Allergy is a chronic inflammatory disease, and its incidence has increased worldwide in recent years. Thalidomide, which was initially used as an anti-emetic drug but was withdrawn due to its teratogenic effects, is now used to treat blood cancers. Although the anti-inflammatory and immunomodulatory properties of thalidomide have been reported, little is known about its influence on the mast cell-mediated allergic reaction. In the present study, we aimed to evaluate the anti-allergic activity of thalidomide and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and passive cutaneous anaphylaxis (PCA) mouse models. Thalidomide markedly decreased the degranulation and release of lipid mediators and cytokines in IgE/Ag-stimulated BMMCs, with concurrent inhibition of FcεRI-mediated positive signaling pathways including Syk and activation of negative signaling pathways including AMP-activated protein kinase (AMPK) and SH2 tyrosine phosphatase-1 (SHP-1). The knockdown of AMPK or SHP-1 with specific siRNA diminished the inhibitory effects of thalidomide on BMMC activation. By contrast, the knockdown of cereblon (CRBN), which is the primary target protein of thalidomide, augmented the effects of thalidomide. Thalidomide reduced the interactions of CRBN with Syk and AMPK promoted by FcεRI crosslinking, thereby relieving the suppression of AMPK signaling and suppressing Syk signaling. Furthermore, oral thalidomide treatment suppressed the PCA reaction in mice. In conclusion, thalidomide suppresses FcεRI-mediated mast cell activation by activating the AMPK and SHP-1 pathways and antagonizing the action of CRBN, indicating that it is a potential anti-allergic agent. Mast cells are well-recognized initiators of IgE-triggered allergic inflammation and anaphylactic responses via their release of a broad array of mediators, including preformed and granule-stored mediators, newly-synthesized eicosanoids, and pro-inflammatory cytokines, upon binding of antigen (Ag) to Ag-specific IgE bound to the high-affinity IgE receptor (FcεRI) ,2.FcεRI-mediated mast cell activation is regulated by coordinated signaling pathways. Ag-induced crosslinking of IgE-bound FcεRI activates FcεRI-associated tyrosine kinases such as Lyn and Fyn, which subsequently recruit and activate Syk. Then, Syk phosphorylates linker for activation of T cells (LAT), resulting in assembly and sequential activation of a downstream signaling complex ,4. TheseThe kinase activity of AMPK is regulated by phosphorylation of the α subunit and allosteric changes in the β and γ subunits ,11,12. IThalidomide was initially used as an anti-emetic drug for pregnant women, although it was withdrawn from the pharmaceutical market due to its serious teratogenic effects ,21,22. HIn the present study, we examined the effects of thalidomide on FcεRI-mediated allergic responses and its possible mechanisms of action, focusing on AMPK, CRBN, and SHP-1 in bone marrow-derived mast cells (BMMCs) and passive cutaneous anaphylaxis (PCA) animal models.v/v) in culture medium. DMSO was used as a vehicle control in all cases.Thalidomide (#T144), dinitrophenyl human serum albumin , DNP-specific monoclonal IgE (#D8406), and p-nitrophenly-N-acetyl-β-D-glucosaminide (#N9376) were obtained from Sigma-Aldrich  and NSC-87877 (#565851) was obtained from Calbiochem . Thalidomide was prepared by dissolving in dimethyl sulfoxide (DMSO) and the final concentration of DMSO was adjusted to 0.1% . After one week of adaptation, animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Daegu Catholic University Medical Center.2 for 4 weeks. RBL2H3 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antifungal agent. For cell stimulation, cells were sensitized with 500 ng/mL of mouse anti-DNP IgE for 2 h and then stimulated with 100 ng/mL of DNP-HSA for different times. Thalidomide was added 1 h prior to the stimulation of cells with DNP-HSA.BMMCs were prepared from Balb/cJ as previously described . Cells w6 cells/mL) with DNP-HSA for 15 min, the culture medium was collected by centrifugation at 1200× g. The same number of control BMMCs were lysed by repeated freezing/thawing and used to measure total levels of degranulation. Twenty-five μL of supernatants were incubated with 50 μL p-nitrophenyl-N-acetyl-β-D-glucosaminide (1.3 mg/mL) in 0.1 M citric acid buffer (pH 4.5) at 37 °C for 1 h. The enzyme reaction was stopped by adding 0.2 M glycine buffer (pH 10.7). The absorbance was measured at 405 nm on a Molecular Devices spectramax iD5 . Data were expressed as a percentage of total values.The activity of β-hexosaminidase was measured to determine the level of degranulation. After stimulation of IgE-sensitized BMMCs (2 × 106 cells/mL) were stimulated with DNP-HSA as indicated above and, then, culture media were collected. The levels of released leukotriene C4 (LTC4), prostaglandin D2 (PGD2), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were assessed using ELISA kits according to the manufactures’ instruction. ELISA kits for LTC4 (#501070) and PGD2 (#512011) were obtained from Cayman Chemicals  and TNF-α (MTA00B) and IL-6 (M6000B) were obtained from R&D Systems .IgE-sensitized BMMCs (1 × 104 cells/well) were seeded into 96-well microplates and stimulated with DNP-HSA for 5 min. The fluorescence was measured at 485/520 nm on a Molecular Devices spectramax iD5 .Calcium mobilization was determined using the Calcium Assay Kit  according to the manufacturer’s instruction . IgE-sen2, 150 mM NaCl, 10 mM KCl, 1 mM Na3VO4, 1 mM DTT, 1 mM benzamide, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 mM β-glycerophosphate, and 1% (v/v) NP-40) on ice for 15 min with vortexing every 5 min. Cell lysates were obtained by centrifugation at 15,000× g for 15 min and protein concentration was measured by detergent-compatible protein assay  Denatured proteins using 4X Laemmli sample buffer  were separated on 10% SDS-PAGE and were transferred to a nitrocellulose membrane (0.45 μm) using a semi-dry transfer cell (Bio-Rad). After blocking with 5% non-fat dry milk in 1x TBST  at room temperature for 1 h, membranes were horizontally cut to probe proteins with different molecular weights. Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The band intensities were analyzed by the Chemi-Doc XRS imaging system . The membranes were stripped and reprobed with anti-GAPDH antibody as needed. The results are expressed as arbitrary units after normalization to GAPDH levels, which was used as loading control for each gel. The primary antibodies used were as follows: Antibodies against p-Akt (#4058), Akt (#9272), p-ACC (#3661), ACC (#3662), p-AMPKα (#2531), AMPKα (#2532), p-LKB1 (#3482), LKB1 (#3050), p-ERK (#4337), ERK (#9102), p-IKK (#2697), p-JNK (#9251), JNK (#9258), p-p38 MAPK (#9215), p38 MAPK (#9212), p-PLCγ1 (#2821), and GAPDH (#2118) were purchased from Cell Signaling Technology . Antibodies against Fyn (#sc-434), IgG , IKK (#sc-7607), LAT (#sc-53550), Lyn (#sc-376100), PLCγ1 (#sc-7290), SHP-1 (#sc-7289), Syk , and p-tyrosine (pY) (#sc-7020) were purchased from Santa Cruz Biotechnology ; anti-CRBN antibody was from Thermo Fisher scientific . The secondary antibodies, goat anti-rabbit IgG antibody (#GTX213110-0), and goat anti-mouse IgG antibody (#GTX213111-01) were purchased from Gene Tex .BMMCs were washed with ice-cold PBS and incubated with lysis buffer  for 2 h at 4 °C. Agarose beads were spin-downed and supernatant was collected for immunoprecipitation. Corresponding antibodies (4 μL) were added to 100 μL of cell lysate (1 μg/μL) and were incubated with gentle agitation at 4 °C overnight. The immunocomplexes were captured by adding 25 μL of protein G/A agarose for 2 h at 4 °C. The mixtures were washed with washing buffer  three times and then resuspended in lysis buffer. The immunocomplexes were subjected to immunoblotting, as described above, as needed. Antibodies used for IP were as follows: Antibodies against AMPKα (#sc-74461), Syk , LAT (#sc-7948), Fyn (#sc-28791), Lyn (#sc-28790), and SHP-1 (#sc-287) were from Santa Cruz Biotechnology .Genetic knockdown was performed using mouse-specific siRNAs  and a non-specific siRNA . IgE-sensitized BMMCs were cultured in serum-free medium for 16 h and transfected with 100 nM of AMPKα (50 nM α1(#sc-29674) and 50 nM α2 (#sc-38924)), CRBN (#sc-142562), and SHP-1 (#sc-29479) siRNAs or a non-specific siRNA (Mock) (#12935300), as a negative control using DharmaFECT3 Transfection reagent . After 48 h, cells were stimulated with DNP-HSA for indicated times .n = 6 per group). Thalidomide and fexofenadine were prepared in 0.5% of carboxymethyl cellulose. Control group was administered an equivalent volume of a vehicle (0.5% of carboxymethyl cellulose). After anesthetization of mice, blood was collected and the levels of serum LTC4 and PGD2 were analyzed as described above. To determine dye extravasation, ears were removed and incubated with 400 μL of formamide at 63 °C overnight, and the absorbance of extracted dye was measured at 630 nm on a Molecular Devices spectramax iD5 .To evaluate the effect of thalidomide on IgE/Ag-mediated type I hypersensitivity reaction in vivo, PCA reaction was induced as described previously . ICR mict test (two-tailed) for comparisons between two groups and one-way ANOVA with Tukey’s post hoc test for multiple comparisons using GraphPad Prism 4.0 [p < 0.05 was considered statistically significant.Statistical significance was determined by unpaired Student’s rism 4.0 . p < 0.04, PGD2, TNF-α, and IL-6, and the intracellular Ca2+ level  in the presence or absence of 20 μM thalidomide. While thalidomide alone had no significant effect on mast cell activation, thalidomide significantly diminished IgE/Ag-stimulated degranulation as well as the production of LTC2+ level A–F. The 2+ level G–I. Thal2+ level . Concurr2+ level . In addi2+ level , suggest4 and PGD2 were significantly higher in AMPKα siRNA-treated BMMCs than in mock-treated BMMCs  for 1 h prior to stimulation with Ag. Thalidomide increased the phosphorylation of LKB1, AMPK, and ACC in a concentration-dependent manner A. The ined BMMCs C–E. The ed BMMCs C–E. Thes4 and PGD2 was significantly lower in CRBN siRNA-treated cells than in mock-treated cells; CRBN siRNA enhanced the inhibitory effects of thalidomide  signaling pathways. Therefore, we investigated the effect of thalidomide on the FcεRI-proximal tyrosine kinases Fyn, Lyn, and Syk in IgE/Ag-stimulated BMMCs. Thalidomide inhibited IgE/Ag-increased tyrosine phosphorylation (pY) of Syk and its target protein LAT; however, it did not affect the phosphorylation of Fyn and Lyn, which are upstream kinases of Syk . CRBN si4 and PGD2)  D–F. Thes4 and PGD2) in a dose-dependent manner  significantly reduced IgE/Ag-increased dye extravasation and the serum levels of eicosanoids (LTCt manner A–C. The Previous reports showing the anti-inflammatory function of thalidomide and inhibitory regulation of AMPK by CRBN indicated to us that thalidomide may have therapeutic effects on mast cell-mediated allergic diseases. In the present study, we showed that thalidomide inhibits FcεRI-mediated allergic reactions in mast cells in vitro and in an in vivo PCA model. Thalidomide decreased the association of CRBN with AMPK and thereby relieved the suppression of the AMPK pathway. In addition, thalidomide inhibited Syk signaling by activating SHP-1 and interfering with the interaction between Syk and CRBN. Thus, relief of the suppression of the AMPK pathway and activation of SHP-1 in parallel with disruption of the interaction between CRBN and Syk may underlie the suppression of FcεRI-mediated mast cell activation by thalidomide.Several lines of evidence demonstrate that thalidomide inhibits CRBN, a binding partner of AMPK ,36,37. C4 and PGD2. However, A770041 has no effect on Syk phosphorylation in IgE/Ag-stimulated BMMCs [CRBN is involved in multiple biological functions including cell metabolism, neuronal action, and autoimmune disorders , and meded BMMCs . This sued BMMCs . Among ted BMMCs . TherefoUpstream tyrosine kinases, such as Fyn and Lyn, and tyrosine phosphatases, such as SHP-1, regulate Syk signaling ,49,50,51In summary, the binding of IgE/Ag to FcεRI promotes the interactions of CRBN with AMPK and Syk. These interactions facilitate mast cell activation by attenuating inhibitory signaling of LKB1 and AMPK and activating stimulatory signaling of the Syk pathway. Thalidomide attenuates FcεRI-stimulated mast cell activation by relieving the suppression of the LKB1/AMPK pathway and activating SHP-1 signaling, while simultaneously interfering with the interaction between CRBN and Syk. These results are summarized in


