr766, and Lys721. The homologation strategy  greatly 3 (ChemPLP fitness: 51.7) adopts an anti-periplanar conformation (regarding the methyl group and carbonyl oxygen atom) that is in accordance with that previously reported for N-methyl-acylhydrazones [The methyl homolog drazones ,40. Cons11) (ChemPLP fitness: 71.1), the benzyl homolog of LASSBio-1586 (2), occupies both regions, resulting in higher scoring. It makes hydrophobic interactions with Ala719, Lys721, and Leu820 in the hydrophobic pocked occupied by LASSBio-1586 (2), and the aniline subunit of erlotinib. Additionally, LASSBio-2070 (10) is also able to interact with Phe699 and Val702 in the Gly-rich loop, comparably to LASSBio-1735 (3).LASSBio-2070 (10). Studies aiming to develop inhibitors of osimertinib-resistant EGFR highlight that a strong non-covalent complex with the enzyme, in which the inhibitor should occupy multiple pockets of the enzyme, might be favorable to overcome resistance [The covalent inhibitor osimertinib , which wsistance ,42,43. 1H NMR, 13C NMR and 13C DEPT 135 NMR spectra were determined in deuterated dimethyl sulfoxide using a Varian NMR at 400 MHz, 500 MHz and 125 MHz. Chemical shifts are given in parts per million (d) from tretramethylsilane as internal standard and coupling constant values (J) are given in Hertz (Hz). Signal multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet) and br . All NMR spectra are found in the All reagents and solvents were purchased from commercial suppliers. The reactions were monitored by thin layer chromatography, which was performed on aluminum sheets pre-coated with silica gel 60  to a thickness of 0.25 mm. The chromatograms were viewed under ultraviolet light (254–365 nm). For column chromatography Merck silica gel (230–400 mesh) was used. −1) and the spectra are found in the Infrared (IR) spectra were obtained with an FTIR Thermo Scientific™ Nicolet™ iS10 spectrophotometer in ATR mode using ruby crystal support, and the absorption values were expressed in inverse centimeters (cm3CN: H2O (6:4 v/v). Mass spectrometry was obtained by positive ionization at BrukerAmaZon SL and data was analyzed in Compass 1.3.SR2 software .The purity of compounds was determined by HPLC (95%) using the Shimadzu—LC20AD apparatus, a Kromasil 100-5 C18 (4.6 mm × 6250 mm) column and the SPD-M20A detector (Diode Array) at 254 nm for quantification of analyte at a 1 mL/min constant flux. The injector was programmed to inject a volume of 20 µL. The isocratic mobile phases used were CH2 was prepared following the methodology previously described by Amaral and coworkers and shown in 1H NMR, 13C NMR and IR data agree with previous reports [Compound  reports . Overall yield: 80%, white solid, m.p. 138–139 °C.1H-NMR  δ (ppm): 11.71 , 8.48 , 7.73 , 7.49–7.45 , 7.24 , 3.87 , 3.73 .13C-NMR  δ (ppm): 163.0 (C-7), 153.1 (C-3/C-5), 148.2 (C-9), 140.9 (C-4), 134.7 (C-10), 130.5 (C-13), 129.3 (C-11/C-15), 128.9 (C-1), 127.5 , 105.6 (C-2/C-6), 60.5 (C-17), 56.5 (C-16/C-18)−1): 3181 (N-H), 2995–2839 , 1646 (-C=O), 1624–1452 (C=C), 1584 (C-N), 1118 (C-O).I.R. ). MS: m/z = 315.1 (M+H)+. 99.4% purity in HPLC   in 7 mL of acetone was added to 3.82 mmol of sodium carbonate. The resultant suspension was stirred at room temperature for 50 min. Then, the appropriate alkyl halide or benzyl bromide  was added to the suspension and the reaction mixture was heated for 24 h at 40 °C. The acetone was removed under reduced pressure and the resulting material was suspended in 2 mL of ethanol, filtered, and washed with petroleum ether. Recrystallization of the target products was performed using a mixture of ethanol/water. Yields and characterization pattern are described below: A solution of LASSBio-1586 (E)-N’-benzylidene-3,4,5-trimethoxy-N-methylbenzohydrazide . . 1H-NMR  δ (ppm): 8.04 , 7.59–7.57 , 7.42–7.34 , 7.00 , 3.77 , 3.75 , 3.50 .13C-NMR  δ (ppm): 169.5 (C-7), 152.2 (C-3/C-5), 140.8 (C-9), 139.5 (C-4), 135.3 (C-10), 130.8 (C-13), 129.9 (C-1), 129.2 (C-11/C-15), 127.2 (C-12/C-14), 108.0 (C-2/C-6), 60.6 (C-17), 56.3 (C-16/C-18), 29.4 (C-8).−1): 2921–2832 , 1641 (-C=O), 1580 (C-N), 1503–1453 (C=C), 1131 (C-O).IR ). MS: m/z = 329.1 (M+H)+.99.8% purity in HPLC -N’-benzylidene-N-ethyl-3,4,5-trimethoxybenzohydrazide . δ (ppm): 8.10 , 7.60–7.58 , 7.42–7.34 , 6.98 , 4.18 , 3.77 , 3.75 , 1.20 .13C NMR  δ (ppm): 169.2 (C-7), 152.2 , 140.3 (C-10), 139.6 (C-4),135.4 (C-11), 131.0 (C-14), 129.9 (C-1), 129.1 , 127.3 , 108.0 , 60.6 (C-18), 56.4 , 36.3 (C-8), 11.3 (C-9)13C DEPT-135 NMR  δ (ppm): 140.43 (s), 129.98 (s), 129.19 (s), 127.30 (s), 108.08 (s), 60.66 (s), 11.38 (s).−1): 2968–2833 , 1633 (-C=O), 1579 (C-N), 1510–1417 (C=C), 1125 (C-O).IR ). MS: m/z = 342.9 (M+H)+.99.1% purity in HPLC -N’-benzylidene-3,4,5-trimethoxy-N-propylbenzohydrazide . δ (ppm): 8.09 , 7.59–7.57 , 7.41–7.31 , 6.96 , 4.09 , 3.77 , 3.74 , 1.66–1.60 , 0.97 .13C-NMR  δ (ppm): 169.6 (C-7), 152.2 (C-3/C-5), 140.4 (C-9), 139.5 (C-4), 135.4 (C-12), 131.1 (C-15), 130.0 (C-1), 129.1 (C-13/C-17), 127.3 , 107.9 , 60.6 (C-19), 56.3 , 42.7 (C-8), 19.0 , 11.67 (C-10).13C DEPT-135 NMR  δ (ppm): 140.43 (s), 130.00 (s), 129.19 (s), 127.30 (s), 107.95 (s), 60.65 (s), 11.68 (s).