In the benzyl alcohol oxidation reaction, we find that the activity increases monotonically with the core size but varies nonmontonically with the shell thickness, where a record-high activity is achieved on a Au@Pd catalyst with a large core size of 6.8 nm and a shell thickness of ~2–3 monolayers. These findings highlight the conjugated dual particle size effect in bimetallic catalysis.Core-shell bimetallic nanocatalysts have attracted long-standing attention in heterogeneous catalysis. Tailoring both the core size and shell thickness to the dedicated geometrical and electronic properties for high catalytic reactivity is important but challenging. Here, taking Au@Pd core-shell catalysts as an example, we disclose by theory that a large size of Au core with a two monolayer of Pd shell is vital to eliminate undesired lattice contractions and ligand destabilizations for optimum benzyl alcohol adsorption. A set of Au@Pd/SiO Tailoring both the core size and shell thickness of core-shell bimetallic nanocatalysts to the dedicated geometrical and electronic properties are vital for catalytic performance optimization. Here the authors demonstrate such conjugated dual particle size effects on Au@Pd catalyzed benzyl alcohol oxidation. In particular, mixing a precious metal with an abundant one in the form of an alloy or core-shell structure improves the cost efficiency necessary for commercialization. Compared to alloy catalysts, core-shell catalysts have peculiar lattice strains and ligand effects to optimize the geometric and electronic properties5. The ligand effects and charge transfers from the core are usually localized at the core-shell interface and are therefore sensitive to the shell thickness. The thin metal shell itself might also exhibit a prominent quantum size effect (QSE). Meanwhile, lattice strains in the metal shell often occur owing to its lattice mismatch with the core at the interface and usually persist over several atomic layers6. These three factors could be entangled with each other, making important impacts on the overall reactivity. This is particularly true for lattice strains, which often show significant modulations of the shell electronic properties and corresponding reactivity9.Bimetallic catalysts have drawn tremendous attention in widespread applications, including fine chemical synthesis, biomass conversion, and fuel cells, for their large phase spaces in various compositions and structures to optimize their activity and selectivity10. Previous studies have mainly focused on the variation of the composition and structure of the metal core to control the lattice strains10. However, the particle size could also play essential roles in bimetallic catalysis. In particular, as the core size is reduced, the metal core often possesses considerable lattice contractions and changes in electronic properties 15. These changes can in fact significantly interfere with the lattice strains and charge transfers in the shell, thus causing remarkable impacts on the bimetallic synergy. Addressing the lattice strain by varying the core particle size to optimize the shell reactivity is inspiring but has not yet been explored to the best of our knowledge. Consequently, investigation of the core size on tailoring the lattice strains and the influence of the shell thickness on the ligand effects and QSE is highly valuable for further improving the overall reactivity of the core-shell catalysts and fundamentally understanding these individual roles.Great efforts have been devoted to tuning lattice strains to boost catalytic performance16. To disentangle the strain and ligand effects modulated by the core size and shell thickness, we first studied the adsorption of BzOH on various Au@Pd catalysts by density functional theory (DFT) calculations. It is found that the adsorption on Au@Pd increases with Au size due to the alleviation of lattice contraction in the core. The ligand effects from the core destabilize the adsorption on the Pd shell but become negligible when the Pd thickness increases from 1 to 2 monolayers (MLs) with increasing adsorption. For a further increase in the shell thickness, however, the adsorption is weakened due to the downshift of the surface Pd 2 support by precisely regulating both the Au core size from 2.8 to 6.8 nm and the Pd shell thickness from approximately 0.3 to 3.2 MLs. In the reaction of solvent-free BzOH oxidation, the trend of the overall activity on the Au size and Pd thickness coincides perfectly with the calculated trend of the BzOH adsorption, and a record high activity of 6.86 × 104 h−1 is achieved for the 6.8 nm Au core and ~2–3 ML Pd shell. The present work provides critical insight to disentangle the involved geometric and electronic properties of core-shell catalysts and highlights the importance of fabricating a dedicated structure and composition to boost the activity and selectivity.Here, we address the above critical questions using Au@Pd core-shell catalysts for their superior performance in the reaction of benzyl alcohol (BzOH) oxidation to benzyl aldehyde, an important industrial process for fine chemical synthesis55 (~1.3 nm), Au147 (~1.7 nm), Au309 (~2.2 nm), Au561 (~2.7 nm) to Au923 (~3.2 nm) in a cuboctahedron shape, where isotropic contractions with respect to bulk are found  structure, the calculated and experimental values of bulk Au (fcc) are 5.5% and 4.9% larger, respectively, and significant stretching of the Pd shell over the Au core is expected. We constructed different Au clusters from Au55 ~.3 nm, Au55 ~.3 nm, Au, Au309 ~.2 nm, Au1ML core-shell catalysts with different sizes, the outmost atomic layer in Au55, Au147, Au309, Au561, and Au923 was replaced with Pd, which was noted as Au13@Pd42, Au55@Pd92, Au147@Pd162, and Au309@Pd252  (2.79 Å). To probe the influence on reactivity, the adsorption of BzOH was considered, and the favorable adsorption site for Au13@Pd42 was identified at the low-coordination corner site with a calculated adsorption energy of −2.59 eV. With an increase in the cluster size, the same adsorption site was adopted to observe the pure size-induced strain effect . Whereas for further increase of the size to Au147@Pd162 and Au309@Pd252, the enhancement becomes small with adsorption energies of −2.96 and −2.90 eV , where the lattice contraction in the Au core is completely diminished, we constructed a pristine four-layer Au(111) surface with a 1 ML Pd overlayer, denoted Au(111)@Pd1ML. Although the Pd–Pd lattice in the corresponding Pd overlayer is approximately 5% larger than that in Au309@Pd252, the calculated adsorption energy is −2.87 eV, which is 0.03 eV weaker. The slight weakening is because the gain in adsorption energy from lattice stretching is counteracted by the higher coordination number on the flat (111) terrace than on Au309@Pd252.To model Au@Pd eV Fig. , respect1ML, the subsurface metal right below the topmost Pd layer is Au, which actually destabilizes the overall adsorption toward BzOH. To see this clearly, we removed the Au substrate underneath from Au(111)@Pd1ML and calculated the corresponding adsorption energy of BzOH without structural optimization  and otherwise weakens to −2.87 eV. In line with Au(111)@Pd1ML, the role of the Au core in AuNP@Pd1ML also destabilizes the BzOH adsorption on Pd1ML. With a decrease in the cluster size, the extent of destabilization induced by the Au substrate becomes smaller for small clusters of Au13@Pd42, Au55@Pd92, and Au147@Pd162. We note that although the corresponding charge redistribution increases slightly @Pdons Fig. .2ML. In fact, for Au(111)@PdnML @Pdrgy Fig. . Neverth2 catalysts with Au particle sizes of 2.8 ± 0.5, 4.3 ± 0.6, 5.6 ± 1.2, and 6.8 ± 0.9 nm were synthesized using the DP method by varying the temperature and pH values (denoted as x nm-Au)17, as verified by transmission electron microscopy (TEM) and X-ray diffraction (XRD)   measurements of Aux@2.9ML-Pd catalysts showed that the average particle size after Pd deposition increased by approximately 1.3–1.6 nm to 4.1 ± 0.6, 5.7 ± 0.9, 7.2 ± 1.1, and 9.4 ± 1.6 nm for Au2.8@2.9ML-Pd, Au4.3@2.9ML-Pd, Au5.6@2.9ML-Pd, and Au6.8@2.9ML-Pd, respectively  elemental mappings showed that the Au signals were surrounded by Pd signals , which is in excellent agreement with theoretical calculations. Similarly, a decrease in the interplanar distance was observed in the outer two and three layers of Au6.8@2ML-Pd and Au6.8@2.9ML-Pd particles, respectively , the downward shift became substantially greater up to 1.3 eV observed on Au6.8@0.3ML-Pd, manifesting the remarkable enhancement of electronic interactions between Au and Pd by increasing the Au core size. In contrast, we found that the Au 4d3/2 binding energy was almost unchanged at 352.5 eV for all these samples. On the other hand, increasing the Pd coverage, the Pd 3d3/2 peak gradually shifted back from 339.3 to 339.8, 340.3, and 340.6 eV on the set of Au6.8@0.3ML-Pd, Au6.8@1.6ML-Pd, Au6.8@2.3ML-Pd, and Au6.8@2.9ML-Pd catalysts, respectively  as a function of Pd coverage  measurements were employed to investigate the electronic properties of these catalysts. As shown in Fig. ely Fig. . A similely Fig. 19. In coage Fig. , indicatage Fig. , 16. At age Fig. . It is c2.8@0.6ML-Pd exhibited a dominant peak at 2087 cm−1 and a much weaker peak at 1975 cm−1, assigned to linear and bridge-bonded CO on Pd, respectively31. The weak bridge-bonded CO feature indicates that the Pd atoms were atomically dispersed and surrounded by Au atoms in the majority in this sample32. As the Au core size increased to 6.8 nm but at a similar low Pd coverage, the linear CO peak on Pd exhibited a continuous redshift, reaching 2077 cm−1 on Au6.8@0.3ML-Pd; meanwhile, a new peak at 2128 cm−1 appeared, assigned to linear CO on low-coordination Au atoms34. The larger redshift of the linear CO peak on Au6.8@0.3ML-Pd than on Au2.8@0.3ML-Pd suggests that the Pd atoms in Au6.8@0.3ML-Pd have more populated 4d state electrons, which strengthens the Pd(4d)-CO(2π*) bonding through π-backdonation, thus endowing a redshift of the CO peak, in excellent agreement with the XPS results in Fig. −1) developed aggressively and became the dominant peak at high Pd coverages  with CO as a probe molecule. As shown in Fig. K and Au L3 edges to reveal the coordination environments of Pd and Au atoms in the Au@Pd catalysts. At the Pd K-edge, the X-ray absorption near edge structure (XANES) curves of all these Au@Pd catalysts exhibited a shape similar