Chlamydomonas reinhardtii. Our results showed that these four mycotoxins have multiple targets, acting mainly on photosystem II (PSII). Their mode of action is similar to that of diuron, inhibiting electron flow beyond the primary quinone electron acceptor (QA) by binding to the secondary quinone electron acceptor (QB) site of the D1 protein, thereby affecting photosynthesis. The results of PSII oxygen evolution rate and chlorophyll (Chl) a fluorescence imaging suggested that fumagillin strongly inhibited overall PSII activity; the other three toxins also exhibited a negative influence at the high concentration. Chl a fluorescence kinetics and the JIP test showed that the inhibition of electron transport beyond QA was the most significant feature of the four mycotoxins. Fumagillin decreased the rate of O2 evolution by interrupting electron transfer on the PSII acceptor side, and had multiple negative effects on the primary photochemical reaction and PSII antenna size. Mevastatin caused a decrease in photosynthetic activity, mainly due to the inhibition of electron transport. Both radicicol and wortmannin decreased photosynthetic efficiency, mainly by inhibiting the electron transport efficiency of the PSII acceptor side and the activity of the PSII reaction centers. In addition, radicicol reduced the primary photochemical reaction efficiency and antenna size. The simulated molecular model of the four mycotoxins’ binding to C. reinhardtii D1 protein indicated that the residue D1-Phe265 is their common site at the QB site. This is a novel target site different from those of commercial PSII herbicides. Thus, the interesting effects of the four mycotoxins on PSII suggested that they provide new ideas for the design of novel and efficient herbicide molecules.Mycotoxins are one of the most important sources for the discovery of new pesticides and drugs because of their chemical structural diversity and fascinating bioactivity as well as unique novel targets. Here, the effects of four mycotoxins, fumagillin, mevastatin, radicicol, and wortmannin, on photosynthesis were investigated to identify their precise sites of action on the photosynthetic apparatus of  Mycotoxins are low-molecular-weight secondary metabolites naturally produced by fungi. These natural products are one of the most important resource repositories for the discovery of new drugs and pesticides because they possess abundant sources, diverse chemical structures, and wide bioactivity. Generally, they exhibit several phytotoxic, cytotoxic, antimicrobial, and even antitumor activities ,2. Some Aspergillus fumigatus. It is a meroterpenoid produced by the esterification of fumagillol and 2,4,6,8-decatetraenedioic acid (Arabidopsis.Fumagillin (CAS No. 23110-15-8) is a natural biomolecule originally isolated from oic acid A 4]. Fu. FuAsperPenicillium citinium [Mevastatin (CAS No. 73573-88-3), also called compactin, was first discovered in citinium  suggesteMonosporium bonorden [Neonectria radicicolas, Colletotrichum graminicola, and other organisms. Radicicol is a 14-membered β-resorcylic acid lactone with a 17R configuration, which is characterized by the resorcyclic acid moiety replaced by a chlorine atom , known as monorden, was first found in bonorden . It is aine atom C. It sho772-57-5,Fusarium oxysporum, P. wortmannii, and P. funiculosum [δ-lactone, acetate ester, and cyclic ketone (Wortmannin (CAS No. 19545-26-7) is a furanosteroid metabolite derived from the endophytic fungi iculosum . It is ac ketone D. WortmaPhotosynthesis is the most important metabolic process in plant growth and development, being very sensitive to changes in environmental conditions. Photosystem II (PSII), as one core of photosynthetic reactions, is a large protein complex located in the thylakoid membranes, including the D1 and D2 proteins as well as several cofactors . Becausea fluorescence signal of C. reinhardtii cells treated with four different mycotoxins were measured to identify their sites of action in photosystems. Because C. reinhardtii is one of the most widely used model systems for molecular biological studies of photosystems including extensive characterization of its PSII reaction center, it was used in this study. The model of the interaction between each mycotoxin and the D1 protein of C. reinhardtii was also constructed by molecular docking to further confirm their possible binding sites in the D1 protein. Finally, it was demonstrated that four mycotoxins have direct effects on PSII and different binding behavior with the D1 protein from the classical herbicide diuron (DCMU).In this study, we hypothesized that these four mycotoxins negatively affect photosynthesis, especially PSII activity. To test this hypothesis, the rate of oxygen evolution and chlorophyll (Chl) C. reinhardtii cells. A concentration-dependent decrease in the rate of oxygen evolution of cells exposed to mycotoxin was observed  of fumagillin-, mevastatin-, and radicicol-treated C. reinhardtii cell shows a gradual decrease with the color fading from blue to dark green. For wortmannin, no obvious effect on the color-coded images of FV/FM was observed at the highest concentration of 600 µM. In the case of the highest concentration of fumagillin (50 µM), mevastatin (200 µM), and radicicol (500 µM), the value of FV/FM decreased by approximately 57%, 58%, and 59% compared with that of the mock, respectively. A nearly 5% decrease in the FV/FM was observed in the presence of 600 µM wortmannin (V/FM was consistent with the change in the fluorescence image presented in V/FM parameter. The electron transport rate (ETR) was also measured to evaluate the effect of these mycotoxins on photosynthetic activity , which is the main change in the OJIP curve relative to the mock. Radicicol and wortmannin at 500 μM, as with DCMU, caused a remarkably fast rise in the J step. Interestingly, the IP phase of the OJIP curve of DCMU- and wortmannin-treated cells disappeared entirely, but still remained in the radicicol-treated cells (A−) in PSII reaction centers (RCs), resulting from the interruption of electron flow beyond QA at the PSII acceptor side [A at the acceptor side of PSII was strongly inhibited by radicicol and wortmannin. This agreed with the ETR results /(FM − FO) (t = Vt(treatment) − Vt(mock)  B and ΔVtVt(mock) C on a loC. reinhardtii cells [A, and the redox status of downstream acceptors was consequently affected. For 500 μM wortmannin treatment, the J peak sharply increased and was similar to the J-step increase for radicicol at the same concentration  of ii cells ,24. For ii cells ,25. Thisntration B,C, indiC. reinhardtii ; radicicol might lead to oxidation of the PQ pool or disruption of PSII antennae (decrease in FM); and fumagillin, radicicol, and wortmannin have different degrees of inhibition of the quantum efficiency of light energy transfer in PSII (decrease in FV/FM). In addition, 200 μM mevastatin showed insensitivity to FV/FM.The JIP test parameters give adequate information about the structure, conformation, and function of the photosynthetic apparatus, especially PSII . Some senhardtii . Both fuFM value A. FumagiJ) and ΔVJ  treatment. In contrast, the ΔVJ values under radicicol (500 μM) or wortmannin (500 μM) treatment were close to those of the DCMU (50 μM) treatment. Thus, the four mycotoxins showed similarities to DCMU in the inhibition pattern of electron transport, but the inhibition effects were significantly different. The inhibition by the four mycotoxins was significantly weaker than that of DCMU, and the inhibition of wortmannin was slightly stronger than that of radicicol. Moreover, radicicol and wortmannin showed an increase in relative variable fluorescence at the I step (VI). The values of both fluorescence parameters VJ and VI are considered to correlate with the state of electron transport on the PSII acceptor side [J values indicated that the four mycotoxins blocked PSII electron transport beyond QA.All four mycotoxins significantly changed the relative variable fluorescence at the J step  by the four mycotoxins, the parameters φEo, ψΕo, and ET0/RC were analyzed  and mevastatin (200 μM) probably do not affect the activity of PSII RCs. Another fluorescence parameter, RJ, explains the inactivation of PSII RCs. It is generally considered that RJ reflects the number of PSII RCs with the QB site filled populated by PSII inhibitors [J increased by 16%, 13%, 74%, and 84% relative to the mock, respectively  generation.The values of the N and S results E. The tu and tFM . Compare of DCMU C,E. A decceptors ,30. Wortcceptors  significcceptors  and patucceptors  have bee centers ,27. The ABS is the most sensitive JIP test parameter, which is used to assess the photosynthetic performance indexes of samples [C. reinhardtii cells were incubated with fumagillin, mevastatin, radicicol, or wortmannin, the values of PIABS decreased by 61%, 31%, 95%, and 93% compared with that of the mock, respectively (ABS are closely related to the antenna size (γRC), the maximum quantum yield of primary photochemistry (φPo), and the probability of electrons on the reduced QA. further entering the electron transfer chain [RC by 15% and 35%, respectively. The φPo of C. reinhardtii cells decreased by 18%, 37%, and 13%, respectively, under fumagillin, radicicol, and wortmannin treatment. Mevastatin showed no significant effect on the parameters γRC and φPo, and wortmannin showed no significant effect on φPo. The ψΕo decreased with fumagillin, mevastatin, radicicol, and wortmannin treatment by 17%, 11%, 72%, and 83%, respectively, as shown in ABS is related to the changes in the parameters γRC, φPo, and ψΕo. In other words, fumagillin harmed the antenna size, primary photochemical reaction, and redox reaction after QA, which together significantly reduced the overall photosynthetic activity of PSII. For mevastatin and wortmannin, the decrease in PIABS was only associated with a significant decrease in the parameter ψΕo. The main mechanism of action of mevastatin and wortmannin is the inhibition of electron transport beyond the QA. As for radicicol, γRC, φPo, and ψΕo were significantly affected, but ψΕo was clearly the dominant factor. This suggested that the main factor for the decrease of PIABS by radicicol as the reduced efficiency of movement in the redox reaction of the electron transport chain. The inhibition of the flow of PSII electrons from QA to QB remained a common feature of the mechanism of action of the four mycotoxins.PI samples . When C.ectively F. Signifer chain ,27. The B site. This further resulted in different degrees of reduction in the number of active PSII RCs and the overall photosynthetic activity of PSII. It has been widely reported that the interruption of electron transport has some negative effects on photosynthesis. For example, reduced electron transport capacity limits the synthesis of ATP and thus inhibits the regeneration of RuBP and the assimilation of CO2 [To summarize, we found that the common and dominant mechanism of action of the four mycotoxins in inhibiting photosynthetic activity was to block electron transfer on the acceptor side of PSII by occupying the Qn of CO2 ,34; the n of CO2 . In addiB niche on the D1 subunit is a binding target for many known PSII inhibitor herbicides [B for the QB binding site during electron transport, ultimately leading to plant death [B-binding pocket is a cavity lined with hydrophobic residues located in the connecting loop between the fourth and fifth transmembrane helices of the D1 protein, which consists of at least 65 amino acids spanning from Phe211 to Leu275 [B site is also conserved in oxygenic photosynthetic organisms [PSII is an important part of photosynthesis, which provides the initial charge separation to generate high-energy electrons for photosynthetic electron transport. The PSII RC core consists of two highly hydrophobic proteins, D1 and D2, which embed most of the redox active components involved in photosynthetic electron transfer through PSII . The QB rbicides , which cnt death . The QB-o Leu275 . All therganisms .C. reinhardtii D1 protein (Protein Data Bank (PDB): 6KAC) was selected to model the position of the four mycotoxins in the QB site with Discovery Studio   of the D1-Phe265 residue instead of amino hydrogen (NH). The simulated modeling distances of the hydrogen bonds