−1): 3050 (NH), 2940–2800 , 1648,3 (-C=O), 1581–1449 (C=C), 1123 (C-O).IR ). MS: m/z = 379.1 (M+H)+.98% purity in HPLC -N’-benzylidene-N-butyl-3,4,5-trimethoxybenzohydrazide . δ (ppm): 8.09 , 7.59–7.56 , 7.41–7.34 , 6.96 , 4.12 , 3.77 , 3.74 , 1.63–1.57 , 1.44–1.22 , 0.95 .13C-NMR  δ (ppm): 169.5 (C-7), 152.2 (C-3/C-5), 140.3 (C-12), 139.6 (C-4), 135.4 (C-13), 131.1 (C-16), 130.0 (C-1), 129.1 (C-14/C-18), 127.3 (C-15/C-17), 108.0 (C-2/C-6), 60.6 (C-20), 56.4 (C-19/C-21), 40.9 (C-8), 27.8 (C-9), 20.1 (C-10), 14.2 (C-11).13C DEPT-135 NMR  δ (ppm): 140.37 (s), 130.01 (s), 129.19 (s), 127.32 (s), 108.00 (s), 60.66 (s), 56.41 (s), 14.22 (s).−1): 3050 (NH), 2940–2800 , 1648,3 (-C=O), 1581–1449 (C=C), 1123 (C-O). IR ). MS: m/z = 371.1 (M+H)+.99.4% purity in HPLC -N’-benzylidene-N-(sec-butyl)-3,4,5-trimethoxybenzohydrazide  δ (ppm): 8.45 , 7.75–7.73 , 7.49–7.43 , 7.05 , 5.13–5.07 , 3.76 , 3.73 , 1.80–1.67 , 1.36 , 0.97 .13C-NMR  δ (ppm): 162. 9 (C-7), 157.0 , 152.4 (C-12), 139.9 (C-4), 135.2 (C-13), 130.7 (C-16), 129.2 , 128.0 , 126.5 (C-1), 108.3 (C-2/C-6), 74.1 (C-8), 60.6 (C-20), 56.3 , 28.7 (C-10), 19.4 (C-9), 10.12 (H-11).13C NMR DEPT 135  δ 157.06 (s), 130.78 (s), 128.29 (s), 128.03 (s), 108.30 (s), 74.16 (s), 60.63 (s), 56.34 (s), 19.42 (s), 10.13 (s).−1): 3050 (NH), 2968–2835 , 1640.2 (-C=O), 1576–1462 (C=C), 1123 (C-O).IR ). MS: m/z= 371.30 (M+H)+. 99.4% purity in HPLC -N-allyl-N’-benzylidene-3,4,5-trimethoxybenzohydrazide . δ(ppm): 7.95 , 7.55 , 7.42–7.35 , 7.03 , 5.95–5.87 , 5.23 , 5.18 , 4.81 , 3.79 , 3.76 .13C-NMR  δ (ppm): 169.4 (C-13), 152.3 (C-17), 135.1 (C-1), 131.3 (C-3), 130.6 (C-2), 130.1 (C-18), 129.2 (C-25), 127.3 (C-21), 117.2 (C-26), 108.1 (C-4/C-6), 60.6 (C-11), 56.4 (C-10/C12), 43.91 (C-24).−1): 3090 (NH), 2997–2831 , 1650.8 (-C=O), 1636–1452 (C=C), 1122 (C-O).IR ). MS: m/z = 355.2 (M+H)+. 98.3% purity in HPLC -N’-benzylidene-3,4,5-trimethoxy-N-(prop-2-yn-1-yl)benzohydrazide . δ (ppm): 8.14 , 7.61–7.58 , 7.45–7.38 , 7.04 , 4.99 , 3.79 , 3.76 .13C-NMR  δ (ppm): 168.9 (C-7), 152.3 , 141.8 (C-11), 140.0 (C-4), 134.8 (C-12), 130.3 (C-15), 129.8 (C-12), 129.3 (C-17), 127.3 , 108.1 , 77.9 (C-9), 75.7 (C-10), 60.6 (C-19), 56.4 , 31.5 (C-10)−1): 3242 (C-H), 2935–2834 (CH3), 1641(C=O), 1583 (C-N), 1126 (C-O).IR ). MS: m/z = 352.9 (M+H)+. 97.6% purity in HPLC -N-benzyl-N’-benzylidene-3,4,5-trimethoxybenzohydrazide . δ (ppm): 7.9 , 7.48–7.46 , 7.40–7.33 , 7.30–7.26 , 7.10 , 5.44 , 3.81 , 3.77 .13C-NMR  δ (ppm): 170.0 (C-7), 152.3 (C-2/C-5), 141.1 (C-15), 139.9 (C-4), 135.8 (C-16), 134.9 (C-9), 130.5 (C-19), 130.1 (C-12), 129.2 , 127.6 (C-1), 127.2 ), 108.3 (C-2/C-6), 60.6 (C-23), 56.5 (C-22/C-24), 45.0 (C-8).13C NMR DEPT 135  δ 141.17 (s), 130.19 (s), 128.25 (s), 127.29 (s), 127.27(s),108.30 (s), 60.70 (s), 56.50 (s). −1): 3090 (NH), 3000–2830 , 1635.8 (-C=O), 1607–1454 (C=C), 1125 (C-O).IR ). MS: m/z = 405.2 (M+H)+. 98.4% purity in HPLC -N-(benzylideneamino)-N-glycinate .Ethyl  δ (ppm): 7.99 , 7.57–7.55 , 7.46–7.37 , 7.04 , 5.00 , 4.22–4.17 , 3.78 , 3.76 , 1.24 .13C-NMR  δ (ppm): 169.5 (C-9), 167.7 (C-7),152.3 (C-5/C-3), 141.1 (C-12), 140.0 (C-4), 134.9 (C-13), 130.2 (C-1), 129.8 (C-16), 129.3 (C-14/C-18), 127.3 (C-15/C-17), 108.1 (C-2/C6), 61.6 (C-8), 60.6 (C-20), 56.4 (C-19/C-21), 43.8 (C-10), 14.5 (C-11).13C DEPT-135 NMR  δ 141.16 (s), 130.31 (s), 129.31 (s), 128.21 (s), 127.38 (s), 108.17 (s), 60.68 (s), 56.40 (s), 40.18 (s), 14.51 (s).−1): 3000 (NH), 2941–2838 , 1787,8 (-C=O), 1664,2 (-C=O), 1125 (C-O).IR ). MS: m/z = 401.0 (M+H)+.98.0% purity in HPLC -N’-benzylidene-3,4,5-trimethoxy-N-(2-morpholinoethyl)benzohydrazide . δ (ppm): 8.12 , 7.58–7.55 , 7.42–7.34 , 6.96 , 4.26 , 3.77 , 3.75 , 3.57 , 2.57 , 2.5 .13C-NMR  δ (ppm): 169.6 (C-7), 152.2 (C-5/C-3), 140.6 (C-14), 139.6 (C-4), 135.3 (C-15), 131.0 (C-1), 130.0 (C-18), 129.2 (C-20/C-16), 127.3 (C-19/C17), 107.9 (C-2/C-6), 66.7 (C-12/C13), 60.6 (C-22), 56.4 (C-10/C-11/C-21/C-23), 54.1 (C-8), 53.9 (C-9).13C DEPT-135 NMR  δ (ppm): 140.66 (s), 130.05 (s), 129.21 (s), 127.35 (s), 107.96 (s), 60.66 (s), 56.40 (s).−1): 3000 (NH), 2958–2843 , 1660.8 (-C=O), 1608–1462 (C=C), 1127 (C-O).IR ). MS: m/z = 428.2 (M+H)+.98.4% purity in HPLC , PC-9  H292 (mucoepidermoid pulmonary carcinoma—EGFR wild type), LoVo  and GM16000 (lymphocyte). All the human cancer cell lines were purchased from Cell Bank of Rio de Janeiro—BCRJ. They were cultured in RPMI 1640 medium with 10% (v/v) FBS, 2 mM glutamine, at 37 °C in a humidified incubator containing 5% CO2. The target compounds were tested (0.0001–100 µM) for cytotoxic activity against selected human cancer cell lines, such as HL-60 (acute promyelocytic leukemia), MCF-7 (mammary adenocarcinoma), PC-3 (prostate adenocarcinoma), H1975 , CA-4 (1) and pelitinib at 37 °C, in triplicate. The medium was incubated with 20 μL of 5 mg/mL MTT solution for 3 h in a humidified incubator containing 5% CO2 [The cytotoxicity of the compounds was evaluated by the MTT assay. The cell lines were cultured in 96 well plates for 72 h with a density of 0.1 × 10g 5% CO2 .50 values were calculated from the means ), using GraphPad Prism®, version 7.The purple-colored formazan crystals formed in the wells were dissolved in DMSO and their absorbance was measured at 595 nm with a microplate reader . The CC1) (0.04 µM) was used as a control for the cell cycle assay and the compounds were tested using a concentration