to that of Pd foil  spectroscopy measurements were further performed at both Pd K-edge extended X-ray absorption fine structure (EXAFS) spectra in the real (R) space demonstrated that at the submonolayer coverage of Pd, the curves of Aux@SubML-Pd showed a doublet peak at ~2.24 and 2.85 Å  of Pd–Au and Pd–Pd are ~8.0 and ~1.0, respectively , in line with the lattice expansion in the Pd shell observed by STEM and theoretical calculations  of approximately 0.32 × 104 h−1, based on the number of Pd surface atoms, along with a benzaldehyde selectivity of ~90% @Pd1ML @Pd2ML was observed  core-shell catalysts at 550 °C in 10% H2 in Ar for 1 h to form Au10.8Pd1, Au1.5Pd1, Au1.2Pd1, and Au1Pd1 alloy catalysts, respectively, yet preserving the similar size, as confirmed by the considerable shifts of the diffraction peaks in the XRD spectra28, TEM and EDS mapping , another important category of catalytic reactions , the specific activity increased by approximately 7 times from 0.53 × 104 to 3.65 × 104 h−1. Meanwhile, with variation of Pt shell thickness, but keeping the same Au core size of 5.3 nm, the specific activity also varied considerably.The conjugated dual size effect found in Au@Pd could be general for core-shell bimetallic catalysis. We further successfully demonstrated it in the Au@Pt-catalyzed selective hydrogenation of 6.8@2.9ML-Pd compared to Pd and AuPd catalysts reported in the literature. We showed that a large Au core size along with a thin Pd shell of ~2 MLs is vital for preserving the large lattice expansion of the Pd shell with a high d-band center but minimizing the destabilization of the ligand effect from the Au core for high activity. The conjugated dual size effect in core-shell bimetallic catalysis disclosed here can be general and was further successfully demonstrated in Au@Pt-catalyzed selective hydrogenation of p-CNB, another important category of reactions. These findings provide deep insight into the essential roles of the dual particle size effect in bimetallic catalysis, paving the way for the rational design of advanced bimetallic catalysts.In conclusion, by combining theoretical calculations with delicate catalyst synthesis and comprehensive structure characterization, we disclosed a conjugated dual size effect in core-shell bimetallic catalysis. In the solvent-free BzOH oxidation reaction, the activities of Au@Pd core-shell catalysts tightly rely on both the Au core size and the Pd shell thickness. By tailoring these two simultaneously, a record high activity was achieved on Au4 · 4H2O, >99%), palladium acetylacetonate (Pd(acac)2, 99.5%), ammonium hydroxide , benzyl alcohol  and ethanol (>99.5%) were purchased from Sinopharm Chemical Reagent Co. Ltd. . Palladium hexafluoroacetylacetate (Pd(hfac)2, >97%) was purchased from Sigma-Aldrich, and trimethyl(methylcyclo-pentadienyl) platinum (IV)  was purchased from Strem Chemicals. All gases, including ultrahigh purity N2 (99.999%), Ar (99.999%), and O2 (99.999%), and mixtures of 10% O2, 10% H2, and 10% CO in Ar (or He), were provided by Nanjing Special Gases. All chemicals were used as received without further purification.Tetrachloroauric acid 2 was dissolved into 50 mL acetylacetone. Then, 400 mg spherical SiO2 was added to the solution and mixed under vigorous stirring at 25 °C for 24 h. The obtained solid was dried at 110 °C overnight and further calcined at 500 °C in 10% O2/He for 3 h followed by a reduction step at 250 °C in 10% H2/Ar for 2 h to obtain the Pd/SiO2 catalyst. The loading of Pd was 3.5 wt% according to ICP‒AES analysis.The spherical SiO2 support for the later synthesis of Au@Pd core-shell bimetallic catalysts, a spherical SiO2 support was calcined in 10% O2/Ar at 700 °C for 3 h to remove most of the surface hydroxyl groups to promote selective Pd deposition.To achieve selective Pd deposition on Au NPs instead of a SiO2 catalysts were prepared using the DP method17. The size of the Au NPs was carefully controlled by varying the amount of HAuCl4·4H2O and adjusting the reduction temperature and pH value by ammonia. These catalysts were denoted as x nm-Au (x is the particle size of Au NPs on average). Typically, for 2.8 nm-Au, 1 mL HAuCl4 aqueous solution (0.0485 M), 1.0 g spherical SiO2, and 150 mL deionized water were co-added into a three-necked bottle and mixed for 30 min under vigorous stirring at 25 °C, and ammonia was used to adjust the pH value between 8 and 9. Then, the system was vigorously stirred for another 12 h. The suspension was then centrifuged and washed with deionized water several times and dried at 80 °C overnight. Finally, the resulting material was calcined at 180 °C in 10% O2/He at a flow rate of 20 mL·min−1 for 1 h to obtain the 2.8 nm-Au catalyst. For 4.3 nm-Au, 5.6 nm-Au, and 6.8 nm-Au, the amount of HAuCl4 solution was increased to 3.0 mL, and the ammonia reduction temperatures were 25, 65, and 65 °C with pH values of 8, 10, and 12, respectively. Finally, these materials were all calcined at 250 °C in 10% O2/He at a flow rate of 20 mL·min−1 for 1 h to obtain 4.3 nm-Au, 5.6 nm-Au, and 6.8 nm-Au catalysts.The Au/SiO2 samples at 150 °C in a viscous flow reactor  Technology) using Pd(hfac)2 and ultrahigh purity H2 (99.999%) as precursors19. Ultrahigh purity N2 (99.999%) was used as a carrier gas at a flow rate of 200 mL/min. The Pd(hfac)2 precursor container was heated to 65 °C to obtain a sufficient vapor pressure. The reaction chamber was heated to 150 °C, and the inlet manifolds were held at 110 °C to avoid precursor condensation. The timing sequence was 120, 200, 60, and 200 s for Pd(hfac)2 exposure, N2 purge, H2 exposure, and N2 purge, respectively. Au@Pd core-shell bimetallic catalysts were fabricated by selectively depositing Pd onto x nm-Au catalysts for different cycles. The resulting samples were denoted as Aux@yML-Pd. As a control experiment, Pd ALD was also applied on the bare SiO2 support with different numbers of ALD cycles under the same conditions as described above. All catalysts were calcined in 10% O2/Ar at 150 °C for 0.5 h and then reduced at 150 °C for another 0.5 h in 10% H2/Ar before any characterization and reaction tests.Synthesis of Au@Pd core-shell catalysts was performed by exclusive deposition of Pd on Au NPs via ALD. Typically, Pd ALD was carried out on the Au/SiO10.8Pd1, Au1.5Pd1, Au1.2Pd1, and Au1Pd1 alloy catalysts with different Au/Pd mole ratios were obtained by annealing the Au6.8@0.3ML-Pd, Au6.8@2.3ML-Pd, Au6.8@2.9ML-Pd, and Au6.8@3.2ML-Pd core-shell bimetallic catalysts at 550 °C in 10% H2/Ar for 1 h, respectively.The silica supported Au2 core-shell bimetallic catalysts were also synthesized by selectively depositing Pt onto x nm-Au samples using Pt ALD40. Here, Pt ALD was carried out on the same ALD reactor at 150 °C. The MeCpPtMe3 precursor was heated to 65 °C to achieve a sufficient vapor pressure, and pure O2 was used as the oxidant. The timing sequence was 30, 200, 6, and 200 s for MeCpPtMe3 exposure, N2 purge, O2 exposure, and N2 purge, respectively. The resulting samples were denoted as Aux@yML-Pt.Au@Pt/SiO6.8@1.1ML-Pd, Au6.8@2ML-Pd, and Au6.8@2.9ML-Pd bimetallic catalysts was carried out on an aberration-corrected HAADF STEM instrument at 200 kV .TEM measurements were performed on a JEM-2100F instrument operated at 200 kV to characterize the morphology of these catalysts. Elemental mapping using EDS was performed on the same equipment. Atomic resolution HAADF-STEM characterization of Auλ = 1.54178 Å), where the operation voltage and current were maintained at 40 kV and 200 mA, respectively. The data were recorded over 2θ ranges of 20–80°.The Pd contents of the resulting Pd catalysts were determined by ICP‒AES by dissolving these samples into hot aqua regia. XRD patterns of these samples were collected on a Rigaku/Max-3A X-ray diffractometer with Cu Kα radiation  detector and a low-temperature reaction cell (Praying Mantis Harrick). After loading a sample into the cell, it was first calcined in 10% Os peak at 284.4 eV as the internal standard. XPS PEAK 4.1 software was used for the deconvolution of XPS spectra. The backgrounds were removed using a Shirley-type integral, and the broadening function was chosen to be a symmetric Voigt function with 80% Lorentzian character. Separated spin-orbit components  and the different area ratios for split peaks  are well fixed for doublet separation. All samples were pretreated with 10% O2/Ar and then 10% H2/Ar at 150 °C for 0.5 h in a tube furnace before UV–Vis and XPS measurements.The UV–Vis spectra were measured on a Shimadzu DUV-3700 spectrophotometer. The XPS measurements were taken on a Thermo-VG Scientific Escalab 250 spectrometer equipped with an Al anode (Al Kα = 1486.6 eV). The binding energies were calibrated using the C 1K-edge (24350 eV) were performed with a Si(311) monochromator at the BL14W1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF), China. The storage ring of SSRF worked at 3.5 GeV in the top-up mode with a maximum current of 210 mA. Ex situ XAFS measurements at the Au L3 edge  were performed with a Si(111) monochromator at the 1W1B beamline of the Beijing Synchrotron Radiation Facility (BSRF), China. The storage ring of the BSRF worked at 2.5 GeV in the top-up mode with a maximum current of 250 mA. Considering the contents of Pd and Au, the XAFS spectra of Aux@yML-Pd catalysts at the Pd K-edge and Au L3 edge were recorded in fluorescence mode and transmission mode, respectively. All the catalyst powders were first pressed into pellets and then loaded into a homemade quartz reaction cell, where Kapton tape was used as the X-ray window material. This quartz reaction cell can be heated to 500 °C with external heating. A K-type thermocouple, protected by a closed end quartz tube, was located near the sample pellet to measure the sample temperature. After loading into the reaction cell, each sample was first reduced in 10% H2/He at 50 °C for 0.5 h (20 mL·min−1). Next, the sample was purged in ultrahigh purity He at 50 °C for 15 min to collect the XAFS spectra.In situ XAFS measurements at the Pd 2/He and then reduced in 10% H2/He at 150 °C for 0.5 h. Then, the catalysts were cooled to room temperature in He, and CO pulses were introduced to the catalyst surface using 10% CO/He until saturation. The amount of chemisorbed CO was calibrated by using a thermal conductivity detector. For dispersion calculations, a stoichiometry of CO:Pd of 1:1 was assumed for Aux@SubML-Pd and AuPd alloy catalysts due to the high ratios of