of fumagillin, mevastatin, radicicol, and wortmannin were 2.53 Å, 2.67 Å, 2.52 Å and 2.39 Å, respectively. The four mycotoxins also established several hydrophobic, Pi-stacking, and hydrogen-bonding interactions with active site residues, as detailed in B site. Compared with the other models, the fumagillin model showed the largest number of bonding interactions with D1. Our previous experimental results also showed that fumagillin had the strongest inhibitory effect on PSII, with the highest affinity in vitro  . The proin vitro C. These B binding site was established to verify the reliability of the binding model. Our model showed that the possible hydrogen binding interaction for DCMU and D1 was formed between the N7 of DCMU and D1-Ser264 Oγ with a 2.37 Å bound distance . C. reinhardtii cells were cultured in liquid tris-acetate phosphate media (TAP) under 100 μmol (photons) m−2 s−1 white light with a 12 h photoperiod at 25 °C. Three-day-old cells in the logarithmic growth phase were collected for further experiments [The eriments .v/v).Fumagillin, mevastatin, radicicol, wortmannin, 3--1,1-dimethylurea (DCMU), dimethyl sulfoxide (DMSO), and other chemical agents were purchased from Sigma-Aldrich . Four mycotoxins and DCMU stock solutions were dissolved in 100% DMSO and further diluted with sterile water. The final concentration of DMSO in the working solutions of all chemical agents was less than 1% , 350 mM sucrose, and 2.0 mM MgCl2 with an A750 value of 0.65. Each mycotoxin was added into 2 mL cell suspensions with the indicated concentrations. The samples were incubated for 3 h in the dark at 25 °C. Subsequently, treated cells containing 45 μg of chlorophylls were added into 2 mL of PSII reaction medium 6, 5mM NH4Cl, and 1 mM p-phenylenediamine). The oxygen evolution rate was determined and recorded during the first three minutes after samples were illuminated with 400 µmol (photons) m−2 s−1 red light.The rate of oxygen evolution of PSII was measured using a Clark-type Oxygen Electrode  according to the method of Guo et al. . After Ca fluorescence imaging, FV/FM, and the ETR of dark-adapted samples were measured using a pulse-modulated Imaging-PAM M-series fluorometer  [−2 s−1) at 25 °C. Then, samples were dark-adapted for 30 min under the imaging system camera. For monitoring fluorescence imaging, the measuring light, actinic light, and saturation pulse light were set to 0.25, 110, and 6000 μmol (photons) m−2 s−1, respectively.Chl Germany) . Cells wa fluorescence rise kinetics OJIP were measured with a Plant Efficiency Analyzer  [−2 s−1 white light at 25 °C. The cells were collected and resuspended in 20 µL of buffer A. After 30 min dark adaptation, 20 µL of the sample was filtered onto a glass microfiber filter  and clamped with a leaf clip. Samples were illuminated with continuous red light  m−2 s−1 maximum light intensity). The raw data were transferred to a computer using Handy PEA software . The experiment was repeated three times with at least 15 repetitions. The detailed parameters and definitions re listed in The Chl ynn, UK) . We incuC. reinhardtii was obtained from the Protein Data Bank . Its dimeric structure was optimized by CHARMm force field using Discovery Studio . The structures of DCMU and the four mycotoxins as ligands were constructed using ChemDraw 18.0 . The ligand structures were energetically minimized using the MM2 energy minimizations tool in Chem3D Pro 14.0 . The possible binding site of ligands docking was set to the QB binding site in the D1 crystal structure of C. reinhardtii (PDB: 6KAC). The molecular docking was performed with CDocker in Discovery Studio. For the setting of the docking parameters, the Top Hits was set to 10, and the Pose Cluster Radius was set to 0.5 Å. We used the default values for the other parameters.The crystal structure information of the D1 protein of One-way ANOVA was carried out, and means were separated by Duncan’s LSD at 95% using SPSS Statistics 20.0 .C. reinhardtii cells. These mycotoxins disrupt PSII RCs by occupying the QB site, then block the electron transport flow from QA to QB, and finally inhibit photosynthetic activity. The four mycotoxins form a hydrogen bond with the QB site via the D1-Phe265 residue, which is different from classical PSII herbicides. The structures of these mycotoxins may provide ideas for the discovery of novel derivatives with stronger herbicide activity. However, the more precise binding conditions of the mycotoxins need to be further explored.In conclusion, we showed that the mycotoxins fumagillin, mevastatin, radicicol, and wortmannin exhibit multiple negative effects on the photosynthetic process of


Acute or chronic exposures to pesticides have been linked to neurotoxicity and the potential development of neurodegenerative diseases (NDDs). This study aimed to consider the neurotoxicity of three widely utilized pesticides: malathion, chlorpyrifos, and paraquat within the hippocampus (HC), corpus striatum (CS), cerebellum (CER), and cerebral cortex (CC). Neurotoxicity was evaluated at relatively low, medium, and high pesticide dosages. All pesticides inhibited acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in each of the brain regions, but esterase inhibition was greatest in the HC and CS. Each of the pesticides also induced greater disruption to cellular bioenergetics within the HC and CS, and this was monitored via inhibition of mitochondrial complex enzymes I and II, reduced ATP levels, and increased lactate production. Similarly, the HC and CS were more vulnerable to redox stress, with greater inhibition of the antioxidant enzymes catalase and superoxide dismutase and increased lipid peroxidation. All pesticides induced the production of nuclear Nrf2 in a dose-dependent manner. Collectively, these results show that pesticides disrupt cellular bioenergetics and that the HC and CS are more susceptible to pesticide effects than the CER and CC. Pesticides are widely utilized industrial and residential chemicals and primarily include herbicides, insecticides, fungicides, and rodenticides. Further sub-classification of pesticides can be made based on their chemical structures; for example, organophosphates (OPs) represent the commonly employed chemical subset of insecticides, estimated at approximately 30% of the global insecticide market and approximately 13% of the European Union market . Experiments were conducted at least 4 times for each concentration of a brain sample. The extinction coefficient was estimated as 6.2 mM−1 cm−1 for NADH, for complex I specific activity measurements. A value of 18.5 mM−1 cm−1 for reduced cytochrome c was used for mitochondrial complex III measurements [For both mitochondrial complex assays, the background was corrected by subtraction of the absorbance of samples without lysate. Specific complex I or III activity for each sample was calculated as the difference between the cuvette readings in the absence and presence of rotenone or antimycin A, respectively. Specific complex enzyme activity . Excess PCA was then precipitated by the addition of ice-cold 2M KOH , and then the homogenate was neutralized with Tris base (pH = 7). ATP levels were then quantified using an ATP assay kit  following the manufacturer’s protocol. The assay is based on the phosphorylation of glycerol to generate a product quantified by a colourimetric method. Absorbance was read at 570 nm via the spectrophotometer , and ATP content was expressed as µmoles/mg of sample homogenate.g for 15 min at 4 °C, and the supernatant was retained. A dilution factor for deproteinization was calculated for each sample; 25 µL of each sample was pipetted into a 96-well plate and incubated with the reaction mixture at room temperature for 30 min. Results were then measured at 450 nm via a spectrophotometer . The absorbance readings from blank wells were subtracted from samples and standards, and a standard curve was plotted. Readings from brain samples were interpolated from the standard curve, and the final concentration of lactate was calculated. Lactate levels were normalized to the protein concentrations. Experiments were performed in triplicates.Quantitation of lactate was performed using a spectrophotometric L-lactate assay kit according to the manufacturer’s guidelines . In brief, 10 mg of brain tissue was washed in ice-cold saline and then homogenized in 400 µL of assay buffer. The homogenate was centrifuged for 5 min at 14,000 rpm at 4 °C to remove pelleted insoluble material. The supernatant was collected and kept on ice. Deproteinization was performed with PCA before samples were centrifuged at 13,000× 2O2 consumed per minute per mg of protein (Ub) according to the method described by Singh et al. (2008) [Catalase (CAT) was assayed colourimetrically at 620 nm and was expressed as µmoles of H. (2008) . Each reAssays for superoxide dismutase-1 (SOD-1) were based on the SOD-mediated inhibition of the reduction of nitroblue tetrazolium to blue formazan by superoxide anions, as described by Beauchamp and Fridovich (1971) . Units oLipid peroxidation was estimated in brain tissues by quantifying the levels of thiobarbituric acid reactive substance (TBARS), according to the method of Niehaus and Samuelson (1968) . In brieNrf2 levels were quantified in nuclear extracts from brain samples according to the manufacturer’s protocol using a commercial kit . Briefly, 40 μL of complete binding buffer (CBB) was added to each well, and then 10 μL of each sample or a blank was added to the wells of a 96-well plate containing an immobilized double stranded DNA consensus binding site for Nrf2. The plate was incubated for 1 h at room temperature on a rocking platform at 100 rpm. Wells were washed 3 times with 200 μL 1× wash buffer, and then immunodetection of bound protein was initiated by incubation with a primary antibody at 1:1000 dilution for 1 h at room temperature. The plate wells were washed before incubation with secondary antibody (1:1000 dilution) for 1 h at room temperature. The plate wells were then washed before the addition of a developing solution (100 μL/well). After 10 min, a stop solution was added, and the absorbance of the wells read at 450 nm . Blank values were subtracted from test sample absorbances.p value of <0.05. The effect of neurotoxins on mitochondrial complex enzyme kinetics was evaluated using nonlinear regression. A MichaelisMenten model was used to determine enzymatic Ki  and Vmax (maximum enzyme velocity).Groups of data were compared using either one-way or two-way ANOVAs with Bonferroni post-tests to compare the effects of the tested pesticides at the three dosing concentrations and for the four different brain regions for enzymatic activities, bioenergetics measurements, and redox status. Statistical analyses were performed using GraphPad Prism 5.0 . Statistical significance was defined as a Although the pesticides MAL, CPF, and PQ decreased AChE activity in a dose-dependent manner, neither the endogenous activity of AChE (controls) nor the response to pesticides was uniform across the brain regions studied  A–C. EndoA similar profile of brain regional responses was observed after dosing with CPF: with only HC and CS regions vulnerable to AChE inhibition at the lower dosing regimens and AChE significantly inhibited within the CC only at the highest dosing treatment. PQ dosing followed the same pattern except that the intermediate dosing also induced significant inhibition of AChE within the CC region A–C.Endogenous NTE activity was relatively uniform across the brain regions, but the vulnerability of the HC and CS brain regions to the effects of pesticides was evidenced by the significant inhibition of NTE by all three pesticides and at all of the pesticide doses A–C. By cThe impact of pesticides on cellular bioenergetics was examined via activity assay measurements of mitochondrial complex I (MC I) and mitochondrial complex III (MC III) from within each of the brain regions. Pesticide dosing reduced MC I and MC III activities in a dose-dependent manner A–F. EndoMC III was more sensitive to inhibition by each of the pesticides, and similar to the serine hydrolase measurements , the HC Cellular ATP levels declined in a dose-dependent manner in response to pesticide treatments, with significant reductions at either the intermediate or high pesticide dosing A–C. The An examination of the production of cellular lactate was used to further evidence altered cellular bioenergetics. Lactate production