equivalent to CC50 value of each compound determined in the MTT assay of 72 h for cell cycle kit: LASSBio-1586 (2) (2.0 µM), LASSBio-1735 (3) (0.08 µM), LASSBio-2070 (10) (3.0 µM) and LASSBio-2074 (11) (1.5 µM. For the EGFR assay osimertinib (0.05 µM) and erlotinib (0.1 µM) were used as standards, with LASSBio-1586 (2) (0.06 µM), and the compounds LASSBio-1735 (3) (0.02 µM) and LASSBio-2070 (10) (0.03 µM). Flow cytometry was applied to help elucidate the mechanism of action of the studied compounds, both to verify the cell cycle phase and to verify the action on EGFR protein kinase. CA-4 (5 per well. The cells were treated with the selected compounds for 72 h. Cells were harvested and stained according to the protocols of The Guava Muse® Cell Cycle Kit (MCH100106) to quantitatively measure the percentage of cells in the G2/M phases of the cell cycle and the EGFR-RTK Activation Dual Detection Kit (MCH200102) (Luminex®) to measure EGFR phosphorylation in relation to total EGFR expression in the cell population, according to the manufacturer’s instructions for use in test kits using a Guava Muse® Cell Analyzer (Luminex®). Each experiment was performed at least three times independently [The anti-tubulin effect was evaluated by seeding H1975, H292 and PC-9 into 6-well plates at a density of 2 × 10endently ,45.® EGFR-RTK Activation Dual Detection Kit  containing the anti-EGFR-PECy5 antibody for measuring total EGFR and the anti-phospho-EGFR antibody (Tyr1173)—Alexa Fluor® 555 for measuring EGFR, activated and performed according to the manufacturer’s instructions. The cells were trypsinized, centrifuged at 300× g for 5 min, and, after the labeling protocol, the samples were taken for reading in the Guava Muse® Cell Analyzer. In this test the compounds were tested and used at concentrations 10 times lower than the CC50 in 72 h.The evaluation of EGFR activation and phosphorylation was performed by the Guava Muse5 cells/well and treated for 48 h with compounds CA-4 (1) (0.3 µM), Taxol (0.1 µM), Vincristine (0.5 µM), LASSBio-1586 (2) (10 µM), LASSBio-1735 (3) (0.5 µM), LASSBio-2070 (10) (4.5 µM); and LASSBio-2074 (11) (20 µM), at concentrations of 5 times the 48 h CC50. After incubation, cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. They were then permeabilized with 0.5% Triton-X 100 in PBS 3 times for 10 min. The same solution was used for all subsequent washing steps. Cells were incubated with an anti-beta-tubulin antibody for 1 h at 37 °C. After incubation, cells were washed for 30 min and incubated with Alexa Fluor® 488, conjugated secondary antibody for 1 h at 37 °C, and nuclei were labeled with DAPI (0.1 mg/mL in 0.9% NaCl) [The cell line H1975 was chosen for this experiment, since the size of the cells would be best to allow the most effective visualization of the effects of the compounds in the microtubules, which were labeled together with the cell nucleus. Cells were plated at a concentration of 5 × 109% NaCl) .http://imagej.nih.gov/ij/ (accessed on 1 September 2021).Live cultured cells grown in coverslips were mounted in Prolong Gold solution  and were imaged on a Leica TCS SPE confocal microscope  using 63x 1.3 N.A. oil immersion and the images acquired by the LAS X software. Cell morphology, focusing on the microtubules was analyzed, qualitatively, using the public domain NIH ImageJ Program , and the lowest energy tautomer was energy-minimized at the semi-empirical PM6 level. Protonation state of docked compounds was determined considering the major species at pH 7.4, using ACD/Percepta program. The EGFR structure co-crystallized with erlotinib was retrieved from the Protein Data Bank server (PDB ID: 1M17) . The tub10 (LASSBio-2017) is suggested as a putative dual inhibitor of tubulin and EGFR, displaying good cytotoxic potency in different human tumor cell lines. The mode of interaction with the target proteins was studied by molecular docking and the information will used in a further lead-optimization step. To the best of our knowledge our work describes, for the first time, the identification by phenotypic models of a compound of low structural complexity that acts by modulating, simultaneously, microtubules and EGFR activities. The benzyl homolog


Correction: Journal of Translational Medicine (2023) 21:52 10.1186/s12967-023-03895-0Following publication of the original article , we have1†, Fengnan Zhi1†, Chunpeng Shi1, Jincheng Xu1, Yuqiu Chao1, Juan Xu3, Yanan Jiang1,2*, Yunlong Bai1,2* and Baofeng Yang1,2,4Xiuyun Shen