linear to bridge-bonded CO in the DRIFTS CO spectra, while a stoichiometry of CO:Pd of 1:2 was applied to 4.6 nm-Pd and Aux@yML-Pd with high Pd coverages according to the literature43.The Pd dispersions of Pd, AuPd alloy, and Au@Pd core-shell catalysts were determined by CO pulse chemisorption, which was conducted on a Micromeritics AutoChem II chemisorption instrument. After loading a sample, the catalyst was first calcined in 10% O2 was carried out in a 25 mL three-necked glass flask equipped with a reflux condenser at 90 °C under atmospheric pressure. Then, 5 mL benzyl alcohol and certain amounts of catalyst  were added to the glass flask. Prior to the reaction, the system was first charged with O2 by bubbling ultrahigh purity O2 at a flow rate of 15 mL·min−1 for 15 min to remove air. Under the continuous flow of O2, the flask was immersed into a dimethylsilicic oil bath at 90 °C to initiate the reaction. During the reaction, the mixture was vigorously stirred at a rate of 1250 rpm to exclude any mass transfer limitation. Finally, the reaction products were analyzed using a Shimadzu GC-2014 gas chromatograph equipped with a Rtx-1 capillary column and an autoinjector.Solvent-free aerobic oxidation of benzyl alcohol using molecular Opara-chloronitrobenzene (p-CNB) was carried out in a 100 mL stainless steel batch reactor . The amount of catalyst used for the reaction test was normalized to have the same Pt to substrate mole ratio of 1:7800. Typically, p-CNB (8 mmol), the catalyst, m-xylene (8/3 mmol) and C2H5OH (40 mL) were loaded into the autoclave. Here, m-xylene was used as the internal standard. Next, the autoclave was flushed with ultrahigh purity Ar to remove the air and then heated to 65 °C under Ar. When the temperature was stabilized at 65 °C, the autoclave was pressurized with 0.3 MPa hydrogen. Then, the mixture was stirred at a rate of 1500 rpm to start the reaction. During the reaction, the liquid sample mixture was removed through a sampling valve and analyzed by gas chromatography .The selective hydrogenation of The initial TOFs of Pd- or Pt-based catalysts were evaluated at conversions below 20% in the kinetic regime unless otherwise noted, according to the following equation Eq. :1\documeHere, the moles of surface metal atoms (Pd or Pt) were determined by CO titration. The specific rates were also calculated using the following equation with the total amount of Pd or Pt Eq. :2\docume44 at the GGA level. The core electrons were described using the projector-augmented wave (PAW) method45. Kohn-Sham valence states  were expanded in a plane wave basis set with a 500 eV kinetic energy cut-off. The electronic states were smeared using the Methfessel-Paxton method with a smearing width of 0.1 eV. The optB86b-vdW functional was used to include dispersive forces47. The equilibrium lattice constants for Au and Pd were calculated to be 4.122 and 3.904 Å, respectively, in close agreement with the corresponding experimental values48.Periodic, spin-polarized DFT calculations were performed using the Vienna Ab initio Simulation Package (VASP)xML, which refers to x MLs of Pd supported on Au(111), the bottom two metal layers were fixed at the bulk positions to mimic the core layers, with the rest of the layers being fully relaxed. The adsorption of BzOH was studied on a (3 × 3) surface unit cell. A Γ-centered 5 × 5 × 1 Monkhorst-Pack k-point grid was used to sample the surface Brillouin zone49. Each slab was separated from its periodic images in the z direction by at least ~10 Å of vacuum. Adsorption was studied only on one side of the slab (adsorbates are fully relaxed), with dipole decoupling applied in the z direction50.For our surface model Au(111)@Pdk-point grid was sampled at the Γ-point only for cluster models and gas-phase molecules. Molecules in the gas phase were optimized in a 14 × 15 × 16 Å3 simulation cell. Calculations for both cluster models and gas-phase molecules have dipole corrections applied in all three directions in the unit cell. The criterion for optimization was set to the extent that the maximum residual force was 0.03 eV/Å or less in all relaxed degrees of freedom.The classic cuboctahedral shape was taken to model Pd partially substituted Au clusters. One of the outermost (111) layers of the pure Au cluster was substituted with Pd to represent the Au@Pd core-shell structure in an ideal manner. The cluster was separated from its periodic images by ~16 Å of vacuum. The ∆Eads = Etotal– Emodel– Egas, where Etotal, Emodel, and Egas refer to the energy of the model with the adsorbate, the energy of the corresponding clean model structure (either a slab model or a cluster model), and the energy of gas-phase adsorbate in a neutral state, respectively. A more negative value indicates stronger adsorption.The adsorption energy was calculated as Supplementary InformationPeer Review File


Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines  located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against The online version contains supplementary material available at 10.1186/s13568-022-01430-w. Bacillus thuringiensis (Bt) during their vegetative growth stage, are important insecticidal proteins that show toxicity against a wide variety of lepidopteran pests , secreted from Spodoptera littoralis, with LC50 of 228.42 ng/cm2, 65.71 ng/cm2, and 388.90 ng/cm2, respectively. By contrast, Vip3Ad showed little insecticidal activity against this species  located on Vip3Aa and the mutation of residue P591 could lead to the decline of stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability of Vip3A toxin might play a key role for its insecticidal activity.Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against lepidopteran pest S. exigua larvae was purchased from Henan Jiyuan Baiyun Industry Co., Ltd, China. S. exigua larvae were fed with artificial diet provided by Hubei Biopesticide Engineering Research Center under the conditions of 26 ± 2 °C, 70% humidity and photoperiod of 14: 10 h (light: dark).A laboratory population of S. exigua midgut proteases were prepared as previously described . Midgut tissues were homogenized with 200 μL of phosphate-buffered saline (PBS) buffer  and centrifuged at 25,000 g for 30 min at 4 °C. The supernatants, which contained midgut proteases, were quantified by the BCA Protein Assay Kit  according to the instructions of the manufacturers.The E. coli BL21 (DE3) cells harboring pET30a-vip3Aa and pET30a-vip3Ad . Cells were lysed by ultrasonication and the soluble protein was purified by a Ni-IDA Prepacked Column  according to the manufacturer’s instructions. The purified Vip3Aa and Vip3Ad were exchanged into Sodium carbonate buffer  by a PD-10 desalination column . Protein concentration was determined using a BCA Protein Assay Kit . Trypsin or S. exigua midgut juice was used to produce Vip3Aa and Vip3Ad activated-toxins as previous described  using PROBIT analysis  for 48 h. After that, the midgut tissues of S. exigua were isolated, immediately fixed in 2.5% glutaraldehyde, and postfixed in 1% OsO4. The fixed midgut tissues were immerged into Epon for embedding. Ultrathin sections were sliced using a Leica ultramicrotome (EM UC7), stained with uranyl acetate and then lead citrate. The ultrastructure of the midgut epithelium was examined using a JEOL JEM-2100HC transmission electron microscope   in 50 μL final volume of sodium carbonate buffer . The mixtures were incubated at 30 °C for different incubation times, with constant shaking (about 40 rpm). The proteolytic reactions were stopped by boiling for 10 min and SDS-PAGE electrophoresis was used to assess the stability of Vip3Aa and Vip3Ad activated-toxins under S. exigua midgut juice.Proteolysis assay was performed to evaluate the stability of Vip3Aa and Vip3Ad activated-toxins under vip3Aa was used as template. Primers used for mutagenesis were listed in Additional file Dpn I (1U) at 37 °C for 1 h and further transformed into E. coli BL21 (DE3) competent to obtain the relevant mutants.Site-directed mutagenesis was carried out to create Vip3Aa variants P591A, P605A and P779A by polymerase chain reaction (PCR) using PrimeSTAR Max DNA Polymerase . Plasmid pET30a-https://www.rcsb.org/). The homology model of and Vip3Ad was constructed by Swiss-Model (https://swissmodel.expasy.org/) with the crystal structure of Vip3Aa protoxin structure (PDB: 6TFJ) as the template (86.1% identity), and evaluated in SAVES (https://servicesn.mbi.ucla.edu/SAVES/).The structure of Vip3Aa (PDB: 6TFJ) was obtained from PDB database (The pretreatment of Vip3Aa and Vip3Ad for molecular dynamic (MD) were performed as previous study with AmberTools18. The system was heated from 0 to 300 K at constant volume in 50 ps with the protein restricted, equilibrated at constant pressure in 50 ps with the protein restricted and equilibrated for 500 ps without restriction of the protein. After equilibration, normal temperature and pressure (NPT) simulation was conducted for 20 ns to produce trajectories of MD simulation The root mean square deviation (RMSD) and the root mean square fluctuation (RMSF) were calculated for the protein backbone atoms using least-square fitting derived from the MD trajectories  expression system. SDS-PAGE indicated that Vip3Aa and Vip3Ad protoxins were both 89 kDa in molecular weight and could be cleaved into 65 kDa activated-toxins by S. exigua midgut juice  could kill S. exigua larvae and low concentration of Vip3Aa (50–200 ng/cm2) could inhibit the growth of larvae. By contrast, it did not have any insecticidal or inhibitory activity under low concentration range of Vip3Ad (50–400 ng/cm2), inhibition effects were observed when the concentration of Vip3Ad increased to 1000 or 4000 ng/cm2   and Root Mean Square Fluctuation (RMSF) were calculated. As shown in Fig. To explore the mechanism for the different stability of Vip3Aa and Vip3Ad activated-toixn, we first compared the amino acid sequence of Vip3Aa and Vip3Ad toxin since they are about 15% differentiation in amino acid sequence. We found that three were three proline  located on Vip3Aa, while the corresponding residues on Vip3Ad were S, A and T, respectively  Spectrum of 19 PTH-amino acids standards; (B)-(F) N-terminal amino acid identification of Vip3Aa activated-toxin. Figure S3. Amino acid sequence alignment of Vip3Aa and Vip3Ad. The sequences in blue box represented the 65 kDa activated-toxins of Vip3Aa and Vip3Ad. The positions of three extra prolines  was indicated as (●).


Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is a biomarker of hepatocellular carcinoma (HCC) progression. Aptamers specifically binding to target biomolecules have recently emerged as clinical disease diagnosis targets. Here, we describe 3D structure-based aptaprobe platforms for detecting GPC3, such as aptablotting, aptaprobe-based sandwich assay (ALISA), and aptaprobe-based imaging analysis.For preparing the aptaprobe–GPC3 platforms, we obtained 12 high affinity aptamer candidates (GPC3_1 to GPC3_12) that specifically bind to target GPC3 molecules. Structure-based molecular interactions identified distinct aptatopic residues responsible for binding to the paratopic nucleotide sequences (nt-paratope) of GPC3 aptaprobes. Sandwichable and overlapped aptaprobes were selected through structural analysis. The aptaprobe specificity for using in HCC diagnostics were verified through Aptablotting and ALISA. Moreover, aptaprobe-based imaging showed that the binding property of GPC3_3 and their GPC3 specificity were maintained in HCC xenograft models, which may indicate a new HCC imaging diagnosis.Aptaprobe has the potential to be used as an affinity reagent to detect the target in vivo and in vitro diagnosing system.The online version contains supplementary material available at 10.1186/s12951-022-01391-z. Liver cancer is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide . HepatocGPC3 is one of six mammalian members of the glypican family (GPC1–GPC6) with heparan sulfate proteoglycans linked to the cell surface through a glycosyl-phosphatidylinositol (GPI) anchor , 15. GPCMolecular probes are powerful tools for identifying disease biomarkers and tracking them in their native environments. SELEX technology has been developed as an in vitro selection method to screen aptamers, which are nucleic acid ligands with prominent functionalities . AptamerThe study includes three major stages: (i) aptamer screening explains the isolation technique of aptamer candidates, (ii) the aptaprobe and aptaprobe–GPC3 complex structural interaction, and (iii) technical platform validations; aptablotting, aptaprobe-based sandwich assay (ALISA), and aptaprobe-based imaging analysis. The first stage has two components: in vitro SELEX and surface plasmon resonance (SPR) affinity analysis. The second stage describes in detail the 3D structural analysis of molecular interactions to provide specific aptaprobes. Next, the platform validation stage describes the experimental results showing the applicability of the selected aptaprobes, and concludes the study by summarizing the results and contributions of HCC-targeted specific diagnosis. The overall flow of this study is shown in Scheme The aptamers used in all the experiments were synthesized by IDT . Recombinant human glypican proteins (GPC3 and GPC5) were purchased from R&D Systems , and anti-human GPC3 monoclonal human antibody was purchased from G&P Bioscience . HSA, BSA, human serum, and anti-mouse IgG peroxidase were purchased from Sigma . Horseradish peroxidase (HRP)-conjugated streptavidin was purchased from Thermo Fisher Scientific . The following buffers were used: TBS-T buffer (0.1% (v/v) Tween 20 in Tris-buffered saline, pH 7.2), blocking buffer (5% non-fat dried milk in TBS-T), and washing buffer (TBS-T buffer).http://mfold.albany.edu/?q=mfold) at 25 ℃ in a 250 mM [Na+] folding algorithm based on nucleotide sequences. A 3D RNA model was constructed from the secondary structure predicted using the Mfold program , pancreatic cancer cell lines , and HeLa cell line (ATCC CCL-2) were cultured on 100Φ dishes. RNA was extracted using the TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific). The extracted RNA was converted to cDNA using a TOPscript™ cDNA Synthesis Kit . GAPDH and GPC3 genes were PCR-amplified using gene-specific primers . PCR was performed for 25 cycles. The results were confirmed using 2% agarose gel electrophoresis.4 cells/well) treated for 1 h with various GPC3_3 aptaprobe concentrations. The OCR and ECAR were measured before and after treating the cells with mitochondrial toxins oligomycin  and carbonyl cyanide-4-phenylhydrazone , and rotenone/antimycin A  to determine the baseline and stress measures of oxidative phosphorylation and glycolysis, respectively. The OCR and ECAR values were normalized to the total protein concentration. All experiments relative to the control were expressed as the mean ± standard error of mean (SEM) from at least triplicate studies.Cell viability and mitochondrial respiratory competency were determined by MTT assay and Seahorse XF analyzer mito stress test assay . For MTT assays, HepG2 cells were seeded in 48-well plates and incubated overnight for cell adhesion. They were then incubated for 24 h with 10–0.125 μM GPC3_3 aptaprobe (two-fold dilutions) and wells containing untreated cells were used as controls. At the end of incubation, the media was replaced with fresh media containing MTT and further incubated at 37 °C for 4 h. Finally, the formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and the absorbance at 570 nm was recorded using a SPECTRAMAX M2e ELISA reader . The Seahorse XF analyzer mito stress test assay was performed on HepG2 cells (1.8 × 106 cells/mL) was monitored using confocal microscopy. After washing twice with cold washing buffer, the cells were incubated with 200 uM of FAM-labeled GPC3 aptaprobes  at 37 °C for 1 h. Subsequently, the cells were washed twice, imaged using a Zeiss LSM 710 Upright confocal microscope , and analyzed using a BD FACS Calibur™ Flow Cytometer .The binding of aptamers to HeLa, HepG2, Hep3B, PANC-1, and MIA-PaCa cell lines . The tumor was allowed to grow till 100–200 mm3 . The tumor volume was calculated using the formula: V(volume) = L(length) × W(width) × H(height)/2. The mice were monitored weekly for tumor growth and GPC3_3 aptaprobe using in vivo imaging system (IVIS) imaging as described below.HCC xenograft models were created using HepG2 cells. Briefly, 1 × 10The analysis was performed using a previously described protocol with some modifications Fig. A 29]. G. G29]. GALISA was performed in streptavidin-coated 96-well plates for quantitative GPC3 protein detection using GPC3 aptamers. Biotinylated GPC3_2 and GPC3_9 aptaprobes (1 μM/well) were used to immobilize a 96-well plate for 1 h at 25 °C. The plate was washed thrice and non-specific binding was blocked with TBS-T containing 2% BSA. The same concentration (500 ng/100 μL) of GPC3, GPC5, BSA, and HSA was then added to each well and incubated for 1 h at 25 °C with general shaking. Then, 5, 10, 25, 50, 100, 250, and 500 ng/100 μL GPC3 protein were added to each well. The unbound proteins were discarded and washed thrice with a washing buffer. To quantify the signaling target protein, the FAM-modified GPC3_3 (1 μM/well) was added to individual wells for 30 min at RT. Following the reaction, the unbound and non-specific bound FAM-modified GPC3_3 was removed and the plate was washed with washing buffer. The fluorescence intensity of each well was then measured at 495 nm extension wavelength and 520 nm emission wavelength using a SPECTRAMAX M2e ELISA reader.5 cells/well) were seeded in 6-well plates onto coverslips (Marienfeld Superior). After incubating for 24 h, the cells were incubated with fresh media containing 200 pM FAM-modified GPC3_2, GPC3_3, and GPC3_9 separately for 4 h at 37 °C in a 5% CO2 incubator. The cells were washed three times with DPBS to remove the unbound aptamers, fixed with 4% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips with fixed cells were mounted on glass slides with a mounting solution. Fluorescence images were obtained using a confocal laser scanning microscope .HepG2 cells (1.5 × 104 cells/well) were cultured in 24 wells onto coverslips. After incubation for 24 h, the cells were incubated with fresh media containing 0–200 pM FAM-modified GPC3_3 for 4 h at 37 °C in a 5% CO2 incubator. Confocal imaging was performed as described above.HCC (HepG2 and Hep3B), pancreatic cancer (PANC-1 and MIA-PaCa-2), and HeLa cell lines (3.0 × 10Cy5.5 (Cy5.5-labeled random sequence); Group II, intravenously injected with 2 mg/kg GPC3_3Cy5.5 (Cy5.5-labeled GPC3_3). The Cy5.5-labeled samples were detected 30 min after injection by placing the mice in the supine position using the IVIS Lumina system III . The mice were euthanized 1 h after injection, laparotomy was performed, and in situ imaged using Cy5.5 filters, which were normalized to the background signal per manufacturer’s protocol. The liver, lung, spleen, kidney, heart, tumor, small intestine, and large intestine tissue of mice were harvested.HCC xenograft model mice were imaged in vivo to minimize the expression of fluorescence in the body by fasting for 12 h. The mice were randomly assigned to two groups (n = 3 per group): Group I, intravenously injected with 2 mg/kg ControlAll statistical analyses were performed using GraphPad Prism or R statistical software . Two-tai, respectively. At the tenth round, the ssDNA aptamer concentration decreased, it means the binding efficiency of ssDNA for the GPC3 was decreased. To provide the binding efficiency of ssDNA for the GPC3, we performed the Post-SELEX and Real-time PCR  of the ssDNA aptamer . The results indicated that the KD values of these ssDNA aptamer candidates could be classified into three distinct groups. Of these ssDNA aptamer candidates, GPC3_1–GPC3_12 had low KD values , which would aid the detection of GPC3 targets.To determine the characteristics of GPC3-binding ssDNA aptamer candidates, an SPR assay was used to analyze the specific binding of 12 aptamers with different sequences to GPC3 and determine the binding affinity  anchor region . The proThese aptamers have complex secondary structures, including protruding loops and stems. The interaction conformation between the GPC3 protein and each aptamer candidate is shown in Additional file Figure Structural analysis of the molecular interaction was performed using MOE-docking-based 3D prediction to determine whether the two aptaprobes occupy distinct GPC3 sites during binding. If an identical binding site is involved, the aptaprobe–GPC3–aptaprobe sandwich configuration would be disturbed. The data on non-ligand residues involved in hydrophobic contact of GPC3 protein–aptaprobe analysis  − 342.75 (R2 = 0.9958)  and Test1–Test3 groups had no effect on signal response , cysteine-rich domain (CRD), and glycosylphosphatidylinositol (GPI). The Notum cleavage site breaks the bond between amino acids 562 and 563 , causingThe aptaprobe-binding capacity can be maintained within the actual cellular environment. We used two pancreatic cancer cells, PANC-1 and MIA-PaCa, as negative controls for aptamer binding, as these cells do not express GPC3. To confirm GPC3 expression, the mRNA expression levels were determined preferentially. GPC3 gene expression was observed in only two HCC cell lines, HepG2 and Hep3B, but not in the pancreatic cancer cells  was mainly distributed in the liver and kidney  analysis of the interaction of GPC3 and GPC3 aptamer candidates (GPC3_1 to GPC3_12) using a Biacore X100. Figure S3. Schematic of the GPC3 protein. Figure S4. Comparing the GPC3 protein and aptamer complex conformation by docking simulation. Figures S5. Ligplot+ molecular interaction analysis of aptaprobes with the GPC3. Figures S6. Aptablotting assay and immunodot blot assay. Figures S7. Confocal imaging and fluorescence intensity quantification of FAM-labeled GPC3 aptaprobes in cell line. Figures S8. mRNA expression test to confirm GPC3 expression of in Hela cells, liver cancer cells (HepG2 and Hep3B), and pancreatic cancer cells (PANC-1 and MIA-PaCa). Figures S9. Cell viability using 3--2,5-diphenyl tetrazolium bromide salt (MTT) assay and Seahorse analysis for HepG2 cells treated with different aptaprobe concentrations. Figures S10. Schematic demonstrating the experimental workflow of in vivo GPC3_3 labeling in an intravenously hepatocellular carcinoma (HCC) xenograft model. Figures S11. Representative images of HCC xenograft model. Table S1. Amino acid residue involved in hydrophobic contact of the between GPC3 protein and aptamer candidate. Table S2. Features of biomolecules for interventional fluorescence imaging of tumors in preclinical studies.


Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from  Bacillus thuringiensis (Bt) are useful for managing some economically important pests. Benefits of Bt crops include better pest control and decreased use of conventional insecticides, which enhances yields and farmer profits, and confers health and environmental benefits6. However, the evolution of resistance to Bt crops by pests decreases these benefits7. Understanding the genetic basis of resistance to Bt crops can improve the monitoring and management of resistance, as well strategies to counter pest resistance.Genetically engineered crops that produce insecticidal proteins from 10. Natural mutations in ABCC2 are genetically linked with resistance to several Bt toxins in the Cry1 family  in seven lepidopterans16. Likewise, resistance to Cry2Ab is linked with natural mutations in another ABC transporter gene, ABCA2, in Helicoverpa armigera17, Helicoverpa punctigera17, Trichoplusia ni18, and in lab- and field-selected populations of Pectinophora gossypiella20. Furthermore, knockout mutations in ABCA2 generated in the laboratory using CRISPR/Cas9 or TALENs caused resistance to Cry2Ab in H. armigera21, T. ni18, Bombyx mori22, and P. gossypiella23. These results provide compelling evidence that ABCA2 plays an important role in the mode of action of Cry2Ab in these lepidopterans.Some ATP-binding cassette (ABC) transporter proteins mediate Bt intoxication in insectsHelicoverpa zea . This lepidopteran is a major pest of many crops in the New World, including corn, cotton, soybean, and sorghum24. It has evolved practical resistance to Bt corn and cotton producing Cry1 and Cry2A toxins throughout much of the southern United States32. Also, early signs of H. zea evolution of resistance to Vip3Aa produced by Bt crops have been reported33. Hence, strategies to delay or overcome the evolution of Bt resistance in H. zea are urgently needed. Although timely identification of genes causing Bt resistance remains challenging, new tools have spurred progress with H. zea and H. armigera36.Here we tested the hypothesis that CRISPR/Cas9-mediated disruption of the gene encoding ABCA2 can cause resistance to Cry2Ab in HzABCA2 gene in a susceptible strain caused resistance to Cry2Ab. We also used CRISPR/Cas9 to introduce disruptive mutations in the HzTO gene , which confirmed previous results showing this causes yellow eye color in H. zea37. All Cry2Ab-resistant individuals tested were found to harbor disruptive mutations in HzABCA2 DNA in the single-guide RNA (sgRNA) target sites used for gene editing. These findings indicate that ABCA2 is important in toxicity of Cry2Ab to H. zea.In this study, we discovered that introducing disruptive CRISPR/Cas9-mediated mutations in the HzABCA2 sgRNAs separately by combining the sgRNA/Cas9 ribonucleotide mixture for each sgRNA (#1–7) with gDNA amplified by PCR from HzABCA2. Cas9 cleavage of the HzABCA2 amplicons in vitro occurred with sgRNA 7, but not sgRNAs 1–6  from the susceptible LAB-S strain injected with the ribonucleotide mixture containing HzTO sgRNA and HzABCA2 sgRNA 7, 97% hatched and 73% pupated. To start the Hz-R2 strain, we pooled 55 G0 pupae  and allowed the adults to eclose and mate. We established the Yellow-R2 strain by pairing two G0 adult females that had yellow eyes  and 93% for Hz-R2 (n = 192), suggesting that CRISPR/Cas9 efficiently introduced germline mutations in HzABCA2 that caused resistance to Cry2Ab. Survival was 0% for LAB-S (n = 32), which is significantly lower than survival of the G1 larvae from either Hz-R2 or Yellow-R2 . In contrast, survival on control diet did not differ significantly between LAB-S (91%) and either Hz-R2 (88%) or Yellow-R2 (100%) . We used the G1 survivors of exposure to Cry2Ab to continue the Yellow-R2 and Hz-R2 strains. In effect, this exposure constituted a single selection for resistance to Cry2Ab after which both strains were reared without exposure to the toxin.For our experimental design, we targeted 4 larvae from Hz-R2 revealed they were highly resistant to Cry2Ab relative to their parent strain LAB-S . The concentration of Cry2Ab killing 50% of larvae (LC50 in μg Cry2Ab per cm2 diet) was > 30 for the G4 larvae from Hz-R2 and 0.14 (95% CI 0.10–0.19) for LAB-S, which yields a resistance ratio of > 217 for Hz-R2 relative to LAB-S.Analysis of the GB-S Fig. . Surviva2 pupae by sex, selected adults with yellow eyes  and allowed them to mate. After six generations of laboratory rearing without additional selection for yellow eyes, all 1,200 individuals examined from the G7-9 Yellow-R2 strain had yellow eyes (n = 400 per generation). This suggests that a HzTO mutation responsible for yellow eyes was likely fixed in the Yellow-R2 strain.To initiate Yellow-R2 containing the desired eye phenotype, we sorted GHzABCA2, we sequenced two clones corresponding to exon 4 (containing HzABCA2 sgRNA 7) from three Hz-R2 and Yellow-R2 G1 survivors reared on diet treated with 1 μg Cry2Ab per cm2  and three LAB-S individuals on untreated diet , which occurred in five of six Hz-R2 clones and all six Yellow-R2 clones. No mutations were found in exon 4 of HzABCA2 in the six clones sequenced from LAB-S.For ite Fig. . In totaHzTO, we sequenced two clones corresponding to exon 6 (the site of the HzTO sgRNA) from four Yellow-R2 G1 moths that had yellow eyes  and two clones from one LAB-S moth. Whereas both clones from LAB-S had the wild-type HzTO sequence, all eight clones from Yellow-R2 harbored disruptive mutations  and the 33-bp insertion introduced a stop codon in the final 3 bp of the inserted sequence .To evaluate inheritance of resistance to Cry2Ab, we used diet overlay bioassays to test Hz-R2, LAB-S, and their Five Fig. . At the h), which varies from 0 for completely recessive resistance to 1 for completely dominant resistance, was determined for the F1 progeny from pooled Hz-R2 X LAB-S mass crosses. The results yield h = 0.0041, indicating recessive inheritance of resistance for Hz-R2 at 1 μg Cry2Ab per cm2 diet.The dominance parameter (H. zea ABCA2 gene (HzABCA2) via CRISPR/Cas9 gene editing caused resistance to Cry2Ab. This finding is consistent with previous results from editing the homologous gene in H. armigera, T. ni, B. mori, and P. gossypiella23. Collectively these data indicate that ABCA2 is important for toxicity of Cry2Ab in at least five species representing three families of Lepidoptera . Despite its evident functional importance, the precise role of this protein in the mode of action of Cry2Ab remains obscure10. Heckel (2021) has proposed that elucidating the three-dimensional structures of the ABC transporters will be critical for progress in understanding how these proteins facilitate pore formation by Bt toxins.The results here show that introduction of disruptive mutations in the H. zea, which harbored CRISPR/Cas9-induced mutations in HzABCA2, was > 200-fold resistant to Cry2Ab compared to its susceptible parental strain LAB-S. Similarly, CRISPR/Cas9 knockout of ABCA2 in two H. armigera lab strains from China revealed Cry2Ab resistance ratios of > 100 relative to the susceptible parental SCD strain21.Here, we determined that the Hz-R2 strain of H. armigera and H. punctigera from Australia is linked with naturally occurring mutations in ABCA217. One of the Cry2Ab-resistant strains of H. armigera from Australia called SP15 was generated from an F2 screen conducted in 200238. Larval survival  at the highest concentration of Cry2Ab tested was 96% for Hz-R2 and 96% for SP1538. The highest concentration tested was 1.6 times higher for Hz-R2 than SP15 (30 versus 19 μg Cry2Ab per cm2 diet), which means the concentration killing 4% of larvae was higher for Hz-R2 than SP15. In the Australian study38, the mean LC50 (in μg Cry2Ab per cm2 diet) of two unrelated susceptible strains of H. armigera was 0.13, which is nearly identical to the LC50 of 0.14 for the susceptible LAB-S strain tested in this study. Thus, the resistance to Cry2Ab relative to conspecific susceptible strains was similar or slightly greater for the CRISPR-generated mutations in Hz-R2 than for the naturally occurring mutations in SP15. Consistent with the results for Hz-R2, resistance to Cry2Ab was recessive in strains of H. armigera from China and Australia as well as in H. punctigera38.The Cry2Ab resistance in lab-selected strains of P. gossypiella from India, mutations disrupting ABCA2 were associated with field-selected resistance to Cry2Ab20. In H. armigera and H. punctigera from Australia, mutations disrupting ABCA2 were associated with resistance to Cry2Ab in lab-selected strains derived from field populations17. However, these naturally occurring mutations affecting ABCA2 were not common in field populations of either species in Australia17, which remain susceptible to Cry2Ab39.Whereas our study using CRISPR/Cas9 and related gene editing studies can rigorously test if mutations in specific genes confer resistance, they cannot determine if mutations in the genes evaluated actually occur in field-selected resistant populations of pests. Accordingly, a key question related to this study is the extent to which mutations disrupting ABCA2 contribute to field-evolved resistance to Cry2Ab. In H. zea make it especially compelling to determine if such mutations are associated with the extensive field-evolved resistance of this pest to Cry2Ab in the southern United States32. In a single-pair family established from H. zea collected from corn producing Cry1A.105 + Cry2Ab in Maryland in 2019, resistance to these two Bt toxins was not associated with chromosome 17 where HzABCA2 occurs40. More extensive screening of H. zea from field populations resistant to Cry2Ab is needed to determine if mutations disrupting ABCA2 are important in this widespread practical resistance.The results here showing that mutations disrupting ABCA2 can cause resistance to Cry2Ab in H. zea eggs from Benzon Research  in July 2021. We maintained this laboratory strain without exposure to Bt toxins and refer to it as LAB-S because of its susceptibility to Cry2Ab and other Bt toxins41. Larvae were reared on Southland diet . All rearing and diet bioassays were done at 28 °C under 