increased according to the pesticide dosage and mirrored the corresponding reduction of ATP levels such that there was significant lactate production in response to intermediate or high pesticide dosing for the HC and CS brain regions, but only at high pesticide dosing for the CER and CS brain regions D–F.Pesticides decreased the activities of the antioxidant enzymes, catalase (CAT) A–C and sIn keeping with the pesticide-induced redox stress, there was a parallel increase in lipid peroxidation measured as TBARS A–C. The Nuclear activation of the transcription factor Nrf2 promotes induction of cytoprotective proteins in response to cellular stress, and nuclear Nrf2 levels increased according to pesticide dose A–C.This study was undertaken to examine the neurotoxic effects of three widely utilized pesticides, the OPs MAL and CPF, and the bipyridilium, PQ. These pesticides share common mechanisms of neurotoxicity via inhibition of the serine hydrolase AChE and through induction of redox stress. However, whether these pesticides have altered capacity to inhibit another serine hydrolase, NTE, or whether they differentially affect cellular bioenergetics and elicit cellular damage that is uniform across different brain regions have not previously been considered. The results of this study indicate that for the four brain regions examined, the HC and CS are more susceptible to the toxic effects of these pesticides, including a disruption of cellular bioenergetics, than the CER and CC.All three pesticides inhibited AChE A–C, the 50) of approximately 8 µM [Inhibition of target AChE is the desired mechanism of action of OP pesticides and, for the relatively inert thions, MAL and CPF, is primarily driven after their bioactivation to oxon forms that are essentially irreversible AChE inhibitors. PQ, although not an OP, is still able to bind and inhibit AChE ,54. CollCollectively at the doses employed, cholinergic toxicity was minimal, and due to the expected hydrolysis of the bound inhibitor as well as AChE turnover, the impact on ACh signalling (cholinergic mechanisms) would seem unlikely to represent a mechanism associated with long-term health consequences in the dosed animals, although an extended time course of animal study was not undertaken.Dosing with these pesticides also led to a mild to moderate inhibition of NTE. NTE is a transmembranous protein ubiquitously expressed within tissues including neurons ,58. SimiAll pesticides resulted in a disruption to the bioenergetic capacity of each brain region with inhibition of mitochondrial complex I and complex III activities A–F. In kIncreased cellular redox stress is mitigated by the antioxidant enzymes CAT and SOD, as well as cellular thiols such as glutathione. SOD catalyses the dismutation of a superoxide radical to form either molecular oxygen or hydrogen peroxide, and the latter can be further decomposed to water and molecular oxygen via the action of catalase. Consistent with the induction of redox stress and depletion of antioxidant enzyme capacities, pesticide dosing resulted in reduced CAT and SOD activities A–F. SimiThe pesticide induction of cellular redox stress will be enhanced through mitochondrial damage and liberation of further ROS that can damage proteins, lipids, and DNA. Oxidative damage to lipids was quantified as TBARS, and these were elevated in response to the pesticides, with the greatest levels of lipid peroxidation within the CS at a high dosing A–C. OtheIn keeping with the stimulation of cellular response to mitigate redox stress, the transcription factor Nrf2 was activated in all brain regions and most notably within the HC and CS A–C. Nrf2In summary, although all three pesticides inhibit AChE and NTE, the relatively mild levels of enzymatic inhibition when coupled to rates of dissociation or spontaneous hydrolysis of the pesticide-protein complex as well as protein turnover, indicate that acute and chronic cholinergic toxicity or induction of OPIDN is unlikely at this level of serine hydrolase inhibition. Furthermore, acute OP toxicity will likely decline due to the activity of xenobiotic metabolizing enzymes, including serine and A-esterases for OPs . Hence, Injury to mitochondria and reduced mitochondrial efficiency and a net reduction of ATP production and concomitant cellular redox stress are common mechanisms of cellular damage from xenobiotic agents, and this includes pesticides. Hence, xenobiotic-induced mitochondrial dyshomeostasis and redox stress are likely a commonality that could contribute to the development and/or progression of NDDs . In this


DM1–DM20) were synthesized and tested for their ability to inhibit monoamine oxidase (MAOs). Compound DM2 exhibited the most significant inhibition against MAO-B with an IC50 value of 0.067 µM, followed by compound DM18 (IC50 = 0.118 µM), with selectivity index (SI) values of 93.88 and >338.98, respectively. However, none of the substances successfully inhibited MAO-A. The MAO-B inhibitors DM2 and DM18 were competitive and reversible, with Ki values of 0.032 ± 0.004 and 0.045 ± 0.001 µM, respectively. DM2 was non-toxic below 100 µg/mL in the cytotoxic test using the Vero epithelial cell line by the MTT method. According to molecular docking studies, DM2 and DM18 formed very similar conformations within the MAO-B binding pocket, with the ortho-chlorine and ortho-fluorine aromatic rings sandwiched between F168 and Y326. These conformations were predicted to show better interactions with the targeted MAO-B than MAO-A. In particular, the induced-fit docking of the dimethoxy phenyl ring of DM2 facing the hydrophobic pocket made up of FAD, Y398, and Y435 had an impact on F168 in the docking pocket. Taken together, DM2 and DM18 may be suitable candidates for treating neurodegenerative conditions such as Parkinson’s disease.Two series of dimethoxy-halogenated chalcones ( Parkinson’s disease (PD) is a neurological condition that affects the patient’s ability to move about and regulate their muscles. The loss of striatal dopaminergic neurons causes motor symptoms of PD, and that of dopaminergic areas causes non-motor symptoms. Bradykinesia, muscle stiffness, and tremors are PD’s motor symptoms, whereas depression, cognitive problems, and sleep disorders are its motor symptoms ,2,3,4. PHistopathological studies explain that the presence of Lewy bodies  and loss of pigmented dopaminergic neurons are significant reasons. The degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which project to the nigrostriatal pathway, is a substantial factor in the loss of dopaminergic function. In the extrapyramidal system, excitatory (D1) and inhibitory (D2) dopamine receptors are responsible for motor activity. Functional dopaminergic loss in PD patients results in the dysfunction of gamma-aminobutyric acid (GABA), and activity was stimulated in the internal globus pallidal segment (GPi) or pars reticulata portion of the substantia nigra (SNpr), which leads to the inhibition of thalamus. Decreased motor activity results from the diminished ability of the thalamus to switch on the frontal cortex ,18.O-methyltransferase (COMT) inhibitors are used for the treatment of motor as well as non-motor symptoms .Several studies have suggested that methoxy-chalcones are multi-targeting scaffolds for the expansion of multi-target ligands for the management of various neurodegenerative disorders ,51,52,53hibition . TherefoBy using Claisen–Schmidt condensation, 2,4-dimethoxy benzaldehyde and 3,4-dimethoxy benzaldehyde were condensed with different substituted halogenated benzaldehydes to synthesize dimethoxy halogenated chalcones . The synDM1–DM10) showed more effective inhibitory activities against MAO-B, compared to 3′,4′-dimethoxy chalcones derivatives (DM11–DM20) . The -Cl atom on ortho position (DM2) increased MAO-B inhibitory activity compared to the parental compound DM1.The present study documented that when compared to MAO-A, two series of dimethoxylated chalcones inhibited MAO-B more potently, and they all had potent MAO-B inhibitory actions with the residual activity of less than 50% at 10 µM . In gene11–DM20) . Compounortho position  improved the MAO-B inhibitory profile less than the -Cl atom in that respective position. MAO-B inhibitory activities increased with ortho-Cl (DM2) > ortho-Br (DM5) > -H (DM1) > ortho-F (DM8). In addition, -Cl atom on ortho position (DM2) was effective on MAO-B inhibitory activity compared to meta and para positions   than ortho-Br (DM15), ortho-Cl (DM12), and -H (DM11). In DM18, -F atom on ortho position inhibited MAO-B 3.75 and four times more, respectively, than -F in the meta and para positions of DM19 and DM20 (DM2 and DM18 were selective for MAO-B with selectivity index (SI) values of 93.88 and >338.98, respectively, over MAO-A, suggesting that DM2 has the highest inhibitory activity, and DM18 has extremely high selectivity for MAO-B  . On the and DM20 . DM2 andor MAO-B . StructuA of chalcones. The 2′,4′- and 3′,4′-dimethoxy substitutions are the only two possible pharmacophores we can generate from this aspect. It is obvious from the selectivity pattern that 3′,4′-dimethoxy substituted chalcones have no effect on the MAO-A inhibition. This theory postulated that the positioning of two methoxy groups separated by an aromatic carbon would maximize the selectivity of MAO-B (DM1–DM10). The 3′,4′-dimethoxy substituted chalcones in close proximity (DM11–DM20) demonstrated strong MAO-B inhibition and weak MAO-A activity.Our design strategy mainly focused on the di-methoxy group in various positions of the second, third, and fourth of ring DM2 and DM18 were competitive inhibitors of MAO-B in the kinetic investigations of MAO-B  plots revealed that of MAO-B A,C, and ectively B,D. ThesDM2, 0.24 µM for DM18, 0.22 µM for lazabemide (a reference reversible inhibitor), and 0.28 µM for pargyline (a reference irreversible inhibitor). To identify the reversibility patterns, the relative activities for samples dialyzed (AD) and for those undialyzed (AU) were compared. The MAO-B inhibitions by DM2 and DM18 were restored from 36.0% (AU) to 74.0% (AD) and from 40.1% to 76.6%, respectively (DM2 and DM18 were reversible MAO-B inhibitors.The concentrations used in the tests were 0.13 µM for ectively . These rDM2. Vero cells were treated to various doses (10–500 µg/mL) for 48 h, and relative cell viability was determined by measuring the absorbance at 570 nm with an ELISA microplate. The results demonstrated that cell toxicity varied with concentration and that at a concentration of 100 µg/mL, 80% of cells were alive  and substituted benzaldehyde (0.01 mol) were dissolved in 50 mL of ethanol and stirred for 6 h at room temperature. After being transferred to water, the solution was acidified with 10% HCl, filtered, washed with water, and dried. In order to obtain pure crystals, the product was recrystallized using ethanol . Throughm of benzylamine in this study was 0.13 mM. Chemicals were from Sigma-Aldrich [Recombinant MAO-A and MAO-B were used to test the MAO inhibitory activity using the substrates kynuramine and benzylamine  . Lazabem-Aldrich .50 values for MAO-A and those for MAO-B. The MAO-B was used to study the kinetics of substances DM2 or DM18 at five different substrate concentrations. By using the LB plots and their secondary plots at three different inhibitor doses, the inhibition patterns were examined.MAO-A At 10 µM, compounds’ inhibitory effects on MAOs were initially recorded. The IC50 values of the compounds were calculated for those that had residual activity that were close to or below 50% . The SI DM2 or DM18 at ~2 × IC50 , dialysis was performed to assess the reversibility of MAO-B inhibition [D) and undialyzed (AU), reversibility patterns were determined.Following a 30 min preincubation with MAO-B and hibition . To servDM2 was carried out as previously described [Cytotoxicity study of the lead molecule escribed ,61 and aThe crystal structure of MAO-B was downloaded from the Protein Data Bank by selecting entry 2V5Z . The docDM1–DM20) with different substituted halogens were synthesized and examined for their ability to inhibit MAOs. All twenty molecules showed greater MAO-B inhibitory action than MAO-A. Chalcones have a significant impact on the MAO-B inhibitory actions depending on the kind and orientation of the halogen atoms on the B phenyl ring of the chalcone. The two sets greatly influenced MAO-B inhibition by substituting halogens at different locations on the phenyl ring B, notably at the ortho position. Compound DM2 most potently inhibited MAO-B with an IC50 value of 0.067 μM, followed by DM18, DM3, DM6, DM17, and DM5 . Relative activities for undialyzed (AU) and dialyzed (AD) were compared, and inhibition of MAO-B by DM2 and DM18 was recovered, similar to the reversible reference inhibitor against MAO-B. The results documented that DM2 and DM18 were reversible inhibitors of MAO-B. The biological safety of DM2 was evaluated by cytotoxic evaluations at different concentrations, and it was found that DM2 was not harmful to Vero cells below a dosage of 100 µg/mL. Induced-fit docking simulations gave a robust and detailed explanation of the binding mode of compounds DM2 and DM18 towards MAO-B. In particular, the two compounds can be assumed to have very similar conformations within the MAO-B binding pocket, with the ortho-chlorine and ortho-fluorine aromatic rings sandwiched between Y326 and F168, and the latter particularly was affected by the induced-fit docking of compound DM2, and the dimethoxy phenyl rings faced the aromatic pocket consisting of FAD, Y398, and Y435. Finally, the presence of hydrogen bonds with the side chain of Y188 can stabilize the two compounds within the binding pocket. The study concluded that the DM2 and DM18 molecules from this series could be considered candidates for the development of a new class of MAO-B inhibitors for the treatment of PD.In this study, two series of twenty dimethoxy-chalcones (