Fallopia multiflora (Thunb.) Harald. contains various pharmacodynamically active glycosides, such as stilbene glycosides, anthraquinone (AQ) glycosides, and flavonoid glycosides. Glycosylation is an important reaction in plant metabolism that is generally completed by glycosyltransferase in the last step of the secondary metabolite biosynthesis pathway, and it can improve the beneficial properties of many natural products. In this study, based on the transcriptome data of F. multiflora, we cloned two Uridine-diphosphate-dependent glycosyltransferases (UGTs) from the cDNA of F. multiflora (FmUGT1 and FmUGT2). Their full-length sequences were 1602 and 1449 bp, encoding 533 and 482 amino acids, respectively. In vitro enzymatic reaction results showed that FmUGT1 and FmUGT2 were promiscuous and could catalyze the glycosylation of 12 compounds, including stilbenes, anthraquinones, flavonoids, phloretin, and curcumin, and we also obtained and structurally identified 13 glycosylated products from both of them. Further experiments on the in vivo function of FmUGT1 and FmUGT2 showed that 2, 3, 5, 4’- tetrahydroxy stilbene-2-dO-β--glucoside (THSG) content in hairy roots was elevated significantly when FmUGT1 and FmUGT2 were overexpressed and decreased accordingly in the RNA interference (RNAi) groups. These results indicate that FmUGT1 and FmUGT2 were able to glycosylate a total of 12 structurally diverse types of acceptors and to generate O-glycosides. In addition, FmUGT1 and FmUGT2 efficiently catalyzed the biosynthesis of THSG, and promoted the production of AQs in transgenic hairy roots.The traditional Chinese medicine plant  Polygonaceae family, Fallopia multiflora is generally used in traditional Chinese medicine and contains various major active components including stilbenes, anthraquinones (AQs), flavonoids, and polysaccharides (dO-β--glucoside (THSG) and AQ glucosides. Researches suggested that THSG may be the attractive candidate for Parkinson’s disease  of ize silk . These rin vitro, and found that GTs in F. multiflora could catalyze the glycosylation of hydroxylated resveratrol to form THSG. In this study, we performed prokaryotic expression, protein purification, and enzymatic product identification studies on FmUGT1 and FmUGT2 and not only discovered novel tool enzymes that promoted the development of different compounds’ glycosylation but also provided important results in the study of THSG the biosynthesis pathway. Additionally, we also constructed a glycosides-rich transgenic system by regulating the transcription levels of FmUGT1 and FmUGT2 in the hairy roots of F. multiflora, which provided a production platform for the biosynthesis of THSG and AQs.F. multiflora were collected from Deqing County, Zhaoqing City, Guangdong Province, China . These materials were well cleaned and dried then flash-frozen with liquid nitrogen and stored at -80°C. The seeds of F. multiflora were obtained from the same location and were sterilized using 0.1% mercuric chloride for 18 min, after which the seeds were flushed thoroughly with sterile distilled water and germinated in MS solid medium -;1H-NMR  δ 7.50 , 6.88 , 6.70 , 6.60 , 6.75 , 7.39 , 4.63 . 13C-NMR  δ 157.1 (C-4’), 154.0 (C-5), 151.1 (C-11b), 136.6 (C-1’’), 132.4 (C-3), 128.6 (C-1), 128.5 (C-β), 128.0 , 121.0 (C-α), 115.5 , 104.7 (C-1’’), 104.0 (C-6), 103.6 (C-1’’’), 102.1 (C-4), 77.1 (C-5’’), 77.0 (C-5’’’), 76.2 (C-3’’), 75.7 (C-3’’’), 74.2 (C-2’’), 73.4 (2’’’), 70.0 (C-4’’), 69.6 (C-4’’’), 61.00 (C-6’’), 60.6 (C-6’’’) (ESI-MS (C-6’’’) .dO-β--glucoside(2) compound 3a : Piceatannol 3’-m/z 405.1198 [M-H]-;1H-NMR  δ 7.44 , 7.06 , 6.89 , 6.85 , 6.79 , 6.39 , 6.12 , 4.76 , 3.77 - 3.11 . 13C-NMR  δ 158.50 , 146.70 (C-3’), 145.66 (C-4’), 139.22 (C-1), 128.91 (C-1’), 127.84 (C-β), 126.42 (C-α), 122.13 (C-6’), 115.94 (C-2’), 114.30 (C-5’), 104.45 (C-6), 102.50 (C-4), 101.90 (C-1’’), 77.41 (C-3’’), 76.02 (C-5’’), 73.44 (C-2’’), 70.18 (C-4’’), 60.98 (C-6’’) (ESI-MS  (C-6’’) .dO-β--glucoside:(3) compound 4a : Emodin 8-m/z 431.1000 [M-H]-; 1H-NMR  δ 7.55 , 7.27 , 7.22 , 6.95 , 5.13 , 3.70 - 3.18 , 2.44 . 13C-NMR  δ 190.06 (C-9), 181.25 (C-10), 163.86 , 161.62 (C-1), 148.60 (C-3), 134.95 (C-11), 132.97 (C-14), 124.31 (C-2), 120.60 (C-4), 113.64 (C-12), 110.89 (C-13), 109.08 (C-5), 108.87 (C-7), 99.94 (C-1’’), 77.28 (C-5’’), 76.23 (C-3’’), 73.11 (C-2’’), 69.50 (C-4’’), 60.55 (C-6’’), 21.60 (CH3-3) (ESI-MS  (CH3-3) .dO-β--glucoside:(4) compound 6a : Kaempferol 3-m/z 447.0950 [M-H]-; 1H-NMR  δ 8.05 , 6.88 , 6.38 , 6.19 , 5.23 , 3.70 , 3.54 , 3.44 , 3.39 , 3.21 – 3.17 . 13C-NMR  δ 179.46 (C-4), 166.58 (C-7), 163.01 (C-5), 161.58 (C-4’), 159.03 (C-8a), 158.55 (C-2), 135.45 (C-3), 132.27 , 122.81 (C-1’), 116.09 , 105.57 (C-4a), 104.17 (C-1’’), 100.11 (C-6), 94.90 (C-8), 78.40 (C-5’’), 75.73 (C-2’’), 71.36 (C-4’’), 62.63 (C-6’’) (ESI-MS  (C-6’’) .dO-β--glucoside:(5) compound 7a : Apigenin 7-m/z 431.1017 [M-H]-; 1H-NMR  δ 12.97 , 10.42 , 7.97 , 6.95 , 6.87 , 6.84 , 6.45 , 5.41 , 5.14 , 5.07 , 4.62 , 3.72 , 3.52 – 3.18 . 13C-NMR  δ 181.99 (C-4), 164.69 (C-7), 162.96 (C-2), 161.35 (C-9), 161.11 (C-4’), 156.94 (C-5), 128.60 (C-2’), 121.03 (C-1’), 116.62 (C-3’), 115.99 (C-5’), 105.33 (C-10), 103.11 (C-3), 99.90 (C-6), 99.52 (C-1’’), 95.26 (C-8), 77.15 (C-3’’), 76.38 (C-5’’), 73.09 (C-2’’), 69.55 (C-4’’), 63.08 (C-6’’) (ESI-MS  (C-6’’) .dO-β--glucoside:(6) compound 8b : Luteolin 7-m/z 447.0977 [M-H]-; 1H NMR  δ 12.99 , 7.45 , 7.43 , 6.91 , 6.79 , 6.76 , 6.45 , 5.09 , 3.71 , 3.50 , 3.51 – 3.23 . 13C-NMR  δ 181.87 (C-4), 164.43 (C-2), 162.92 (C-7), 161.12 (C-5), 156.94 (C-9), 149.82 (C-4’), 145.69 (C-5’), 121.40 (C-1’), 119.16 (C-2’), 115.95 (C-3’), 113.54 (C-6’), 105.33 (C-10), 99.89 (C-1’’), 99.52 (C-6), 94.72 (C-8), 77.14 (C-3’’), 76.32 (C-5’’), 73.06 (C-2’’), 69.50 (C-4’’), 60.58 (C-6) (ESI-MS 58 (C-6) .dO-β--glucoside:(7) compound 9a : Myricetin 3-m/z 479.0887 [M-H]-; 1H-NMR  δ 12.63 , 10.91 , 9.18 , 8.95 , 7.20 , 6.38 , 6.20 , 5.48 , 3.60 - 3.11 . 