20–40% humidity and 14 h light:10 h dark. Moths were reared in separate incubators from larvae and had access to a 10% sugar water solution for feeding and cheese cloth for oviposition41.We bought 42 and provided by Corteva Agriscience. Cry2A.127 is 98.6% identical with Cry2Ab1 and Cry2Ab2 (9 substitutions out of 633 amino acids) and is referred to here as Cry2Ab.We used Cry2A.127, a variant of Cry2Ab protoxin that was prepared, purified, and solubilized as described previouslyHzABCA2 encoding the ATP-binding cassette protein ABCA2 (from the H. zea genome36); and (2) vermilion  MG976796.1. The latter gene was previously shown to result in yellow mutant eye color in CRISPR/Cas9 mutant knockouts of H. zea37. One sgRNA located in HzTO exon 6 and seven sgRNAs located within 5' exons of HzABCA2 were made   that contained the T7 RNA polymerase binding site, a 20-bp gene-specific target sequence, and the 80-bp common stem-loop tracrRNA sequence. DNA templates were used to synthesize sgRNA using the HiScribe T7 High Yield RNA synthesis Kit . Transcribed sgRNAs were treated with DNase I for 20 min at 37 °C and purified using RNAClean XP (Thermo Fisher Scientific) following the manufacturer’s protocol.We designed sgRNAs targeting two genes: (1) HzABCA2 gDNA in vitro, we used the Guide-it sgRNA Screening Kit  as previously published23. We first extracted gDNA from LAB-S 3rd instar larvae using the Gentra Puregene Tissue Kit , which served as DNA template for PCR. Primer pairs 1HzABCA2-5 + 2HzABCA2-3, 3HzABCA2-5 + 4HzABCA2-3, and 5HzABCA2-5 + 6HzABCA2-3  and aliquots of each digestion reaction and corresponding negative controls were analyzed by 1.5% agarose gel electrophoresis stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific).To test if each sgRNA complexed with Cas9 was able to cut PCR-amplified HzABCA2 and HzTO were singly complexed with the Alt-R Streptococcus pyogenes HiFi Cas9 nuclease V3 (Integrated DNA Technologies), at 50 ng µL−1 of sgRNA to 100 ng µL−1 of Cas9, then incubated at room temperature for 15 min to generate the Cas9-ribonucleoprotein (RNP) complex. The two RNP complexes were combined to create a mixture of HzABCA2 and HzTO RNP, then the mixture was placed on ice and immediately used for injections.sgRNA targeting H. zea females naturally affix eggs to the coverslips and no additional manipulation of the embryos was needed. An IM-300 microinjector  equipped with an Olympus IMT-2 inverted microscope  was used to inject newly laid eggs (less than 1 h old) with approximately 100–200 picoliters of the HzTO + HzABCA2 RNP solution. Quartz needles  were beveled using a Model EG-44 micropipette grinder (Narishige) at a 30° angle and a rotor speed of ~ 1800 rpm or 90% of the maximum speed. Needles were backfilled with 3 µL of the RNP mix using Eppendorf Microloader pipette tips. Following injection, the coverslips containing embryos  were placed into 100 X 15 mm Petri dishes containing 1% agarose and held at 28 °C until G0 neonates emerged.Embryos were collected from LAB-S by placing microscope glass coverslips  on top of screened lids for several cages containing 5 male and 5 female adults for 45 min. Adult 0 neonates were transferred to individual 30 mL translucent polystyrene cups containing 10 mL of Southland diet and reared at 28 °C (14:10 L:D) to pupation. Pupae (n = 125) were sexed and transferred to individual 30 mL cups until adult eclosion. Two G0 adult females showing full yellow eyes were allowed to mate with two G0 males showing mosaic eyes to generate Yellow-R2 G1 neonates used in diet selection bioassays (below). The remainder of the surviving G0 adults  were placed into cages with 8–10 males and 8–10 females and used to generate Hz-R2 G1 neonates used for selection (below).Newly emerged G1 larvae from the unselected Yellow-R2 and Hz-R2 strains for susceptibility to Cry2Ab using our standard 7-d diet overlay bioassays41. We tested one neonate per well in bioassay trays  on diet treated with 1 μg Cry2Ab per cm2 diet (n = 192 for Hz-R2 and n = 224 for Yellow-R2) or on control diet (n = 16) treated with 40 µl of 0.1% Triton X-100 (no Cry2Ab). Trays were covered with Pull N' Peel covers . Survivors (≥ 4th instar larvae at 7 days) on Cry2Ab-treated diet were used to further propagate the Hz-R2 and Yellow-R2 strains. We calculated adjusted survival (%) as the survival (%) on diet containing 1 μg Cry2Ab per cm2 divided by survival (%) on untreated diet. We used a two-tailed Fisher's exact test  to determine if a significant difference occurred between LAB-S and the G1 of each CRISPR-edited strain in the proportion of live larvae on treated diet and on untreated diet.We tested G1 larvae of Yellow-R2 and Hz-R2 that survived on diet surface treated with 1 μg Cry2Ab per cm2 harbored mutations corresponding to sgRNA target sites, we amplified, cloned, and DNA sequenced the relevant HzABCA2 and HzTO gDNA corresponding to sgRNA target sites. We extracted gDNA separately from LAB-S, Yellow-R2, and Hz-R2 larvae using the DNeasy Blood and Tissue kit . Phusion Green High-Fidelity DNA Polymerase (Thermo Fisher Scientific) was used with oligonucleotide primers . DNA bands were excised, cloned into the pJET1.2 vector (Thermo Fisher Scientific), and transformed into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific). Purified plasmid DNA was Sanger sequenced by Retrogen . Multiple sequence alignments were performed using MUSCLE43. The full-length HzABCA2 sequence from LAB-S is deposited in the GenBank public database (Accession number OP186036.1).To determine if G41 to determine the concentration of Cry2Ab necessary to kill 50% of larvae (LC50) for Hz-R2 and LAB-S. Newly emerged neonates (n = 48 larvae per concentration) were placed into separate wells of bioassay trays. For LAB-S these contained 0, 0.1, 0.3, or 1 μg Cry2Ab per cm2 diet, and for Hz-R2 (G4) they contained 0, 1, 3, 10, and 30 μg Cry2Ab per cm2 diet. Trays were held at 28 °C (14 h light:10 h dark). After 7 days, we scored live larvae that were third or higher instars as survivors. We adjusted mortality for control mortality using Abbott’s correction and calculated LC50 using R (v 3.6.3)44 and the publicly available script https://github.com/JuanSilva89/Probit-analysis/commit/2eaaff05da0f89294788bd0bed564e1bf257acf2. The resistance ratio was calculated by dividing the LC50 for Hz-R2 by the LC50 for LAB-S.We used diet overlay bioassays1 neonates from reciprocal Hz-R2 X LAB-S crosses in diet overlay bioassays on 1 μg Cry2Ab per cm2 diet (n = 130 neonates per cross) and control diet (n = 32 neonates per cross). Survivors (≥ 4th instar larvae) were determined after 7 days of diet exposure and adjusted % survival was calculated by dividing survival on treated diet by survival on diet without Cry2Ab. We evaluated the dominance parameter h, which varies from 0 for completely recessive resistance to 1 for completely dominant resistance45. We calculated h for Hz-R2 as: h = /, where the S is LAB-S, R is Hz-R2, and F1 is larvae resulting from reciprocal crosses between Hz-R2 and LAB-S.We evaluated the mode of inheritance of resistance to Cry2Ab for Hz-R2 by testing FSupplementary Information.


The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (M In addi021 (pro . Current021 (pro .in vitro effectiveness of nirmatrelvir against variants Alpha (B.1.1.7), Beta (B.1.351), Delta (B1.617.2), Gamma (P.1), Lambda (B.1.1.1.37/C37), and Omicron (B.1.1.529) has already been demonstrated  relative to Mpro. We identified 389 distinct polymorphisms in Mpro with a n ≥ 10 individuals. From those and based on available structural information, we selected mutants that were within an 8.0-Å radius of each inhibitor structure, which would allow us to investigate how near active site mutations affect drug effectiveness against the enzyme. We identified 16 polymorphisms, which are summarized in e.g., M49, G143, M165, and Q189). To investigate the impacts of the polymorphisms on Mpro activity and calculate inhibitors constants, we were able to express, purify, and characterize 15 of those mutants. Mutant N142L did not generate any soluble protein and was therefore not characterized further.For this study, we selected active site polymorphic versions of Mdatabase  (containpro mutants were able to recognize and cleave the fluorogenic substrate. The turnover number (kcat) and Michaelis–Menten constant (KM) were determined for all mutants (cat/KM) relative to the wildtype (WT). The WT enzyme showed a KM value of 22 ± 2 μM and a kcat value of 31 ± 1 s−1, which agrees with previous characterizations of Mpro for this substrate   . The mutand 205% .Figure 2A). Mutants N142S, R188S, A191V, A193S, and A193T showed similar relative catalytic efficiencies to the WT (A). Mutants G143S, Q189K, and A193V showed diminished relative catalytic efficiencies in comparison with the WT, with a reduction of 33-, 3-, and 2-fold, respectively (A).Mutants M49I, M49T, N142D, M165I, R188K, T190I, and A191T exhibited a catalytic efficiency significantly higher than the WT, with increments up to 2-fold, with N142D and R188K being the ones with the higher relative catalytic efficiencies A. Mutanto the WT A. Mutantectively A.Figure pro, with an IC50 value of 0.022 ± 0.004 μM and calculated inhibition constant (Ki) of 0.006 ± 0.0005 μM, in agreement with the previous reported value 0.003 μM for the Ki . Mutants N142D, R188S, A191V, A193S, and A193T showed modest diminished inhibitory constants for nirmatrelvir, with 2- to 3-fold increase in Ki values relative to the WT. Mutants Q189K and G143S exhibited the highest diminished inhibitory constants for nirmatrelvir, showing 16- and 147-fold increase in the Ki values relative to the WT, respectively . Mutants N142D and T190I showed modest diminished inhibitory constants for ensitrelvir, with 2- to 3-fold increase in Ki values relative to the WT. Mutants M49T, N142S, A191T, and A191V showed significantly diminished inhibitory constants against ensitrelvir, with 3- to 5-fold increase in Ki values relative to the WT. Mutants M49I, G143S, and R188S showed the highest diminished inhibitory constants of ensitrelvir, exhibiting 6-, 15-, and 5.7-fold increase in the relative Ki values, respectively . The WT structure in complex with nirmatrelvir shows key interactions between protein and ligand, including three polar contacts between the pyrrolidone group and amino acids F140 (3.3 Å), H163 (2.6 Å), and E166 (3.1 Å), as well as other two hydrogen bonds between the tert-butyl moiety of nirmatrelvir and E166 N (2.9 Å) and O (2.8 Å), and a salt bridge between nirmatrelvir O atom with Q189 side chain NE2 (4.5 Å)  .Figure 4D). The structure of the Q189K mutant in complex with nirmatrelvir showed that the replacement of Q189 with lysine resulted in the disruption of a salt bridge between the NE2 of glutamine and the O atom from the carbonyl group of nirmatrelvir, dislocating the ligand position compared with the WT crystal structure and causing the loss of a productive hydrogen contact between the pyrrolidone moiety and the O from amino acid F140 in S1 .In nirmatrelvir WT complex, the S2 subsite is occupied by the dimethyl-cyclopropyl-proline substituent, which interacts mainly by hydrophobic interactions with the main chains of R188 and the side chains of H41, M49, M165, and Q189 . The samFig. 