Ki value of 13.0 nm). Further mutational analyses of the peptide resulted in the development of a short peptide inhibitor against subtilisin BPN′ that showed high inhibitory activity and binding affinity . This activity was found to be derived from the conformational rigidity caused by the intramolecular disulfide bond and the small residue at the P1′ site and from the interaction of the P4 and P6′ residues with subtilisin BPN′.Proteinaceous protease inhibitors can strongly and specifically inhibit cognate proteases, but their use as pharmaceuticals is limited by their size. As such, the development of effective protease peptide inhibitors would be beneficial for biochemical studies and drug discovery. In this study, we applied a phage display system to select subtilisin BPN′‐binding peptides and evaluated their inhibitory activities against subtilisin BPN′. A 12mer peptide with an intramolecular disulfide bond inhibited subtilisin BPN′ ( Ki) of 13 nm against subtilisin BPN′. Subsequent mutation analyses on this peptide led to the development of an improved inhibitory peptide with Ki of 0.3 nm.We identified several potential inhibitory peptides for subtilisin BPN′, a typical serine protease, by phage display method. Of these, a 12mer peptide, named ASIP, showed inhibitory activity with an inhibition constant ( ASIPartificial subtilisin inhibitor peptideCI‐2hiproly barley chymotrypsin inhibitor 2EGLleech eglin‐CMSTmarinostatinOMTKY3turkey ovomucoid third domainSSIStreptomyces subtilisin inhibitorTI‐IIwound‐induced tomato protease inhibitor‐IIProtein–protein interactions are involved in various cellular processes, such as signal transduction, electron transfer, protein modification, cellular metabolism, and immune system functions. Protein functions including enzyme activities can be modulated via complex formation through protein–protein interactions. Among these, the interactions between proteases and their inhibitors have been well studied for many decades. In addition, short peptides, including cyclic forms, are attractive compounds for drug discovery because they regulate disease‐related protease functions .Proteases are enzymes that catalyze the hydrolysis of peptide bonds in peptides and proteins. Serine proteases are the most studied protease family, and they have the Ser‐His‐Asp catalytic triad in their active site and are widely involved in biological processes. For example, matriptase is associated with cancer cell metastasis . TherefoNumerous inhibitors of serine proteases have been discovered in nature or developed through chemical synthesis, and they have a wide size distribution from small molecules to proteins. Peptides could stand out as inhibitors because of their variability, productivity, and efficacy. Proteinaceous protease inhibitors can enhance the interaction with proteases or alter the specificity of proteases through the introduction of amino acids into the reactive site loops , 6, 7. PPhage display is a powerful screening method to detect interactions between a target molecule  and a peptide or protein genetically inserted into a coat protein gene of a bacteriophage . In combN‐succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide (N‐suc‐AAPF‐pNA) were purchased from Sigma‐Aldrich . The oligopeptides used in this study were synthesized by Eurofins Genomics . The formation of an S‐S bond in each peptide was accomplished by incubating an equimolar amount of cystine with the peptide, and the concentrations of the oxidized peptides were determined by amino acid analyses after hydrolysis of the peptide with 6 m HCl at 110 °C for 24 h in vacuo.Subtilisin BPN′ and Phage display was performed using a Ph.D.‐12 Phage Display Peptide Library Kit . According to the protocol, phages were co‐incubated with biotin‐labeled subtilisin BPN′, and then, the mixture was poured onto a streptavidin plate. The plate was washed to remove nonspecifically bound phages, and phages specifically bound to subtilisin BPN′ were amplified. After three rounds of panning, the phages were collected. The nucleotide sequences of the selected phages were determined by dideoxy sequencing of phage DNA.Ki values, the proteolytic activities of subtilisin BPN′ in the presence of various molar ratios of synthetic peptides were measured using a synthetic substrate, N‐suc‐AAPF‐pNA, as described previously [Ki values were calculated by nonlinear curve fitting using the Morrison equation [The inhibitory activities of peptides against subtilisin BPN′ were preliminarily measured using the casein‐Folin method. First, casein was incubated with subtilisin BPN′ in the absence or presence of various molar ratios of synthetic peptides at 37 °C and pH 7.0 for 10 min. Trichloroacetic acid was added to the reaction mixture to precipitate the undigested casein, and the supernatant was mixed with sodium carbonate and Folin's phenol reagent. The absorbance at 660 nm was then measured to determine the amount of peptide obtained by digestion. Inhibitory activity was defined as the reduction in the amount of peptides derived from the enzymatic digestion of casein in the presence of a synthetic peptide when compared to the amount of peptides in the absence of synthetic peptides. To determine the eviously , and theequation .Based on the phage display experiment with three cycles of panning, eight 12‐residue sequences were identified from the 16 phages analyzed . The inhibitory properties of the oxidized form of ASIP were then investigated. Oxidized ASIP clearly exhibited inhibitory activity toward the digestion of a synthetic substrate of subtilisin in a concentration‐dependent manner, and the inhibition constant was determined to be 13.0 nTo investigate the required minimum length of ASIP for strong inhibition of subtilisin BPN′, several truncated derivatives of ASIP were constructed and their inhibitory activities toward subtilisin BPN′ were preliminarily measured using casein as a substrate. However, the inhibitory activities obtained by this method were generally lower than those obtained using a synthetic substrate. ΔS1, Δ(S1 + D2), and Δ(S1 + D2 + F3) represent peptides truncated by one, two, and three residues from the N‐terminus of ASIP, respectively, and ΔT12 and Δ(R11 + T12) represent peptides truncated by one and two residues from the C‐terminus, respectively. As shown in Fig. To evaluate the contribution of each amino acid of ASIP to its inhibitory activity, a single‐alanine scanning experiment for ASIP was carried out , and then, the inhibitory activities were measured using casein as a substrate similar to that with the truncated ASIP. As shown in Fig. F3A, L6A, E8A, and R11A showed relatively weaker inhibitory activities than the prototype ASIP. In particular, Leu6 and Phe3 were presumed to be the P1 and P4 residues, respectively, as indicated by the specific cleavage of the linear form of ASIP between Leu6 and Ser7 by subtilisin BPN′. The side chains of these residues were thought to be likely responsible for the strong binding of ASIP to subtilisin BPN′. Moreover, Glu8 and Arg11 were assumed to form salt bridges or hydrogen bonds with subtilisin BPN′ via polar side chains.Ki values for subtilisin BPN′ using N‐suc‐AAPF‐pNA as a synthetic substrate. As shown in Table Ki value of 13.0 nm. The S7A and S7G substitutions showed stronger binding and inhibition , whereas the S7V, S7F, S7D, and S7K mutations significantly decreased inhibition. These results suggest that a residue with a large side chain is inappropriate at the P1′ position, even if the residue is hydrophobic or hydrophilic, which is probably because of steric hindrance in binding with subtilisin BPN′.To examine the possibility of improving the inhibitory activity and binding ability of ASIP to subtilisin BPN′, we focused on Ser7 and Thr12, which showed increased inhibitory activity in alanine scanning experiments. These two residues were substituted with various amino acids, and their inhibitory activities were evaluated by determining the Ki = 17.4 nm) compared with that of the prototype ASIP, although T12A showed slightly increased inhibition of subtilisin when casein was used. In addition, the Ki values of T12G and T12L were calculated as 10.6 and 12.9 nm, respectively, indicating that the substitution of Thr12 with uncharged residues did not significantly improve the inhibitory activity. By contrast, drastic changes in the inhibitory activity were obtained by substituting charged residues. T12K and T12R showed significantly higher inhibition and binding affinity, as shown by the Ki value, which presented as a more than a two‐fold reduction, whereas T12D decreased the inhibitory activity compared to that of the prototype (Thr at the 12th position). These results indicate that a basic residue at the 12th position of ASIP enhances the inhibitory activity against subtilisin BPN′, which might be due to an electrostatic interaction between the positively charged C‐terminal residue of the inhibitor peptide and the negatively charged surface of subtilisin BPN′.For Thr12, the replacement with alanine (T12A) resulted in a slight decrease in inhibitory activity  [Ki value of 0.3 nm. Its length might be among the shortest observed for peptides composed of common amino acids that show strong inhibitory activity with Ki less than 1 nm. Such a short length is associated with the formation of the disulfide bond and might be due to the presence of a specific P4–S4 interaction in addition to the P1–S1 interaction in well‐known proteinaceous inhibitors. As described, further structural analyses of the complex will clarify these points, and such analyses will also provide valuable insights for the further development of a functional peptide inhibitor.The results showed that the linear form of the chemically synthesized peptide (ASIP) with a sequence selected by the phage display system was specifically cleaved at Leu6‐Ser7 by subtilisin BPN′ whereas the oxidized form of the peptide with an S‐S bond between Cys5 and Cys10 inhibited subtilisin BPN′. These findings indicate that the P1 residue of ASIP is Leu6, which is consistent with the substrate specificity of subtilisin BPN′, and suggest that the rigid conformation around the P1 residue caused by the S‐S bond is required for the inhibitory activity of ASIP, which is consistent with many other proteinaceous inhibitors. The binding ability of the prototype ASIP was subsequently increased 40‐fold based on the Ser7 → Ala and Thr12 → Lys mutations. Then, we compared the amino acid sequence of the resulting strong inhibitor with that of other subtilisin inhibitors for which the three‐dimensional structures are known  , hiprolyor (SSI) , and maror (SSI) ). As sho , hiprolor (SSI) . The Thror (SSI)  can alsoThe authors declare no conflict of interestSK conceived and supervised the study; SK designed the experiments; MS, AW, and YM performed the experiments; KI, MS, AW, YM, AN, and SK analyzed the data; KI, AN, and SK wrote the manuscript; KI, AN, and SK made the manuscript revision.