13C-NMR  δ 177.41 (C-4), 164.02 (C-7), 161.25 (C-5), 156.25 , 145.36 , 136.56 (C-4’), 133.51 (C-3), 120.06 (C-1’), 108.54 , 103.97 (C-10), 100.90 (C-1’’), 98.58 (C-6), 93.31 (C-8), 77.60 (C-3’’), 76.56 (C-5’’), 73.92 (C-2’’), 69.93 (C-4’’), 61.09 (C-6’’) (ESI-MS  (C-6’’) .dO-β--glucoside:(8) compound 9c : Myricetin 5’-m/z 431.0891 [M-H]-; 1H-NMR  δ 12.45 , 10.79 , 9.40 , 9.31 , 8.84 , 7.54 , 7.53 , 6.47 , 6.19 , 4.72 , 3.77 – 3.19 . 13C-NMR  δ 175.82 (C-4), 163.89 (C-7), 160.62 (C-5), 155.60 (C-9), 146.30 (C-2), 145.94 (C-3’), 145.50 (C-4’), 137.31 (C-3), 136.03 (C-1’), 121.01 (C-5’), 110.74 (C-2’), 107.55 (C-10), 102.99 (C-6’), 102.85 (C-1’’), 98.14 (C-6), 93.55 (C-8), 77.21 (C-3’’), 75.85 (C-5’’), 73.28 (C-2’’), 69.58 (C-4’’), 60.59 (C-6’’) (ESI-MS  (C-6’’) .d-O-β--glucoside:(9) compound 10a : Hesperetin 7m/z 463.1314 [M-H]-; 1H-NMR  δ 12.04 , 9.13 , 6.97 – 6.85 , 6.16 , 6.13 , 5.49 , 4.97 , 3.77 , 3.66 – 3.16 , 3.16 – 3.09 , 2.76 . 13C-NMR  δ 197.09 (C-4), 165.31 (C-7), 162.93 (C-5), 162.63 (C-9), 147.99 (C-4’), 146.48 (C-3’), 130.91 (C-1’), 117.85 (C-6’), 114.17 (C-2’), 111.98 (C-5’), 103.29 (C-10), 99.57 (C-1’’), 96.48 (C-6), 95.49 (C-8), 78.50 (C-2), 77.08 (C-3’’), 76.32 (C-5’’), 73.03 (C-2’’), 69.48 (C-4’’), 60.56 (C-6’’), 55.69 (OCH3-4’), 42.19 (C-3) (ESI-MS 19 (C-3) .dO-β--glucoside:(10) compound 11a : Phloretin 4’-m/z 435.1352 [M-H]-; 1H-NMR  δ 7.02 , 6.66 , 6.04 , 4.87 , 3.67 – 3.28 , 3.21 – 3.16 , 2.77 . 13C-NMR  δ 205.07 (C=O), 163.80 (C-4’), 163.40 (C-2’), 155.43 (C-4), 131.52 (C-1’), 129.18 (C-3), 115.11 (C-2), 105.30 (C-1), 99.55 (C-1’’), 95.08 , 77.13 (C5’’), 76.44 (C-3’’), 73.06 (C-2’’), 69.45 (C-4’’), 60.51 (C-6’’), 46.30 (C-α), 29.33 (C-β) (ESI-MS 33 (C-β) .dO-β--glucoside:(11) compound 12b : Curcumin 4’-m/z 529.1787 [M-H]-;1H-NMR  δ 7.58 , 7.56 , 7.47 – 6.94 , 6.84 , 6.77 , 6.09 , 5.00 , 3.84 , 3.68 - 3.14 . 13C-NMR  δ 185.71 (C-3), 183.77 (C-5), 151.06 (C-4’), 150.78 (C-4’’), 150.10 (C-3’), 149.61 (C-3’’), 142.62 (C-1), 141.52 (C-7), 130.31 (C-1’), 127.89 (C-1’’), 124.84 (C-2), 124.14 (C-6), 124.03 (C-6’), 122.74 (C-6’’) 117.30 (C-5’), 116.67 (C-5’’), 113.00 (C-2’), 112.93 (C-2’’), 102.65 (C-4), 101.23 (C-1’’’), 78.69 (C-2’’’), 78.39 (C-3’’’), 74.71 (C-4’’’), 71.18 (C-5’’’), 62.20 (C-6’’’), 57.37 (OCH3-3’), 57.32 (OCH3-3’’) (ESI-MS CH3-3’’) .FmUGT1 and FmUGT2 and att B adapter from pDONR-221, and the RNAi fragments contained specific 244 and 247 bp fragments of FmUGT1 and FmUGT2, respectively, with att B adapter. The primers are shown in FmUGT1, pDONR-FmUGT2, pDONR-FmUGT1i, and pDONR-FmUGT2i were validated by sequencing.OE primers corresponding to the open reading frame (ORF) of the FmUGT1, pK7WG2D-FmUGT2, pK7GWIWG2D-FmUGT1i and pK7GWIWG2D-FmUGT2i by PCR analysis and sequencing. These recombinant expression vectors were then transformed into the Agrobacterium rhizogenes (C58C1) by liquid nitrogen freeze-thawing to induce the production of F. multiflora transgenic hairy roots.We constructed the recombinant OE/RNAi expression vectors with pK7WG2D and pK7GWIWG2D (II) using Gateway LR Clonase™ II Enzyme Mix  and validated the recombinant expression vectors pK7WG2D-F. multiflora as explants and precultured them on MS medium for 2 d. For inoculation, the control/OE/RNAi groups of C58C1 were cultured in the dark in a liquid TY medium supplemented with 100 mg/L rifampicin and 50 mg/L spectinomycin (spectinomycin was not required for the control group) at 200 rpm for 15 h at 28°C. A 500 μL aliquot of the cultures was then sub-cultured in a 50 mL liquid TY medium at 200 rpm and 28°C until the OD600 ≈ 0.6-0.8, after which the subculture could be used for the genetic transformation. We then immersed the explants in different groups of C58C1 under shaking at 130 rpm for 25 min. Then the explants were picked out and patted dry on sterile filter paper, and co-cultured on solid MS medium in the dark at 25°C for 2 d. Next, the explants were transferred to solid MS medium supplemented with 400 mg/L Cefotaxime sodium (Cef) for sterilization. The hairy roots grew from the leaf margins and reached 3-5 cm in length in two weeks at which time the OE and RNAi group hairy roots needed to be transferred onto the solid MS medium containing 400 mg/L Cef and 50 mg/L Kanamycin for selective culture. During this period, we gradually reduced the concentration of Cef  every 3 d to ensure that the selected hairy roots could be continually grown. The Selected hairy root lines that remained strong after the selective culture were transferred into liquid MS medium  in the dark at 25°C and 130 rpm for 3 weeks and then sub-cultured in new liquid MS medium two times repeatedly to obtain putatively transformed hairy root lines with stable growth.For genetic transformation, we used the leaves of Accurate Taq Master Mix . Additionally, the pK7WG2D and pK7GWIWG2D (II) plant expression plasmid both carry the enhanced green fluorescent protein (eGFP) gene, which can induce the transgenic organs to produce green fluorescence, meaning that the expression of eGFP in the transgenic hairy roots could be detected using the OLYMPUS BX53 fluorescent microscope .Different groups of the putatively transformed hairy roots were twice weighed out to 0.1000 g each and ground into powder with liquid nitrogen for PCR and RT-qPCR analysis. TaKaRa MiniBEST Plant Genomic DNA Extraction Kit  was used