4A). For both crystal structures with mutations of residue 193, A193S and A193T, even though being the most distant mutations of the active site, the additional steric volume and polar features of the serine and threonine side chains caused a displacement in the ligand binding mode in comparison with the binding mode to the WT (E and F). The binding modes of nirmatrelvir to both A193S and A193T mutants were similar, but the displacement is more severe in A193S, causing the disruption of pyrrolidone hydrogen bound with E166 (E).The tert-butyl substituent at P3 position remained solvent exposed for all mutants, as observed for the WT crystal structure. The S4 subpocket accommodates the trifluoroacetamide substituent at position P4, where the main chain oxygen of the E166 residue makes a hydrogen bond with nirmatrelvir amide nitrogen N1 A. For boo the WT , E and Fith E166 E.A). For the mutant G143S complex, we observed that the pyrrolidone group from nirmatrelvir that occupies the S1 pocket appears in four distinct orientations, one to each chain in the two dimmers composing the asymmetric unit , G143 (3.2 Å), S144 (3.2 Å), C145S (3.3 Å), H163 (3.1 Å), and E166 (3.0 Å) through productive hydrogen bonds . A charaitrelvir . Howeverand C145 .Figure 6pro, indicating that these mutations would likely be able to generate versions of Mpro with high fitness for selection . The structure of M49I in complex with nirmatrelvir showed that the mutation has also a minor effect on ligand orientation relative to the WT, where all key molecular contacts were maintained (D). More distant mutations affecting subsites P3-P4 small residues, such as A191T, A191V, A193T, and A193S (but not A193V) have shown significantly increased Ki values for nirmatrelvir when compared with P1 and P2 mutations. The structure of A193T and A193S mutants showed that the increased steric volume and polar features of the side chains caused a displacement of the inhibitor related to the WT (E and F). As expected, we also notice that less conserved mutations in a specific amino acid seemed to have a greater effect on inhibitory constants of compounds, as exemplified by R188K versus R188S (1.4- versus 2.9-fold increase in Ki). Another potentially impactful resistance mechanism depicted here was the one caused by the Q189K mutant, in which the crystal structure revealed that the absence of a productive bond between the oxygen atom of the amide group of nirmatrelvir and the side chain of Q189 caused a rearrangement of the inhibitor’s binding mode (B), leading to an increase of 16-fold in the Ki value (B).The mutant panel tested herein explored the capacity of nirmatrelvir to retain activity B. The stintained D. More do the WT , E and Fing mode B, leadinKi value B.versus a substrate containing Gln side chain at P1. Despite the high decrease in inhibitory constants generated by this mutation, the low catalytic efficiency exhibited by this polymorphism might limit its ability to be selected over other strains, unless it is associated with secondary mutations that compensate the deleterious effect.The mutation of G143 to serine shifted the charge distribution and/or stereochemistry on S1, preventing the stabilization of the pyrrolidinone group into a single conformation . A similpro, developed by Shionogi using an intense structure-based drug design program (B). Another key difference between ensitrelvir and nirmatrelvir resistance profiles is that the former seems to retain near full activity against the Q189K mutant.Ensitrelvir is a nonpeptidic noncovalent inhibitor of SARS-CoV-2 M program . Their c program . This sc program . In cont program . The str program B. Anothepro. These results are important not only in the monitoring of emergence of resistant strains of SARS-CoV-2 but also for planning a more suitable treatment in the event of one of these polymorphisms becoming a strain of concern. Moreover, the depicted complexes between inhibitors and mutants help us to understand the structural features involved in resistance, which should assist the development of the next generation of Mpro inhibitors.The combined results suggest that these two distinct inhibitors have a different resistance profile against a panel of mutants, which can be explained by the distinct binding modes to Mpro that have been already identified in circulation. To do this, we sorted sequencing data available from the GISAID hCoV-19/SARS-CoV-2 sequences database (http://cov-glue.cvr.gla.ac.uk) relative to Mpro. Then, we selected all mutants that were within an 8.0-Å radius of the drugs and with n ≥ 10 individuals.We selected polymorphic versions of Mdatabase  in CoV-Get al. (pro coding region (residues 3264–3569) was inserted into the pET_M11/LIC vector using the ligation-independent cloning method (pro residues (YFQGAMSAVLQ↓SGFRK). For site-directed mutagenesis, the pET_M11/LIC-Mpro vector was used as template for inverse PCR . Cells were disrupted by sonication. The lysate was clarified by centrifugation at 15,000g, at 4 °C for 30 min. After expression, Mpro was obtained in the native form after autocleavage. The protein was obtained by precipitation with addition of 1.5 M ammonium sulfate followed by incubation on ice for 10 min. The precipitated protein was isolated by centrifugation at 15,000g at 4 °C for 15 min. The protein was resuspended in lysis buffer and injected into a Superdex 200 26/100 column  pre-equilibrated with gel filtration buffer . After size exclusion chromatography, the protein was buffer exchanged to 20 mM Tris pH 8.0, 1 mM DTT and further purified by ionic exchange chromatography using a Mono-Q column . The protein was eluted with a linear gradient of a buffer containing 20 mM Tris pH 8.0, 1 M NaCl, and 1 mM DTT. Fractions containing the purified protein were collected and quantified using the measured absorbances at 280 nm and the theorical extinction coefficient of 32,890 M−1 cm−1. Protein purity was analyzed by SDS-PAGE. For enzymatic assays, protein was aliquoted at 0.5 mg/ml and flash-frozen using liquid nitrogen. Samples were stored at −80 °C until use.We used a self-cleavable construct of SARS-CoV-2 M culture . The sam2 medium  until A6pro mutants were crystallized using the sitting-drop vapor diffusion method. A volume of 1 μl of protein at 5 to 14 mg/ml was mixed with 1 μl of precipitating solution containing 0.1 M Mes pH 6.7, 8% PEG 4K, and 5% dimethyl sulfoxide (DMSO)   and 0.2 pro mutants in complex with nirmatrelvir and ensitrelvir were obtained by soaking the compound into Mpro mutants apo crystals grown as described previously. A volume of 2 μl of a solution containing 80% PEG 400, 20% DMSO, and 10 mM of compound was added directly into the 2 μl crystallization drops. Crystallization plates were incubated at 16 °C for 24 h. Crystals were manually harvested and flash-cooled in liquid nitrogen for data collection.Crystals of SARS-CoV-2 M1/2 (pro (PDBid 7MBG). Ligand and covalent link restraints were generated using AceDRG through the CCP4i2 program suite (Diffraction data collection was performed at MANACA beamline at the Brazilian Synchrotron SIRIUS using a Pilatus 2M detector (Dectris). Data were processed and scaled using autoPROC/STARANISO from Global Phasing , 33. Res1/2 , 35. For1/2  was usedam suite , 38. Refam suite  or Pheniam suite . Structuam suite . Figurespro mutants were diluted to a final concentration of 40 nM. To determine the kinetics parameters , the substrate was diluted to a range of concentrations from 200 μM to 0.1 μM. Reactions were previously incubated at 37 °C for 10 min and started by addition of substrate in the respective concentrations. Fluorescence measures were monitored in SpectraMax Gemini EM Microplate Reader with λexc/λemi of 360/460 nm, every 60 s over 60 min at 37 ° C. The initial velocity was derived from the slope of linear phase of each time-course reaction, and Michaelis–Menten fitting was obtained using Origin Pro 9.5.1 Software (OriginLab). Relative efficiency of Mpro mutants was calculated by comparing the kcat/Km relative to WT Mpro. For IC50 determination, reactions containing nirmatrelvir or ensitrelvir from 10 μM to 0.0006 nM were previously incubated at 37 °C for 10 min and started by addition of 10 μM of substrate. Inhibition percentages were determined by comparison with the DMSO control. All assays were performed in independent triplicates, and presented values were determined from average values. For determining the Ki values, nirmatrelvir was considered an uncompetitive inhibitor, whereas ensitrelvir was considered a competitive inhibitor, and values were calculated using IC50-to-Ki -NH2 in assay buffer  in Corning 384-well white microplates. M50-to-Ki .8DZ2, 8E25, 8E26, 8DZ9, 8DZ6, 8E1Y, 8DZA, 8DZ0, and 8DZ1. Other data are available from the corresponding author upon reasonable request.Structure factors and atomic coordinates have been deposited with the Protein Data Bank with accession codes PDB IDs This article contains The authors declare that they have no conflicts of interest with the contents of this article.