In addition, four implantation methods with different regions based on the three parallel lines in horizontal plane were simulated as types 1–2, 1–3, 2–2, and 2–3 . L3‐4 ROM, interbody cage stress, screw‐bone interface stress, and L4 superior endplate stress were tracked and calculated for comparisons among these models.A validated L2‐4 finite element (FE) model was modified for simulation. The L3‐4 fused segment undergoing LLIF surgery was modeled. The area between the superior and inferior edges and the anterior and posterior edges of the vertebral body (VB) is divided into four zones by three parallel lines in coronal and horizontal planes. The L3‐4 ROM of Models A, B, and C decreased with the extent ranging from 47.9% (flexion‐extension) to 62.4%  with no significant differences under any loading condition. Types 2–2 and 2–3 had 45% restriction, while types 1–2 and 1–3 had 51% restriction in ROM under flexion‐extension conditions. Under lateral bending, types 2–2 and 2–3 had 70.6% restriction, while types 1–2 and 1–3 had 61.2% restriction in ROM. Under axial rotation, types 2–2 and 2–3 had 65.2% restriction, while types 1–2 and 1–3 had 59.3% restriction in ROM. The stress of the cage in types 2–2 and 2–3 was approximately 20% lower than that in types 1–2 and 1–3 under all loading conditions in all models. The peak stresses at the screw‐bone interface in types 2–2 and 2–3 were much lower (approximately 35%) than those in types 1–2 and 1–3 under lateral bending, while no significant differences were observed under flexion‐extension and axial rotation. The peak stress on the L4 superior endplate was approximately 30 MPa and was not significantly different in all models under any loading condition.in situ screw implantation in LLIF surgery.Different regions of entry‐exit screws induced multiple screw trajectories and influenced the stability and mechanical responses. However, different implantation angles did not. Considering the difficulty of implantation, the ipsilateral‐contralateral trajectory in the lateral middle region of the VB can be optimal for  in situ screw implantation in LLIF.Different angles of entry‐exit screw influenced the stability and mechanical responses of the fused segment. Ipsilateral‐contralateral trajectory in lateral middle region of the VB can be an optimized selection for  Over the past 15 years, significant interest has arisen in minimally invasive anterolateral approaches to the lumbar spineet al. reported that spinal movement with stand‐alone LLIF could compromise intervertebral fusion and might present a higher risk of cage subsidenceet al. reported a 1‐year follow‐up after stand‐alone LLIF; 70% of the cases had mild to moderate cage subsidence, and 30% were considered severe subsidenceSuccessful fusion is conducive to a satisfactory clinical outcome. Since the anterior and posterior longitudinal ligaments are preserved, stand‐alone LLIF  was once considered to have the ability to stabilize the mobile segmentet al. showed that lateral stabilization added to the vertebra and the spinous process could achieve stiffness under all loading conditions, similar to pedicle screwset al. reported that the lateral plate increased stiffness under bending and axial rotation and reduced cage stress in all motion modesin situ screws as supplemental instrumentation in LLIFin situ screws avoids damage to the posterior bony elements, conducive to withstanding greater compressive loads, resulting in an important bearing function due to its mechanical propertiesSupplemental internal fixation can be performed in different ways. Spinal surgeons accustomed to traditional posterior lumbar interbody fusion (PLIF)/transforaminal lumbar interbody fusion (TLIF) surgery might prefer posterior pedicle screw fixation. However, previous studies have demonstrated that lateral instrumentation could provide similar stability of the fused segment and reasonable biomechanical protection of the interbody cage and endplate compared to pedicle screwset al. put forward a new division theory in 2010In situ screw implantation and disc space preparation are both performed through the lateral retroperitoneal approach in LLIF surgery, and both have a risk of nerve injuries. Therefore, referring to the classification proposed by Uribe et al., the area between the anterior and posterior edges and the superior and inferior edges of the vertebral body (VB) is divided into four zones by three parallel lines in the coronal and horizontal planes. Theoretically, anterolateral in situ screws could be implanted in different regions and at different angles. However, to date, no study has been reported on whether different in situ screw implantation regions and angles have any effect on segmental stability. Therefore, the goal of this study was (i) to introduce screw‐setting strategies for anterolateral in situ screws following LLIF and (ii) to explore the biomechanical effect on different screw‐setting strategies following LLIF using finite‐element analysis (FEA). It was hypothesized that different implantation regions and angles of screws possess various degrees of mechanical stability and protective effects.To investigate the anatomically safe zones relative to the disc spaces for the prevention of nerve injuries during the lateral retroperitoneal transpsoas approach, Uribe A 3D FE model of L2‐L4 was constructed in this study Figure . The imaThen, preprocessing FE software Simlab  was used to construct the spinal ligaments. The anterior/posterior longitudinal ligament (ALL/PLL), ligamentum flavum (LF), interspinous ligament (ISL), supraspinous ligament (SSL), and intertransverse ligament (ITL) were constructed in the FE model.The vertebra consisted of the cortical layer with a thickness of 1 mmet al.The inferior surface of the L4 vertebra was completely constrained in all directions, and the loading condition was applied to the superior surface of the L2 vertebra. Utilizing an approach similar to that of Shim MAE) and the root mean squared error (RMSE).RMSE and MAE are methods that attempt to determine the relationships between input variables and one or more response variables. These criteria are as follows:yi are predicted values that were obtained from the finite element model, xi are actual values, and n are the numerical data that were analyzed, as shown in Formulas MAE and RMSE means better matching of the finite element model and experimental sample.The kinematic behavior of the FE model was verified under the conditions of flexion, extension, lateral bending, and axial rotation. The range of motion (ROM) and intradiscal pressure (IDP) were the parameters chosen for validation. To validate the present model, a few criteria were applied. They were the mean absolute error  of one side of the VB and penetrated the cortex from the middle (Line 2) of the other side of the VB, we defined the fixation method as type 1–2. Similarly, after penetrating the cortex from the posterior (Line 3) of the other side of the VB, we defined the fixation method as type 1–3. When an in situ screw was inserted from the middle (Line 2) of one side of the VB and penetrated the cortex from Line 2 and Line 3, we defined it as 2–2 and 2–3, respectively  and polyetheretherketone L3/4 ROM, interbody cage stress (von Mises stress), screw‐bone interface stress, and L4 superior endplate stress were tracked and calculated to compare the models.et al., as shown in Figure MAE (0.50) and RMSE (0.48) values are minor, which indicates that the present FE model possesses a good predictive capacity. Moreover, under 300 N and 1000 N compressive forces of L2/3 IDP, the results are consistent with the results of Brinckmann et al.A mesh convergence test indicated that the FE solution used a model with 99,948 nodes and 241,923 elements. A mesh quality assessment in Simlab software showed that 1% of the elements had an aspect ratio of less than 5.0 (maximum 10.0), while 97% and 96% of all shell and solid elements had Jacobian values larger than 0.6 (minimum 0.24). We compared the intact lumbar spine model with a previous cadaverbiomechanical study under the same loading conditions. The ROM values of the L2/3 and L3/4 segments are well‐correlated with the results of Shim The percentage differences in the ROM and IDP between the linear basic model and the linear low‐value model and between the linear basic model and the linear high‐value model under flexion and compression are shown in Figure Since ROM is a stress sensitivity index, this study compared the ROM among the three models with different angles. Compared with the intact model under all loading conditions, the ROM of Models A, B, and C at L3/4 was significantly reduced, as shown in Figure All models provided the lowest ROM restriction under flexion‐extension. No ROM difference existed among the three models in types 1–2 and 1–3 or types 2–2 and 2–3. However, in Model A, types 1–2 and 1–3 (51% restriction) restricted ROM more than types 2–2 and 2–3 (45% restriction) compared with the intact model, and this trend existed in Models B and C.For lateral bending, types 1–2 and 1–3 reduced the ROM of the intact model by 61.2% in Model A, and the ROM was 1.3 times greater than that of types 2–2 and 2–3 (70.6% restriction). There was no significant difference in type 2–2 and type 2–3. This trend also existed in Models B and C. All models had the largest ROM restriction under lateral bending than flexion‐extension and axial rotation.Similar to lateral bending, types 2–2 and 2–3 reduced the ROM of the intact model by 65.2% more than types 1–2 and 1–3 (59.3% restriction) in Model A. Similar ROM restrictions existed between types 1–2 and 1–3 and types 2–2 and 2–3 of Model B together with Model C.The maximum von Mises stress in the interbody cage for the implanted models is shown in Figure in situ screws of four types in different models under all loading conditions are shown in the supplemental materials.The peak stress at the screw‐bone interface was also investigated. It could reflect the load distribution between the vertebrae and the spinal implants. It is important to assess the risk of screw loosening and migration at the fusion segment, one of the important complications of internal fixation. Figure The peak stress of the L4 superior endplate, which was associated with endplate fracture and cage subsidence, was also investigated, as shown in Figure Our study showed that the ROM of all models with implants was restricted under all loading conditions compared with the intact model, and the lowest ROM restriction was under flexion‐extension, while the largest was under lateral bending. In terms of the screw implantation angle in the coronal plane, the difference in ROM restriction was not significant among the different models. From the aspect of the screw implantation region in the horizontal plane, types 2–2 and 2–3 were restricted more than types 1–2 and 1–3, with no significant difference. The maximum stress of the interbody cage in types 2–2 and 2–3 was approximately 20% lower than that in types 1–2 and 1–3 under all loading conditions. For the peak stress of the screw‐bone interface, the models did not differ significantly in terms of flexion‐extension and axial rotation. However, in types 2–2 and 2–3, it was much lower  than that in types 1–2 and 1–3 under lateral bending.in situ screw fixation through the LLIF incision can avoid changes in the patient's position during the operation, the need for a second incision, and injury to the spinal cord and nerve by the pedicle screwsin situ screw implantation on the stability of the fused segment, our study first used FEA to explore screw‐setting