SARS-CoV-2 variants are constantly emerging,hampering public health measures in controlling the number of infections.While it is well established that mutations in spike proteins observed for the different variants di...SARS-CoV-2 variants are constantly emerging,hampering public health measures in controlling the number of infections.While it is well established that mutations in spike proteins observed for the different variants directly affect virus entry into host cells,there remains a need for further expansion of systematic and multifaceted comparisons.Here,we comprehensively studied the effect of spike protein mutations on spike expression and proteolytic activation,binding affinity,viral entry efficiency and host cell tropism of eight variants of concern(VOC)and variants of interest(VOI).We found that both the full-length spike and its receptor-binding domain(RBD)of Omicron bind to hACE2 with an affinity similar to that of the wild-type.In addition,Alpha,Beta,Delta and Lambda pseudoviruses gained significantly enhanced cell entry ability compared to the wild-type,while the Omicron pseudoviruses showed a slightly increased cell entry,suggesting the vastly increased rate of transmission observed for Omicron variant is not associated with its affinity to hACE2.We also found that the spikes of Omicron and Mu showed lower S1/S2 cleavage efficiency and inefficiently utilized TMPRSS2 to enter host cells than others,suggesting that they prefer the endocytosis pathway to enter host cells.Furthermore,all variants'pseudoviruses we tested gained the ability to enter the animal ACE2-expressing cells.Especially the infection potential of rats and mice showed significantly increased,strongly suggesting that rodents possibly become a reservoir for viral evolution.The insights gained from this study provide valuable guidance for a targeted approach to epidemic control,and contribute to a better understanding of SARS-CoV-2 evolution.展开更多
Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low to...Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.展开更多
G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization ...G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization of inhibitors targeting GRK2 are highly meaningful. Therefore, in order to design GRK2 inhibitors with better performance, the most active molecule was selected as a reference compound from a data set containing 4-pyridylhydrazone derivatives and triazole derivatives, and its scaffold was extracted as the initial scaffold. Then, a powerful optimization-based framework for de novo drug design, guided by binding affinity, was used to generate a virtual molecular library targeting GRK2. The binding affinity of each virtual compound in this dataset was predicted by our developed deep learning model, and the designed potential compound with high binding affinity was selected for molecular docking and molecular dynamics simulation. It was found that the designed potential molecule binds to the ATP site of GRK2, which consists of key amino acids including Arg199, Gly200, Phe202, Val205, Lys220, Met274 and Asp335. The scaffold of the molecule is stabilized mainly by H-bonding and hydrophobic contacts. Concurrently, the reference compound in the dataset was also simulated by docking. It was found that this molecule also binds to the ATP site of GRK2. In addition, its scaffold is stabilized mainly by H-bonding and π-cation stacking interactions with Lys220, as well as hydrophobic contacts. The above results show that the designed potential molecule has similar binding modes to the reference compound, supporting the effectiveness of our framework for activity-focused molecular design. Finally, we summarized the interaction characteristics of general GRK2 inhibitors and gained insight into their molecule-target binding mechanisms, thereby facilitating the expansion of lead to hit compound.展开更多
The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptoti...The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.展开更多