These compounds exerted antiproliferative activity against cell lines derived from solid tumors. Herein, the antileukemic activities of these compounds (4a–n) against two different leukemia cell lines (Jurkat acute T cell and THP-1 acute monocytic) were investigated. Our investigation included examining their activity in vivo in a zebrafish embryo model. Remarkably, compounds 4a and 4d were the most potent in suppressing cell proliferation, with an IC50 value range of 4–6.5 µM. Flow cytometry analysis indicated that both compounds halted cell progression at the G2/M phase and induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analyses also showed that both compounds effectively induced apoptosis by upregulating the expression of proapoptotic factors while downregulating that of antiapoptotic factors. In vivo animal toxicity assays performed in zebrafish embryos indicated that compound 4d was more toxic than compound 4a, with compound 4d inducing multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration. Moreover, both compounds perturbed the hematopoiesis process in developing zebrafish embryos. Collectively, our data suggest that compounds 4a and 4d have the potential to be used as antileukemic agents.Many quinazoline derivatives with pharmacological properties, such as anticancer activity, have been synthesized. Fourteen quinazoline derivatives bearing a substituted sulfonamide moiety ( According to the GLOBOCAN estimate of 2020 global cancer statistics, leukemia caused around 311,594 deaths and is expected to cause approximately 474,519 new cases ) 8-methoxy and hydrophobic electron-donating (+I) 4-methyl group at the 2- and 3-position, respectively, showed higher activity than the unsubstituted phenyl distal moiety 4a.A preliminary structure activity relationship (SAR) analysis focused on the impact of the hydrophobic and electronic nature of the substituent used in this study. In addition, it was focused on the impact of the type and length of the linkers used and the distal moieties. From the obtained results, it was found that compound 4a  led to a significant increase in the population of cells at G2/M . As with 4a, the incubation of Jurkat cells with compound 4d resulted in a significant increment in the G2/M cell population  compared to the control cells (34.2 ± 0.82%). Similarly, the exposure of THP-1 cells to compound 4a . In the same manner, THP-1 cells treated with 2.5, 5 and 10 µM of compound 4d increased the cell population at the G2/M phase to 35 ± 0.44%, 41.45% ± 1.14 and 52.1 ± 0.42% of the control cell value. Our data is in line with Zhang et al. (2021), who found that quinazoline derivatives caused a G2/M cell cycle arrest in H1975 lung cancer cells [Cell cycle arrest is a crucial mechanism by which anticancer drugs suppress cancer cell proliferation . Therefopound 4a  resulteder cells ,17,18.4a and 4d on cell death by detecting phosphatidylserine exposure on the outer membrane of apoptotic cells. Apoptosis stimulation is a major objective in the development of antineoplastic drugs. In fact, most of the currently utilized anticancer drugs in clinical oncology trigger cancer cell death through apoptotic signaling pathways [Flow cytometry was also employed to assess the impact of compounds pathways ,20.4a and 4d. The percentage of early apoptotic cells in 4a-treated Jurkat cells increased from 6.7 ± 0.28% in the control cells to 11.2 ± 0.71%, 12.85 ± 0.49% and 27.6 ± 0.49% at 2.5, 5 and 10 µM, respectively  and caspase-3  in comparison to the untreated cells, while a significant decrease in the expression levels of Bcl-2 was only detected with the 10 µM treatment . On the other hand, treatment with compound 4d slightly enhanced the levels of Bax by 1.12-, 1.52- and 10.2-fold and markedly increased the expression of caspase-3 by 1.35-, 2.04- and 2.15-fold, respectively. Similarly, the exposure of THP-1 cells to compound 4a resulted in a significant increase in the expression of Bax with the 5 and 10 µM treatments  and a significant downregulation of Bcl-2 at the same concentration . The expression levels of caspase-3 were also noticeable after the exposure of THP-1 cells to various concentrations of each compound  even at the highest concentration used (50 µM). These compounds neither killed the embryos nor induced any teratogenic effect (data not shown). Compounds 4a and 4d affected the development of zebrafish embryos. Compound 4d turned out to be more toxic than compound 4a; 4d induced toxicities and its LD50 (the concentration at which 50% of the embryos died) value was just 1.67 µM. Moreover, compound 4d also induced multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration (0.3–0.76 µM). The treated embryos showed severe scoliosis, enlarged yolk, cardiac hypertrophy and an absence of pigmentation  looked very similar to the control embryos, and no obvious teratogenesis was detected  of compound  results , the zeb4a-treated embryos. The embryos were exposed to 4a at the shield stage (6 hpf) and fixed at 48 and 96 hpf with O-dianisidien. As shown by the black arrows in 4a on zebrafish hematopoiesis was also time dependent. Prolonged exposure until 96 h completely abolished hematopoiesis and no hemoglobin staining was observed in 15.88 µM 4a-treated embryos , which expresses green fluorescent protein in the blood. The transgenic embryos were treated with 4a (1.58 µM) at the shield stage (6 hpf) and the effect on blood vessels was evaluated at 72 hpf. The formation of intersomatic blood vessels (isv) and sub-intestinal veins (sib) were investigated in the treated embryos. The results are shown in 4a did not affect the number or structures of blood vessels in the treated embryos.During embryonic development, the hematopoietic (which make various blood cells) and endothelial cells (which make blood vessels) differentiate from common hemangioblasts . In orde4a and 4d induced a significant level of cytotoxicity in leukemia cell lines, and 4d was the most toxic compound; however, the in vivo study suggested that compound 4d induced toxicity in normal cell proliferation during zebrafish embryonic development. For this reason, compound 4a was concluded to be a favorable anti-leukemia target molecule, as it was the least toxic toward normal cells while still being toxic toward leukemia cell lines and hematopoiesis in zebrafish embryos. Flow cytometry analysis demonstrated that both compounds caused G2/M cell cycle arrest and induced apoptotic cell death. In vivo data revealed that 4a disrupted hematopoiesis in less than the micro molar range without inducing gross teratogenicity or toxicity. Hematopoietic defects were observed in embryos treated with 4a at a concentration between 0.2 and 1.00 μM, whereas more than 17 μM was required to induce lethality or toxicity in zebrafish embryos. In general, the presented data offer new insights into the development of quinazoline–sulfonamide derivatives as potential antileukemic agents.Among all the newly synthesized compounds, only 4a, 2-methyl phenyl 4b, 3-methyl phenyl 4c, 4-methyl phenyl 4d, 2-ethyl phenyl 4e, 3-ethyl phenyl 4f, 4-ethyl phenyl 4g, 4-methoxy phenyl 4h, 4-ethoxy phenyl 4i, 3,5-dimethoxy phenyl 4j, 3,4,5-trimethoxy phenyl 4K, 2-methyl-4-nitro phenyl 4l, 2-methyl-6-nitro phenyl 4m and 2,4-dinitro phenyl 4n, were detailed and characterized as the compounds 4a-n in our previous work [1H-NMM, 13C-NMR and mass spectral data.A series of quinazoline compounds containing a biologically active substituted sulfonamide at the 3-position and substituted aromatic moieties at the 2-position, including phenyl ous work . The str2 and maintained in RPMI 1640 media  supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin .Human acute T-cell leukemia  and THP-1 human monocytic (ATCC-TIB202) cells were grown at 37 °C in a humidified incubator  with 5% CO5 cells/well on a 24-well plate and treated with various concentrations  of each compound. After the incubation period (24 h), the MTT reagent,  (5 mg/mL) was added to each well. Then, the plates were incubated for 4 h at 37 °C and the formazan was dissolved in DMSO . Thereafter, the optical density was measured at 570 nm using an ELISA reader  and cell viability was calculated according to the equation % V = A − Ao/Ac − Ao × 100. The IC50 (compound concentration required for the inhibition of 50% of cells) values were calculated from the dose–response curves using the OriginPro 8.5 .For the detection of the viability of Jurkat and THP-1 cells, an MTT assay was employed as previously described . Briefly4a and 4d. After a 24 h incubation time, the cells were washed with 1× phosphate-buffered saline (PBS), fixed with 70% ice-cold ethanol and kept for 4 h at 4 °C. Thereafter, the cells were precipitated, washed with 1x PBS and resuspended in 500 μL of PBS containing propidium iodide  with 0.1 mg/mL of Ribonuclease A and further incubated for 30 min in the dark. The analysis of the cell cycle was performed using flow cytometry  and CXP software V. 3.0.Both Jurkat and THP-1 cells were cultured in a 12-well plate and were treated with different concentrations of compounds 4a and 4d  for 24 h, apoptotic cells were detected using an apoptosis detection kit according to the supplier’s instructions . In brief, both untreated and treated cells were collected, washed with PBS and then resuspended in 0.1 mL Annexin binding buffer(1x). Thereafter, the cells were stained with Annexin V-FITC and PI (5 µL each) for 15 min in the dark. The samples were then quantified by a FACS Scan Flow Cytometer .After treatment with compounds RT-PCR was used to assess the level of mRNA in proportion to β-actin, the housekeeping gene. An RNA stabilization reagent , was used to extract total RNA from treated and untreated THP-1 and Jurkat cells, and the RNA was quantified by measuring the absorbance at 260 nm . Using the Superscript IV Vilo Master mix , a total of 1 µg of RNA from each group was utilized to synthesize complementary DNA (cDNA). The cDNA samples were kept at a temperature of −20 degrees Celsius. Bax, Bcl2, caspase-3 and β-actin mRNA assays were performed with RT-PCR using the newly generated cDNA. An aliquot of 1 µL of cDNA from each group was amplified in a 25 µL reaction mixture with 5X FIREpol master mix . The oligonucleotide sequences were designed using Integrated DNA Technologies (IDT). The forward and reverse primers for Bax were 5′-TTTGCTTCAGGGTTTCATC-3′ and 5′-ATCCTCTGCAGCTCCATGTT-3′, respectively. The primers for Bcl2 were 5′-TGATGCCTTCTGTGAAGCAC-3′ and 5′-ACAGGCGGAGCTTCTTGTAA-3′; 5′-TGGAATTGATGCGTGATGTT-3′ and 5′-GGCAGGCCTGAATAATGAAA-3 for caspase-3; and 5′-CATCGTGATGGACTCTGGTG-3′ and 5′-TTTGATGTCACGCACGATTT-3′ for β-actin. The sample was denatured for 5 min at 95 °C, then amplified using 32 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 60 s and elongation at 72 °C for 60 s, followed by final elongation at 72 °C for 5 min using a SimpliAmp Thermal Cycler . After that, a 1.2 percent agarose gel containing ethidium bromide was used to electrophorese 20 µL of the PCR-amplified material, and the bands on the gel were photographed.The THP-1 and Jurkat cells were treated with different concentration of each compound for 24 h, and untreated cells were taken as a control. After 24 h of treatment, cells were harvested and washed twice with 1× PBS, and the harvested cells were lysed by incubation in a lysis buffer (20 mM Tris-HCl (PH 7.5), 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.25% Triton X-100, protease inhibitor mixture, 2 mM PMSF and 1 mM DTT) for 2 h on ice. The obtained lysate was clarified by centrifuging at 1300 rpm at 4 °C for 15 min. The protein content of the supernatant was measured using a Bradford protein assay . The expression levels of Bax, Bcl2, caspase-3 and β-actin were detected with a Western blot by resolving 25 µg of protein on 12% SDS-PAGE. After electrophoresis, the proteins on the gel were transferred to a PVDF membrane  and the membrane was blocked with 5% BSA for 1h at room temperature, followed by washing with Tris-buffered saline, 0.1% Tween-20 (TBST) and incubation with primary antibodies  against Bax, Bcl2, caspase-3 and β-actin overnight at 4 °C. β-actin was used as an internal control. Following this, an incubation with horseradish peroxidase-conjugated secondary antibodies diluted to 1:4000 in PBS containing 5% BSA was completed at room temperature for 30 min. The binding of immunoproteins was detected using an enhanced chemiluminescent assay  and X-ray films .Wild type (AB Tubingen) and transgenic “Tg (Fli-1: EGFP)”  zebrafisV/V)) was used as a mock control. Embryos at identical developmental stages were sorted at the shield stage, transferred to 35 mm sterile glass dishes and exposed to a serial dilution (0.1–10 µM) of each compound in a total volume of 5 mL of embryo medium. The embryos remained exposed to the compound until the end of the experiment (3 days), where the embryo medium, including the compound, was changed daily. The embryonic mortality and teratogenic effects were monitored every 24 h until the end of the experiment. The treatment was repeated at least three times . The LC50 values for zebrafish embryonic toxicity were calculated using an updated Probit analysis [The compounds were dissolved in molecular biology grade DMSO (Sigma Aldrich) to prepare a stock solution of 25 mM. DMSO  was added to the embryo medium. The embryos were cultured and fixed at 48 and 72 h post-fertilization (hpf) for O-dianisidien staining. The staining was performed in the dark for 30 min at room temperature within a solution containing O-dianisidine (0.6 mg/mL), 0.01 M sodium acetate (PH 4.5), 0.65% hydrogen peroxide and 40% ethanol. Once stained, the embryos were washed with distilled water and fixed in 4% paraformaldehyde for 1 h at room temperature. The embryos were washed with phosphate-buffered saline (PBS) containing 0.1% Tween-20.p value of less than 0.05.All data are represented as the mean ± standard deviation (SD). Statistical significance was determined by a paired t-test using the OriginPro software. Statistical significance was defined as a