strategies for anterolateral in situ screws following LLIF from a biomechanical point of view.Previous studies have reported that stand‐alone LLIF was insufficient to provide stability under any loading condition, and this resulted in various complicationsin situ screw fixation could effectively offer sufficient stability for the segment fused in LLIF surgery. In addition, the in situ screw effectively reduced the ROM under lateral bending and axial rotation, and the effect was the most obvious in the former condition. These results are consistent with the findings of Shasti et al., who found that the reduction in lateral bending ROM was more remarkable when supplemented by an in situ screw in LLIFet al. also believed that ROM was an external response that was sensitive to the material properties of the spineLess ROM does not necessarily mean more stability. A stable system is one that does not undergo large displacements under small perturbations. According to the FDA definition, a less than 5° ROM is considered to be a successful fusion in clinical practicein situ screw. Instead, the difference might be caused by different regions of implantation in the horizontal planes. These findings are only useful for describing the static effect on immediate postoperative stability, but they do reflect an overall trend.Although all reconstructive models increased the stability of the fused segment compared to the intact model under all loading conditions, their magnitudes were not the same. Our results showed that types 2–2 and 2–3 had less ROM restriction than types 1–2 and 1–3 in flexion‐extension. In contrast, under both lateral bending and axial rotation, types 2–2 and 2–3 had more ROM restriction than types 1–2 and 1–3. Types 2–2 and 2–3 were more similar than types 1–2 and 1–3. Moreover, in the same implantation region, there was no significant difference in the stability of Models A, B, and C. Therefore, we believe that the stability of the fused segment is not correlated with the implantation angle of the in situ screw is made of titanium alloy, and its typical mechanical properties are 1380–2070 MPa for ultimate bearing strength and 825–895 MPa for yield strengthin situ screw was in the range between the yield strength and ultimate strength.In clinical practice, screw loosening and breakage are common reasons for revision of internal spinal fixation. Reducing the stress on the screws could reduce the risk of screw breakage and looseningCage subsidence with endplate injury is another complication that can occur after spinal fusion surgery. Although severe osteoporosis is a clear risk factor, the effect of stress on the cage should also be considered. From the aspects of biomechanics, excessive stress on the interbody cage could lead to cage subsidence. In our study, for Models A, B, and C, the stress on the cage in types 2–2 and 2–3 was lower than that in types 1–2 and 1–3 under all loading conditions. In addition, the stress in type 2–3 was less than that in type 2–2. Therefore, types 2–2 and 2–3 might have relatively low incidences of cage subsidence and endplate injury. Of note, although type 2–3 showed the advantages of less stress on the screw‐bone interface compared with type 2–2 from the perspective of biomechanics, it required more technical skill by the spinal surgeons for implantation and might increase the risk of encroachment on the foramina and spinal canal in clinical practice.In addition, endplate stress is also associated with endplate injury and cage subsidence. Previous biomechanical and clinical studies have demonstrated that supplemental pedicle or lateral screw fixation could significantly decrease endplate stress and effectively reduce the incidence of cage subsidence with endplate injuryThe intact finite element model was validated in this study, which was in good agreement with the results of previous studies. Stress sensitivity analysis can provide accurate insight into FE models investigating stress or strain. Our results showed that the 25% difference in modulus led to a total difference in ROM of 13.8% and 21.5% and IDP of 9.9% and 13.4% under flexion and compression, respectively. Actually, the tendency of the predicted results under various fixation options would not be materially changed depending on the individual geometric model and simplified material properties. Therefore, the intact FE model in this study was reasonable and sensitive for investigating biomechanical behavior. Of course, since there are many parameters of the material properties of the lumbar spine, a stress sensitivity analysis could not compare the individual effects of each parameter on the biomechanical behavior. In future research, we will analyze each parameter of the spinal components in detail.Although the findings in this study might be meaningful for clinical practice, its limitations should not be ignored. Bone tissues, ligaments, and implants all have linear‐elastic material properties. Since the focus of this study is not to predict the mechanical behavior of implants, isotropic linear‐elastic material models can be used to simulate the preyield mechanical behaviorin situ screws had little effect on the stability of LLIF. Types 2–2 and 2–3 could both provide better mechanical stability under all loading conditions and provide superior protective effects on the interbody cage and screw‐bone interface under lateral‐bending conditions. Compared with type 2–2, type 2–3 might have a higher risk of injury to the spinal cord or nerve. Therefore, the lateral middle region of the VB can be selected as the entry point for in situ screws in clinical practice. Moreover, type 2–2 might be a safer implantation strategy for in situ screws. Of course, additional clinical studies are needed to evaluate and confirm these findings.This study found that the angle of the No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.Table S1. The material properties of spinal componentsClick here for additional data file.


Chrozophora oblongifolia aerial parts resulted in the isolation of five phenolic compounds. The isolated metabolites were tested for their antioxidant and advanced glycation end-products (AGEs) formation, α-glucosidase, and lipase inhibitory activities. 1,3,6-Trigalloyl glucose exhibited the highest activity as an antioxidant and AGEs inhibitor as well as an α-glucosidase inhibitor. It showed promising binding affinity and stability towards the human intestinal maltase-glucoamylase α-glucosidases, as revealed through coupled molecular docking and dynamics studies that could encourage the utilization of this compound in the management of diabetes and its complications.The chemical investigation of C. oblongifolia aerial parts resulted in the isolation of five major compounds: apeginin-7-O-β-D-glucoside (1), quercetin-3-O-β-D-glucuronic acid (2), quercetin-3-O-β-D-galacturonic acid (3), rutin (4), and 1,3,6-trigalloyl glucose (5). The isolated compounds were tested for their antioxidant and AGEs formation, α-glucosidase, and lipase inhibitory activities. Compound 5 revealed the highest antioxidant and AGEs inhibitory activity in bovine serum albumin (BSA)-methylglyoxal, BSA-fructose, and arginine-methylglyoxal models. Moreover, it exhibited a potent inhibitory profile on Saccharomyces cerevisiae α-glucosidases compared to the positive control, acarbose. Compound (5) further depicted promising binding affinity and stability towards the human intestinal maltase-glucoamylase α-glucosidases, which is a diabetes-related therapeutic target, through coupled molecular docking and dynamics studies. The obtained results encourage the usage of 1,3,6-trigalloyl glucose in the management of diabetes and its complications. However, detailed in-vivo studies for this compound should be performed.Modern life is associated with low physical activity that leads to the accumulation of fats, gaining more weight, and obesity. Accumulation of fat in the abdomen region contributes to diabetes via insulin resistance and hyperglycemia. Polyphenols are major plant constituents that exert antidiabetic activity through different mechanisms, including radicle scavenging activity, regulation of glucose uptake, and inhibition of fat and polysaccharide hydrolysis in addition to their inhibitory role regarding the formation of advanced glycation end products (AGEs). Chemical investigation of  Diabetes is a complex chronic metabolic disease resulting from insulin deficiency or resistance of receptors to released hormone . The disProtein glycation represents one of the critical pathways involved in the progress of diabetic complications. Chronic hyperglycemia triggers postsynaptic protein alteration through a series of reactions called Maillard reactions . Non-enzGarcinia cambogia, Nigella sativa, Hibiscus sabdariffa, Caralluma fimbriata 8-barrel catalytic domain.From a therapeutic standpoint, the above profound activity of the aromyces . In thisaromyces . The ado1 family ,53,54,552H3-half-chair conformational structure as being lodged within the −1 subsite. This depicted orientation has directed the non-hydrolyzable inter-glycosidic nitrogen atom towards the catalytic center for hydrogen bond pairing with the Asp542 sidechain. The stability of acarbose at +1 subsite was mediated via the ligand’s C2 and C3 hydroxyl groups through polar interactions with Arg526, Asp542, and Asp203, where the last residue originated from the N-terminal loop at the β-sheet domain. Notably, loop residues at such β-sheet domain exhibited invariable interactions with several carbohydrate ligands within all available GH-31 crystalline structures . De. De41]. 1), quercetin-3-O-β-D-glucuronic acid (2), quercetin-3-O-β-D-galacturonic acid (3), rutin (4), and 1,3,6-trigalloyl glucose (5) were purified and characterized from C. oblongifolia. Their antioxidant and AGEs formation, α-glucosidase, and lipase inhibitory capacities were estimated. Compound 5 possessed the highest antioxidant and AGEs inhibitory potential. The furnished molecular docking and dynamics simulations illustrated the preferential stability and binding affinity of compound 5 towards human α-glucosidase in relation to its reported potent inhibitor, acarbose. The latter findings suggest the potential therapeutic utility of the C. oblongifolia-isolated compound for managing diabetes mellitus as well as other obesity-related conditions.Five major phenolic constituents: apeginin-7-O-β-D-glucoside (