for the DNA extraction of the hairy roots for molecular biological detection. The PCR primers are shown in Table S6. We performed PCR analysis using the 2 x FmUGT1 and FmUGT2 genes using a CFX96 Touch  and extracted the total RNA of the hairy roots using an RNAprep Pure Plant Kit . After this, an Evo M-MLV RT Kit with gDNA Clean for qPCR II  was used to reverse transcribe the total RNA and erase the genomic DNA. The F. multiflora PPAP gene was then used as the internal control to normalize expression levels of the target genes among different groups of samples, and the primers are shown in Table S6. Next, RT-qPCR was performed using a SYBR® Green Premix Pro Taq HS qPCR Kit II . The RT-qPCR programs were as follows: pre-denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 5 s, and annealing/extension at 60°C for 30 s with fluorescence reading. Finally, we calculated three biological replicates and three technical replicates using the 2-ΔΔCt method  in the dark at 25°C and 130 rpm for 45 d prior to compounds contents assay. The stilbenes were then extracted as described by Zhu et al. (2013). First, the samples (0.2000 g each) were finely ground under liquid nitrogen and dried at 45°C. They were then soaked with 25 mL of diluted ethanol  for 12 h, and were resuspended every 2 h during this period. These extracts were centrifuged at 5,500 rpm for 20 min to remove impurities, and the supernatant was filtered through a 0.45 μm nylon filter  as test solution. Analysis of the test solution was performed on a Waters 1525 HPLC system equipped with a PDA detector, and separation was achieved using a Phenomenex 00G-4252-E0  with the gradient program set to: acetonitrile (A): water (B)  for 20 min. The flow rate was 1.0 mL/min, and the injection volume was 20 μL, with a detection wavelength of 320 nm. Finally, we dissolved THSG standard in diluted ethanol and diluted it to 8 gradient concentrations . The standard curve was then plotted with the concentrations and peak areas as the abscissa and ordinate, respectively.We extracted the AQs compounds using the method described by FmUGT1 and FmUGT2 gene expression, THSG, and AQs contents of different groups of transgenic hairy roots were analyzed using one-way ANOVA from three independent biological replicates with three technical replicates each. All data are shown as mean ± SD. We used SPSS  for data analysis and statistical calculations, and generated graphs with GraphPad Prism .The data from our LC-MS/MS analysis were completed using SCIEX OS software . Comparisons of the levels of FmUGT1 and FmUGT2 (Table S7) using ExPASY ProtParam, and both of them were predicted to be unstable hydrophilic proteins. The SignalP 5.0 predicted that there were no transmembrane region for FmUGT1 and FmUGT2, and the Cell PLOC 2.0 subcellular localization predicted that FmUGT1 was located in the cell membrane and that FmUGT2 was located in the chloroplast. The predicted secondary structures of FmUGT1 and FmUGT2 are shown in Table S8. The three-dimensional models of FmUGT1 and FmUGT2 were constructed by SWISS-MODEL, using 6l90.1.A  boxes containing highly conserved sequences “HCGWNS” were also indicated . We thus conclude that FmUGT1 and FmUGT2 were indeed successfully induced in this study by the process described above. Using Ni-NTA to purify the soluble proteins induced from FmUGT1 and FmUGT2, we successfully eluted the purified FmUGT1 and FmUGT2 using Ni-NTA columns with the 0.05 M Tris-HCl buffer (pH= 7.4) containing 150 mM and 200 mM imidazole, respectively, and the SDS-PAGE results show that the purified protein bands were consistent with those of the induced proteins  and quercetin 3-O-glucoside standard by comparing their retention time in the HPLC spectrum  were both upregulated (P<0.001) by 12.61-fold and 4.15-fold compared to the pK7WG2D-0, respectively and that the relative expression of FmUGT1 and FmUGT2 in the RNAi groups (pK7GWIWG2D (II)) were 36.25-fold (P<0.01) and 17.33-fold (P<0.05) lower than in pK7GWIWG2D-0, respectively. Moreover, compared to the OE groups, the relative expression levels of FmUGT1 and FmUGT2 were 0.13% and 0.62% of the level in the RNAi groups (P<0.001), respectively  and 7.37-fold higher (P<0.001) than pK7WG2D-0 and pK7GWIWG2D-FmUGT1i, respectively. In addition, the content of THSG in the pK7WG2D-FmUGT2 hairy roots was 2027.37 μg/g, which was 16.79% (P<0.05) and 3.88-fold higher (P<0.01) than in pK7WG2D-0 and pK7GWIWG2D-FmUGT2i, respectively. In contrast, the THSG content in pK7GWIWG2D-FmUGT1i and pK7GWIWG2D-FmUGT2i was 78.61% and 72.26% lower, respectively, than in pK7GWIWG2D-0 (P< 0.001).The results of the assay of the content of stilbene in the hairy roots of each group are shown in FmUGT1 was 164.15 μg/g, which was higher (P<0.001) by 2.00-fold and 1.26-fold compared to pK7WG2D-0 and pK7GWIWG2D-FmUGT1i, respectively. For pK7WG2D-FmUGT2, the emodin content was 147.62 μg/g, which was 1.70-fold and 5.21-fold higher (P< 0.001) than in pK7WG2D-0 and pK7GWIWG2D-FmUGT2i respectively. Physcion content in the pK7WG2D-FmUGT1 hairy roots was 337.36 μg/g, which was higher (P< 0.001) by 1.07-fold and 1.49-fold compared to pK7WG2D-0 and pK7GWIWG2D-FmUGT1i, respectively, and for the pK7WG2D-FmUGT2 hairy roots the concentration was 170.75 