This novel study identifi es the effective anti-inflammatory phenolic compounds in dandelion and provides mechanistic insights into their interactions with receptor proteins(toll-like receptor 4,TLR4;co-receptor myelo...This novel study identifi es the effective anti-inflammatory phenolic compounds in dandelion and provides mechanistic insights into their interactions with receptor proteins(toll-like receptor 4,TLR4;co-receptor myeloid differentiation protein-2,MD-2)using UHPLC-ESI-MS/MS,lipopolysaccharide(LPS)-stimulated THP-1 cell line,fluorescence quenching and anisotropy,molecular docking(single ligand and multi-ligand docking)and molecular dynamics simulation.A 50%aqueous methanol extract had a greater anti-inflammatory effect and higher chicoric acid content,compared with the 100%water and 100%methanol extracts.Chicoric acid,chlorogenic acid,methylophiopogonone A,caffeic acid,gallic acid monohydrate and 4’-O-demethylbroussonin A had relatively high binding energies and contents in all extracts.Chicoric acid competed with chlorogenic acid,4’-O-demethylbroussonin A and quercetin for MD-2.Among dandelion’s phenolics,chicoric acid most effectively hindered TLR4-MD-2 complex formation,with a quenching constant of 0.62×10^(6) L/mol for MD-2 or TLR4 at 320 K,and binding energies of-6.87 and-5.97 kcal/mol,respectively,for MD-2 and TLR4.展开更多
Phosphatidylinositol polyphosphates (PIPx) are related with tyrosine kinase activation, cell proliferation and carcinogenesis. In order to investigate the action mechanism of PIPx, it is desirable to synthesize affini...Phosphatidylinositol polyphosphates (PIPx) are related with tyrosine kinase activation, cell proliferation and carcinogenesis. In order to investigate the action mechanism of PIPx, it is desirable to synthesize affinity column of PI-3,4-P-2, which is expected to be able to isolate the binding proteins of PI-3,4-P-2. Thyramine reacted with CH-Sepharose 4B giving column 13. The p-amino group of 3'-(1',2'-distearoyl-glyceryl)-1-(2-p-aminobenzyl)-3,4-di-O-phosphoryl-myo-inosityl phosphate (12) was diazotized, then diazo-coupled with column 13 to give PI-3,4-P-2 affinity column 14. This PI-3,4-P-2 affinity column is an effective tool to pick up binding proteins of PI-3,4-P-2.展开更多
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No.XDB29050100)the National Key Research and Development Program of China (Grant No.2022YFC0869900,2022YFC2303501,2020YFC0861100)the Program of the Chinese Academy of Sciences (Grant No.2020YJFK-Z-0150).
文摘SARS-CoV-2 variants are constantly emerging,hampering public health measures in controlling the number of infections.While it is well established that mutations in spike proteins observed for the different variants directly affect virus entry into host cells,there remains a need for further expansion of systematic and multifaceted comparisons.Here,we comprehensively studied the effect of spike protein mutations on spike expression and proteolytic activation,binding affinity,viral entry efficiency and host cell tropism of eight variants of concern(VOC)and variants of interest(VOI).We found that both the full-length spike and its receptor-binding domain(RBD)of Omicron bind to hACE2 with an affinity similar to that of the wild-type.In addition,Alpha,Beta,Delta and Lambda pseudoviruses gained significantly enhanced cell entry ability compared to the wild-type,while the Omicron pseudoviruses showed a slightly increased cell entry,suggesting the vastly increased rate of transmission observed for Omicron variant is not associated with its affinity to hACE2.We also found that the spikes of Omicron and Mu showed lower S1/S2 cleavage efficiency and inefficiently utilized TMPRSS2 to enter host cells than others,suggesting that they prefer the endocytosis pathway to enter host cells.Furthermore,all variants'pseudoviruses we tested gained the ability to enter the animal ACE2-expressing cells.Especially the infection potential of rats and mice showed significantly increased,strongly suggesting that rodents possibly become a reservoir for viral evolution.The insights gained from this study provide valuable guidance for a targeted approach to epidemic control,and contribute to a better understanding of SARS-CoV-2 evolution.
基金supported by the National Key R&D Program of China(2017YFD0200400)the National Natural Science Foundation of China(31272115)
文摘Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.
基金supported by the National Natural Science Foundation of China Excellent Young Scientist Fund(22422801)the National Natural Science Foundation of China General Project(22278053)+1 种基金the National Natural Science Foundation of China General Project(22078041)Dalian High-level Talents Innovation Support Program(2023RQ059).