The recently available analogs showed a wide variety of inhibitory potentials that ranged between 1.31 ± 0.05 and 38.60 ± 0.70 µM (against α-amylase) and between 2.71 ± 0.10 and 42.31 ± 0.70 µM (against α-glucosidase) under the positive control of acarbose (IC50 = 10.30 ± 0.20 µM against α-amylase) (IC50 = 9.80 ± 0.20 µM against α-glucosidase). A structure–activity relationship (SAR) study was carried out for all analogs based on substitution patterns around both rings B and C respectively. It was concluded from the SAR study that analogs bearing either substituent(s) of smaller size (−F and Cl) or substituent(s) capable of forming hydrogen bonding (−OH) with the catalytic residues of targeted enzymes enhanced the inhibitory potentials. Therefore, analogs 2 (bearing meta-fluoro substitution), 3 (having para-fluoro substitution) and 4 (with ortho-fluoro group) showed enhanced potency when evaluated against standard acarbose drug with IC50 values of 4.10 ± 0.10, 1.30 ± 0.05 and 1.90 ± 0.10 (against α-amylase) and 5.60 ± 0.10, 2.70 ± 0.10 and 2.90 ± 0.10 µM (against α-glucosidase), correspondingly. On the other hand, analogs bearing substituent(s) of either a bulky nature (−Br) or that are incapable of forming hydrogen bonds (−CH3) were found to lower the inhibitory potentials. In order to investigate the binding sites for synthetic analogs and how they interact with the active areas of both targeted enzymes, molecular docking studies were also conducted on the potent analogs. The results showed that these analogs adopted many important interactions with the active areas of enzymes. The precise structure of the newly synthesized compounds was confirmed using several spectroscopic techniques as NMR and HREI-MS.In this study, hybrid analogs of benzimidazole containing a thiazole moiety ( Hyperglycemia, which can be characterized as a high blood glucose level, describes the metabolic disorder known as diabetes mellitus (DM), which is non-infectious and chronic. The alarming incidence of diabetes mellitus already includes 415 million cases worldwide ,3. CarboProton pump inhibitors, anti-histaminic, anti-convulsant, anti-analgesic, anti-coagulant, anti-hypertensive, anti-cancer, anti-fungal and antiviral are only a few of the biological properties that benzimidazole and its derivatives are known to exhibit ,17,18,19The thiazole motif bearing scaffolds displayed a diverse range of biological profile including anti-bacterial, anti-diuretic and anti-Alzheimer’s . AdditioKeeping in mind the biological significance of previously reported benzimidazole ,31,32 an1–17) was completed in two steps. In the first step, 2-mercapto benzimidazole thiol (I) was treated with 4-nitro-substituted phenacyl bromide (II) in ethanol along with a catalytic amount of tri-ethyl amine under reflux condition to afford an intermediate (III) [1–17). A thin-layer chromatographic (TLC) plate was used to monitor the conversion of reactants into products. All of the synthesized derivatives were purified by washing with petroleum ether and recrystallized from ethyl acetate to afford a purified form of the targeted benzimidazole-based thiazole analogs (1–17), which were then characterized through 1HNMR, 13CNMR and HREI-MS techniques (The preparation of benzimidazole-based thiazole derivatives (te (III) . This inchniques .1–17) were verified by NMR and HRMS. To better understand the spectral analysis of synthesized scaffolds (1–17), the synthesized compounds were divided into three major parts, including a benzimidazole part, a thiazole part and a 4-nitro benzene ring B and ring C respectively. The 1H-NMR results showed that protons of thiazole and benzimidazole (-NH) were resonated as a singlet at a chemical shift value of δH 7.28 and 12.55 ppm (for benzimidazole). In addition, a singlet was observed for the -NH proton present between benzimidazole and thiazole rings at δH11.98ppm. Besides that, the remaining protons of benzimidazole were recorded as multiplets at approximately δH 7.43–7.12 ppm, and the characteristic -S-CH2- groups between the benzimidazole and thiazole heterocyclic rings were observed at δH 3.782 ppm asa singlet. The remaining protons of ring C were resonated at approximately δH 7.36–8.35 ppm as multiplets , depending on the attached electron-withdrawing (EW) or electron-donating (ED) groups. In the 13C-NMR spectra, the typical shifts near δ13C 171.7 (C-thiazole), 155.6 (C=N), 150.2 (C-thiazole), 105.0 (CH-thiazole) and 37.5 (-S-CH2-) ppm reveal the presence of a thiazole ring, C=N and (-S-CH2-) moieties of the targeted benzimidazole-based thiazole analogs.The precise structures of all newly afforded derivatives  and 2.70 ± 0.10 to 42.30 ± 0.70 µM (α-glucosidase) when compared to standard acarbose (IC50 = 10.30 ± 0.20 µM for α-amylase) (IC50 = 9.80 ± 0.20 µM for α-glucosidase)  . Based oosidase) . ortho-fluoro substitution on ring C along with a para-nitro group on ring B, with analogs 2 and 4 bearing a fluoro group at the meta- and ortho-positions of ring C; analog 3 displayed better activity toward α-amylase and α-glucosidase than its counterparts, 2 and 4. The difference in the inhibitory potentials of these three fluoro-substituted analogs toward α-amylase and α-glucosidase may be caused by different positions of the fluoro group around ring C , 15 (bearing an ortho-chloro group) and 16 (with a meta-chloro group) bearing only one chloro group. This higher inhibitory potential of analog 1 might be due to greater numbers of attached chloro groups in comparison to other analogs 14, 15 and 16. However, analog 15 (IC50 = 5.20 ± 0.10 µM) bearing ortho-chloro substitution on the phenyl ring showed a superior activity for both alpha-amylase and alpha-glucosidase enzymes than its structurally similar counterparts 14 (IC50 = 16.40 ± 0.30 µM) and 16 (IC50 = 12.50 ± 0.20µM), demonstrating that altering the location of the substituent(s) around the phenyl ring has a significant impact on the inhibitory potentials  were found to display considerable inhibition against both these targeted enzymes (α-amylase and α-glucosidase). Among chloro-substituted analogs, analog 1 bearing di-Cl substitutions at the 3,4-position of the phenyl ring showed enhanced inhibitory potentials against both α-amylase and α-glucosidase enzymes when compared to other analogs, 14  and 17 (bearing meta-nitro substitution on ring C), analog 10exhibited better inhibitory potential than its structurally similar analogs 9 and 17. This suggests that the inhibitory potentials are increased by the nitro group’s ortho-position, which is more efficient for interactions with the active sites of both amylase and glucosidase enzymes. By moving the ortho-nitro group of ring C to its para-position, like in the case of analog 9, the inhibitory potential of analog 10 was drastically reduced. The potency was further decreased by shifting the ortho-nitro group to the meta-position as in analog 17. This difference in potency of these nitro-substituted analogs might be due to a different position of the nitro group around ring C  at various position of ring C along with the nitro-substitution at the 4-position of ring B resulted in decreased inhibitory potentials against both α-amylase and α-glucosidase. Therefore, analogs 6 (bearing para-bromo on ring C), 7 (having meta-bromo on ring C) and 8 (with an ortho-bromo moiety on ring C) showed many-fold less potency when compared to either chloro-substituted analogs 16 (meta-chloro-substitution on ring C), 14 (having para-chloro-substitution on ring C) and 15 (with ortho-chloro-substituted ring C) or fluoro-substituted analogs 2 (meta-fluoro-substitution on ring C), 3 (having para-fluoro-substituted on ring C) and 4 (with ortho-fluoro-substitution on ring C) respectively (It was noteworthy that the attachment of substituent(s) of either a bulky nature (–Br group) or substituent(s) incapable of interactions through hydrogen bonding  of smaller size was found to be a better competitor of both targeted α-amylase and α-glucosidase, compared to analogs that bear substituent(s) of larger size. Moreover, it was also noted that inhibition properties for both α-amylase and α-glucosidase enzymes were greatly influenced by varying the number(s), positions and natures (electron-donating or electron-withdrawing groups) of substituent(s) around both rings B and C, respectively.Understanding how synthetic analogs interact with enzymes (both α-amylase and α-glucosidase) was the main goal of the molecular docking study. After using a command prompt, a docking procedure was completed, and nine different poses of each ligand was obtained in a log file in which the top-ranked conformations (having the lowest binding affinity) were selected in order to conduct a more complete visualization of protein–ligand interactions (PLI).https://www.rcsb.org/ (18 May 2022)). α-Amylase and α-glucosidase protein 1b2y and 3w37, respectively, were downloaded in PDB format. A different step procedure was adopted for the exploration of protein–ligand interactions (PLI). In the first step, the retrieved protein was opened in Auto Dock Vina; the water molecule was removed, and, in addition, polar hydrogen and Kollman and Gasteiger charges were added. This was followed by the addition of a ligand molecule wherein charges added, and coordinates for configuration  and the dimension of these coordinates were 80 and grid box center X = 25.555Å, Y = 61.538Å and Z = 51.515Å with exhaustiveness = 8. The saved file in text format and both protein and ligand were also saved in PDBQT format. 1b2y  and 3w37 .To explore the binding modalities of the ligand with the active site of the protein, a molecular docking study was carried out . Varied Interactions were discovered by using a command prompt in which the location of the target file was mentioned, and then processes were carried out. A total of nine poses for each ligand were obtained, which were explored in DSV to visualize binding interactions. A protein–ligand interaction (PLI) profile was summarized in . The Bruker AM 500 MHz machine NMR was used for the characterization of new compounds, and all necessary chemicals and reagents were bought from Sigma Aldrich, St. Louis, MO, USA. The splitted pattern of the peak was recorded as follows: dt, doublet of triplets, dd, doublet of doublets; sextet; sext, quintet; quint, q, quartet; t, triplet; d, doublet; m, multiplet, s, singlet. The coupling constant (J) was measured in hertz (Hz). High-resolution electron impact mass spectra (HREI-MS) were recorded on a Finnigan MAT-311A mass spectrometer (Germany). On precoated silica gel aluminium plates, thin-layer chromatography (TLC) was carried out . TLC plates were visualized by a UV lamp with a wavelength of 254 and 365 nm; the melting point was recorded with a Buchi M-560.3N (a few drops) to afford the formation of substrate III [1–17) in appropriate yield. The precise structures of all of the newly synthesized derivatives were confirmed by using NMR and HREI-MS spectroscopic methods. Initially, benzimidazole-2-thiol I  was reacted and stirred with 4-nitro-substituted phenacyl bromide II  in ethanol (10mL) and Etrate III . In the Appendix: 3.2. α-amylase inhibition assay, 3.2. α-glucosidase inhibition assay, 3.4. Doc king protocol  and 3.5.1–17), which were then tested for their inhibition properties against α-amylase and α-glucosidase. All of the synthesized analogs were found to display a varied range of inhibition properties against both enzymes, with IC50 values of 1.31 ± 0.05 to 38.60 ± 0.70 µM (for α-amylase) and 2.71 ± 0.10 to 42.31 ± 0.70 µM (for α-glucosidase) when compared to standard acarbose (IC50 = 10.30 ± 0.20 µM for α-amylase) (IC50 = 9.80 ± 0.20µM for α-glucosidase). Among the synthesized series, seven analogs such as 1, 2, 3, 4, 5, 10 and 15 were found to be more potent than standard acarbose, with IC50 values of 2.20 ± 0.10, 4.10 ± 0.10, 1.30 ± 0.05, 1.90 ± 0.10, 3.60 ± 0.20, 7.20 ± 0.20 and 5.20 ± 0.10 (against α-amylase) and 3.90 ± 0.20, 5.60 ± 0.10, 2.70 ± 0.10, 2.90 ± 0.10, 4.30 ± 0.30, 9.60 ± 0.20 and 6.30 ± 0.10, respectively (against α-glucosidase). Besides that, the remaining ten analogs also exhibited considerable inhibitory potentials but were found to be less potent than standard acarbose. For active analogs, molecular docking was devised in order to examine the binding locations of synthetic analogs and how they interact with the catalytic cavity of amino acids in enzymes. The findings showed that these analogs adopted a number of significant interactions with the active regions of enzymes. In addition, various spectroscopic tools such as HREI-MS and NMR were employed to confirm the precise structure of the synthesized compounds. In conclusion, by employing acarbose as a standard drug, an approach was established for the synthesis of hybrid analogs of benzimidazole containing thiazole (