μg/g, which was 1.61-fold higher (P<0.05) than in pK7GWIWG2D-FmUGT2i. However, we found no statistically significant difference between pK7WG2D-FmUGT1 and pK7WG2D-0.Results for the AQs contents in the hairy roots of each group are shown in FmUGT1 and FmUGT2 showed substrate promiscuity through in vitro catalytic reaction and successfully catalyzed the glycosylation of 12 substrates with different structures. Through structural analysis of the glycosylation products, we found that FmUGT1 and FmUGT2 could catalyze the 3-O-glycosylation of THSG and resveratrol to form polygonimitin C and polydatin, as well as could also cause the 3’-O-glycosylation of piceatannol to form piceatannol 3’-O-glucoside. Unfortunately, when we tried to prepare the 2,3,5,4’- tetrahydroxystilbene compound from THSG using acidic hydrolysis method, it was found to be severely degraded within 24 h, which was also demonstrated by in vitro glycosylation catalysis studies. When using emodin as the substrate, FmUGT1 and FmUGT2 both tended to transfer sugars from glycoside scaffolds to an 8-O position at the aglycon to form emodin 8-O-glucoside. According to the glycosylation sites of compound 5b, 6a, and 9a, FmUGT1 and FmUGT2 most frequently attached glycosyl at 3-O. However, in the absence of the 3-OH, FmUGT1 and FmUGT2 tended to form 7-O-glycosylation of flavonoids .FmUGT1 and FmUGT2 were both more prone to attaching glycosyl at the 4’-O position, which is similar to the 7-O position at the A-ring of flavonoids, suggesting that FmUGT1 and FmUGT2 might have stereoselectivity during glycosylation. Additionally, FmUGT1 could catalyze 5’-O-glycosylation of myricetin to form compound 9c, and FmUGT1 and FmUGT2 could both catalyze the 4’-O of curcumin to form compound 12a, which helped improve the solubility of curcumin. These results indicate that FmUGT1 and FmUGT2 demonstrated substrate promiscuity and possibly stereoselectivity, which may further supplement the glycosylation tool enzyme library and provide a new method for the synthesis of glycosides.Interestingly, phloretin, a dihydrochalcone, is structurally similar to apigenin, and FmUGT1 and FmUGT2 in the synthesis of secondary metabolites of F. multiflora further, we constructed pK7WG2D-FmUGT1, pK7GWIWG2D-FmUGT1i, pK7WG2D-FmUGT2, and pK7GWIWG2D-FmUGT2i recombinant plant expression vectors for genetic modification. Molecular biology detection and RT-qPCR detection from these efforts showed that FmUGT1 and FmUGT2 were successfully overexpressed or interfered with in corresponding groups of hairy roots. Moreover, our RT-qPCR results displayed that the relative expressions of FmUGT1 and FmUGT2 in the RNAi group were significantly downregulated compared with the control groups and that their THSG contents were correspondingly decreased. However, the results of OE-FmUGT1 and OE-FmUGT2 were quite opposite to those of the RNAi groups. In the former groups the relative expressions were upregulated significantly compared to those of the control groups, and their THSG contents were also significantly increased, indicating that FmUGT1 and FmUGT2 play a key role in the THSG biosynthesis pathway in F. multiflora.To study the role of FmUGT1 and OE-FmUGT2 were significantly higher than those in the control and RNAi groups, but after interfering with the expression of FmUGT1 and FmUGT2, the contents of emodin and physcion were not significantly different from those in the control groups, showing that although promoting the transcription of FmUGT1 and FmUGT2 could help enhance the production of AQs in the hairy roots of F. multiflora, FmUGT1 and FmUGT2 were not key members of the AQs biosynthesis pathway. Additionally, we also tried to detect the flavonoid content of different groups of hairy roots using HPLC-PDA, but could not find any relevant flavonoids.Similarly, the contents of emodin and physcion in OE-FmUGT1 and FmUGT2 showed substrate promiscuity and could catalyze 12 substrates of stilbenes, AQs, flavonoids, phloretin, and curcumin to produce corresponding glucosides. Our analysis of the in vivo function of FmUGT1 and FmUGT2 further showed that FmUGT1 and FmUGT2 could efficiently catalyze the glycosylation of THSG and promote the production of AQs in F. multiflora transgenic hairy roots. Taken together, the above results will provide a crucial foundation to help clarify the molecular pathway of THSG biosynthesis.In summary, we found that https://www.ncbi.nlm.nih.gov/nuccore/ON262204.1; https://www.ncbi.nlm.nih.gov/nuccore/ON262205.1.The datasets presented in this study can be found in online repositories. The names of the repositories and accession numbers can be found below: LZ, and QY conceived and designed the experiments. QC, CL and LL performed the experiments and drafted the manuscript. QC, CL, YG, XC and BL analyzed the data. All authors contributed to the discussion and revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.This work was supported by National Key Research and Development Program (2017YFC1700704), Special Fund for the Protection of Lingnan Chinese Medicinal Materials in Guangdong Province in 2017 (Yue cai she [2017] No. 60), and Guangdong Provincial Youth Innovation Talents Program (2018KQNCX133).The 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.