文摘G protein coupled receptor kinase 2 (GRK2) is a kinase that regulates cardiac signaling activity. Inhibiting GRK2 is a promising mechanism for the treatment of heart failure (HF). Further development and optimization of inhibitors targeting GRK2 are highly meaningful. Therefore, in order to design GRK2 inhibitors with better performance, the most active molecule was selected as a reference compound from a data set containing 4-pyridylhydrazone derivatives and triazole derivatives, and its scaffold was extracted as the initial scaffold. Then, a powerful optimization-based framework for de novo drug design, guided by binding affinity, was used to generate a virtual molecular library targeting GRK2. The binding affinity of each virtual compound in this dataset was predicted by our developed deep learning model, and the designed potential compound with high binding affinity was selected for molecular docking and molecular dynamics simulation. It was found that the designed potential molecule binds to the ATP site of GRK2, which consists of key amino acids including Arg199, Gly200, Phe202, Val205, Lys220, Met274 and Asp335. The scaffold of the molecule is stabilized mainly by H-bonding and hydrophobic contacts. Concurrently, the reference compound in the dataset was also simulated by docking. It was found that this molecule also binds to the ATP site of GRK2. In addition, its scaffold is stabilized mainly by H-bonding and π-cation stacking interactions with Lys220, as well as hydrophobic contacts. The above results show that the designed potential molecule has similar binding modes to the reference compound, supporting the effectiveness of our framework for activity-focused molecular design. Finally, we summarized the interaction characteristics of general GRK2 inhibitors and gained insight into their molecule-target binding mechanisms, thereby facilitating the expansion of lead to hit compound.
基金financially supported by Postdoctoral Applied Research Project of Qingdao(No.861605040085,to CZ,SW)Grant of Innovation Plan in Biomedical Research of Qingdao City(No.15-10-3-15-(28)-zch,to SW)
文摘The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.
基金supported by the funding“Innovation Project of Shandong Province Agricultural Application Technology”(2130106)“Key Technology Research and Development Program of Shandong”(2019GNC106004).
文摘This novel study identifi es the effective anti-inflammatory phenolic compounds in dandelion and provides mechanistic insights into their interactions with receptor proteins(toll-like receptor 4,TLR4;co-receptor myeloid differentiation protein-2,MD-2)using UHPLC-ESI-MS/MS,lipopolysaccharide(LPS)-stimulated THP-1 cell line,fluorescence quenching and anisotropy,molecular docking(single ligand and multi-ligand docking)and molecular dynamics simulation.A 50%aqueous methanol extract had a greater anti-inflammatory effect and higher chicoric acid content,compared with the 100%water and 100%methanol extracts.Chicoric acid,chlorogenic acid,methylophiopogonone A,caffeic acid,gallic acid monohydrate and 4’-O-demethylbroussonin A had relatively high binding energies and contents in all extracts.Chicoric acid competed with chlorogenic acid,4’-O-demethylbroussonin A and quercetin for MD-2.Among dandelion’s phenolics,chicoric acid most effectively hindered TLR4-MD-2 complex formation,with a quenching constant of 0.62×10^(6) L/mol for MD-2 or TLR4 at 320 K,and binding energies of-6.87 and-5.97 kcal/mol,respectively,for MD-2 and TLR4.
文摘Phosphatidylinositol polyphosphates (PIPx) are related with tyrosine kinase activation, cell proliferation and carcinogenesis. In order to investigate the action mechanism of PIPx, it is desirable to synthesize affinity column of PI-3,4-P-2, which is expected to be able to isolate the binding proteins of PI-3,4-P-2. Thyramine reacted with CH-Sepharose 4B giving column 13. The p-amino group of 3'-(1',2'-distearoyl-glyceryl)-1-(2-p-aminobenzyl)-3,4-di-O-phosphoryl-myo-inosityl phosphate (12) was diazotized, then diazo-coupled with column 13 to give PI-3,4-P-2 affinity column 14. This PI-3,4-P-2 affinity column is an effective tool to pick up binding proteins of PI-3,4-P-2.