Pseudomonas species as Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov. was not in accordance with the rules of the International Code of Nomenclature of Prokaryotes (ICNP) and as such cannot be validly published. This corrigendum is prepared to change the previous names to Pseudomonas triticifolii sp. nov. and Pseudomonas folii sp. nov., respectively, based on ICNP rules.The naming of the two new Also, the stereoisomers of the carbon sources used are now correctly indicated with capital letters, e.g., D-melibiose, D-arabitol, D-glucose, L-malic acid … etc. instead of d-melibiose, d-arabitol, d-glucose, l-malic acid...Pseudomonas species, Description ofPseudomonas triticumensis sp. nov. and Description ofPseudomonas foliumensissp. nov:A correction has been made to the protologues of the two novel Description ofPseudomonas triticifoliisp. nov.Pseudomonas triticifolii sp. nov. .16:0, C16:1 ω6c/C16:1 ω7c (summed feature 3), C18:1 ω7c/C18:1 ω6c (summed feature 8), C17:0 cyclo; C12:0 2-OH, C12:0 3-OH and C10:0 3-OH. Based on Biolog GENIII microplate assays, strain 32L3A utilized 37 carbon sources including D-melibiose, D-arabitol, α-D-glucose, stachyose, myo- inositol, D-mannitol, D-sorbitol, D-cellobiose, gentiobiose, L-serine but not L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, L-malic acid, α-keto-butyric acid, acetoacetic acid, propionic acid, acetic acid, D-glucuronic acid, glucuronamide, mucic acid, quinic acid, D-saccharic acid. Using API ZYM assays, these bacteria are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and β-glucosidase, but negative for α-glucosidase, α-galactosidases, β-galactosidases and β-glucuronidases. The most abundant fatty acids are C16:0, C16:1ω7c and/or C16:1ω6c (summed feature 3) and C18:1ω7c and/or C18:1ω7c (summed feature 8). These bacteria are resistant to troleandomycin, rifamycin SV, vancomycin, lincomycin but sensitive to nalidixic acid and minocycline. The type strain is 32L3AT (= DOAB 1067T = CECT 30249T = LMG 32140T), isolated from necrotic wheat leaf tissues naturally infected by Xanthomonas translucens from Alberta, Canada. The DNA G + C content of type strain 32L3AT is 59.3 %.Cells are aerobic, Gram-reaction-negative, non-spore-forming rods (0.6–1.0 μm wide and 2.0–4.5 μm long), motile with one, or multiple polar flagella. After 48 h on KB, colonies are white-yellowish and circular , convex with regular margins and produce fluorescent pigments. Growth in different NaCl concentrations optimal at 4%, and grows at 4°C with optimal growth at 28–30°C and no growth at 40°C. The major cellular fatty acid peaks of the bacteria were CDescription ofPseudomonas foliisp. nov.Pseudomonas folii sp. nov. .Pseudomonas strains were C16:0, C16:1 ω6c/C16:1 ω7c (summed feature 3), C18:1 ω7c/C18:1 ω6c (summed feature 8), C17:0 cyclo; C12:0 2-OH, C12:0 3-OH and C10:0 3-OH. Based on Biolog GENIII microplate assays, these bacteria readily utilize 47 carbon sources e.g., α-D-glucose, D-mannose, gelatin, pectin, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-rhamnose, D-raffinose, D-fucose, α-D-Lactose, D-melibiose, β-methyl-D-glucoside, D-salicin, dextrin but not gentiobiose, sucrose, L-aspartic acid, L-glutamic acid, L-histidine, D-glucuronic acid, glucuronamide, mucic acid, quinic acid, citric acid, α-keto-glutaric acid, D-malic acid and L-malic acid. Using API ZYM assays, these bacteria are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and β-glucosidase, but negative for α-glucosidase, α-galactosidases, β-galactosidases and β-glucuronidases. The most abundant fatty acids are C16:0, C16:1ω7c and/or C16:1ω6c (summed feature 3) and C18:1ω7c and/or C18:1ω7c (summed feature 8). These bacteria are resistant to troleandomycin, rifamycin SV, vancomycin, lincomycin but sensitive to nalidixic acid and minocycline. The type strain is 10L4BT (= DOAB 1069T = CECT 30250T = LMG 32142T), isolated from necrotic wheat leaf tissues naturally infected by Xanthomonas translucens from Alberta, Canada. The DNA G + C content of the type strain 10L4BT is 57.2%.Cells are aerobic, Gram-reaction-negative, non-spore-forming rods (0.6–1.0 μm wide and 1.5–2.5 μm long), motile with one or two polar flagella. After 48 h on KB, colonies are white-yellowish and circular , convex with regular margins and do not produce fluorescent pigments. Growth in NaCl concentrations optimal at 4%, and grows at 4°C with optimal growth at 28–30°C and no growth at 40°C. Strong growth at pH 6 but very weak growth at pH 5. The major cellular fatty acid peaks of the The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.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.