Conformational Dynamics of the Activated GLP‑1 Receptor‑Gs Complex Revealed by Cross-Linking Mass Spectrometry and Integrative Structure Modeling
Shijia Yuan, Lisha Xia, Chenxi Wang,Fan Wu, Bingjie Zhang, Chen Pan, Zhiran Fan, Xiaoguang Lei, Raymond C. Stevens, Andrej Sali, Liping Sun,* and Wenqing Shui* ACS Cent. Sci. 2023, 9, 992−1007
Despite advances in characterizing the structures and functions of G protein-coupled receptors (GPCRs), our understanding of GPCR activation and signaling is still limited by the lack of information on conformational dynamics. It is particularly challenging to study the dynamics of GPCR complexes with their signaling partners because of their transient nature and low stability. Here, by combining cross-linking mass spectrometry (CLMS) with integrative structure modeling, we map the conformational ensemble of an activated GPCR-G protein complex at near-atomic resolution. The integrative structures describe heterogeneous conformations for a high number of potential alternative active states of the GLP-1 receptor−Gs complex. These structures show marked differences from the previously determined cryo-EM structure, especially at the receptor−Gs interface and in the interior of the Gs heterotrimer. Alaninescanning mutagenesis coupled with pharmacological assays validates the functional significance of 24 interface residue contacts only observed in the integrative structures, yet absent in the cryo-EM structure. Through the integration of spatial connectivity data from CLMS with structure modeling, our study provides a new approach that is generalizable to characterizing the conformational dynamics of GPCR signaling complexes.
Diversity-oriented synthesis of cyclohexenes by combining enzymatic intermolecular DielsAlder reactions and decarboxylative functionalizations，
Jin Wang, Han Ke, Jun Yang, Nianxin Guo, Kangdelong Hu, Ruyao Tang,Qi Ding, Lei Gao*and Xiaoguang Lei*
Chem Catalysis, 2023, 3(1), 100451
Substituted cyclohexanes are common scaffolds found in both natural products and drug molecules. Diels-Alderases that can efficiently catalyze intermolecular Diels-Alder reactions to generate cyclohexene ring systems have received considerable interest. However, the synthetic power of Diels-Alderases is incomparable with chemo-catalysts due to their limited substrate scopes. Here, we report a new chemo-enzymatic strategy for the diversity-oriented syntheses of functionalized cyclohexenes. We first applied focused rational iterative site-specific mutagenesis to generate a natural Diels-Alderase variant M3, which shows a 34-fold increase in catalytic efficiency, broad substrate scope, and good to perfect stereoselectivity. Then, we used diverse transition-metal-catalyzed decarboxylative coupling reactions to functionalize the enzymatic Diels-Alder products. This work offers an efficient synthetic route to structurally diverse cyclohexenes that are not accessible by solely using biocatalysis or chemo-catalysis and illustrates how chemo-catalysis can cooperate with biocatalysis to expand the synthetic application of biocatalysts.
C-Glycosides are critical motifs embedded in many bioactive natural products. The inert C-glycosides are privileged structures for developing therapeutic agents owing to their high chemical and metabolic stability. Despite the comprehensive strategies and tactics established in the past few decades, highly efficient C-glycoside syntheses via C−C coupling with excellent regio-, chemo-, and stereoselectivity are still needed. Here, we report the efficient Pd-catalyzed glycosylation of C−H bonds promoted by weak coordination with native carboxylic acids without external directing groups to install various glycals to the structurally diverse aglycon parts. Mechanistic evidence points to the participation of a glycal radical donor in the C−H coupling reaction. The method has been applied to a wide range of substrates (over 60 examples), including many marketed drug molecules. Natural product- or drug-like scaffolds with compelling bioactivities have been constructed using a late-stage diversification strategy. Remarkably, a new potent sodium-glucose cotransporter-2 inhibitor with antidiabetic potential has been discovered, and the pharmacokinetic/ pharmacodynamic profiles of drug molecules have been changed using our C−H glycosylation approach. The method developed here provides a powerful tool for efficiently synthesizing C-glycosides to facilitate drug discovery.
Total Synthesis of Diverse Tetramic Acid Bearing cis-Decalin Natural Products Haoran Dong, Dachao Hu, Benke Hong, Jin Wang, and Xiaoguang Lei*
Angew. Chem. Int. Ed. 2023, e202301872
We here report the first total syntheses of four natural antibiotics vermisporin (1), PF1052/AB4015-A (2), AB4015-L (3), AB4015-B (4) and one hydrogenated natural product derivative AB4015-A2 (5) that all feature a tetramic acid bearing cis-decalin ring. The construction of the functionalized cis-decalin ring was achieved by a diastereoselective intramolecular Diels-Alder (IMDA) reaction which underwent a rare endo-boat transition state. Through an intramolecular neighboring-group-oriented strategy, the sterically hindered epoxy group in vermisporin (1), PF1052/AB4015-A (2) and AB4015-L (3) was installed efficiently. A one-pot aminolysis/Dieckmann-condensation cascade using L-amino acid derivatives afforded the desired tetramic acid structure. The total synthesis led to the unambiguous verification of the absolute configuration of these natural products
Xueping Wang, Victor Tomilin, Merve Ertem, Abigail McKernan, Xiaoguang Lei, Oleh Pochynyuk, Ossama B Kashlan* J. Physiol. 2022, 600, 4695-4711.
Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel.
Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H+ gradient across the plasma membrane (PM) established by PM H+-adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H+-ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H+-ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H+-ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H+-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H+-ATPases. Our results support the chemiosmotic model in which PM H+-ATPase itself plays a fundamental role in polar auxin transport
The Diels-Alder (D-A) reaction is one of the most critical chemical transformations to construct C–C bonds with predictable regio- and stereo-selectivities. It has been widely used as a retrosynthetic disconnection in synthetic chemistry. Although significant advances have been made, synthetic challenges remain in how to precisely control the stereochemistry of intermolecular Diels-Alder reaction. Enzymes are well known for their remarkable catalytic efficiency and selectivity compared with chemo-catalysts. Therefore, identifying and designing intermolecular Diels-Alderases that can be used in organic synthesis have received considerable attention from the synthetic community. In this review, we review all the enzymes capable of catalyzing formal intermolecular Diels-Alder reactions in natural product biosynthetic pathways, discuss their catalytic mechanisms in detail, and highlight their synthetic potential in the precise and efficient synthesis of enantiopure D-A products. We also discuss the different strategies that can be used to create new artificial Diels-Alderases, especially RNA-based Diels-Alderases.
Current Opinion in Chemical Biology 2022, 70, 102185
The reversible phosphorylation of substrates mediated by kinases and phosphatases affects their subcellular localization, catalytic activity, and/or interaction with other molecules. It is essential for signal transduction and the regulation of nearly all cellular processes, such as proliferation, apoptosis, metabolism, motility, and differentiation. Small molecule kinase inhibitors (SMKIs) have served as critical chemical probes to reveal the biological functions and mechanisms of kinases and their potential as therapeutic targets. In this review, we focused on a few novel SMKIs and their recent application in biological and preclinical studies to showcase how highly selective and potent SMKIs can be developed and utilized to propel the investigations on kinases and the biology behind.
Yange Niu, Wenhao Cui, Rui Liu, Sanshan Wang, Han Ke, Xiaoguang Lei, Lei Chen*
Nature Commun. 2022, 13, 6440
Sodium glucose co-transporters (SGLT) harness the electrochemical gradient of sodium to drive the uphill transport of glucose across the plasma membrane. Human SGLT1 (hSGLT1) plays a key role in sugar uptake from food and its inhibitors show promise in the treatment of several diseases. However, the inhibition mechanism for hSGLT1 remains elusive. Here, we present the cryo-EM structure of the hSGLT1-MAP17 hetero-dimeric complex in the presence of the high-affinity inhibitor LX2761. LX2761 locks the transporter in an outward-open conformation by wedging inside the substrate-binding site and the extracellular vestibule of hSGLT1. LX2761 blocks the putative water permeation pathway of hSGLT1. The structure also uncovers the conformational changes of hSGLT1 during transitions from outward-open to inward-open states.
Background TNF-α elicits a cascade amplification effect in psoriasis. Macromolecule drugs targeting TNF-α are widely used for the clinical treatment of psoriasis. However, there are currently no effective small-molecule inhibitors that can be used in the clinic.
Objective Novel TNF-α inhibitor was identified via high-throughput screening (HTS) and its anti-inflammatory activity was evaluated.
Methods Two cell death models were established to identify inhibitors of TNF-α through HTS from a library of 3256 compounds. The effect of the inhibitor of TNF-α was tested by HaCaT cells in vitro and IMQ-induced psoriasis-like mouse model in vivo.
Results Tiamulin fumarate (TF) was identified as an effective inhibitor of TNF-α. TF significantly blocked the NF-κB and MAPK signaling pathways in TNF-α-stimulated HaCaT cells. Additionally, systemic and topical administration of TF improved IMQ-induced psoriasis-like dermatitis in the mouse model.
Conclusion Our study established a HTS method to identify TF as an inhibitor of TNF-α. The protective roles of TF in psoriasis-related inflammation reveal the potential therapeutic value of TF for psoriasis.
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Conformational Dynamics of the Activated GLP‑1 Receptor‑Gs Complex Revealed by Cross-Linking Mass Spectrometry and Integrative Structure Modeling
Conformational Dynamics of the Activated GLP‑1 Receptor‑Gs Complex Revealed by Cross-Linking Mass Spectrometry and Integrative Structure Modeling
Shijia Yuan, Lisha Xia, Chenxi Wang,Fan Wu, Bingjie Zhang, Chen Pan, Zhiran Fan, Xiaoguang Lei, Raymond C. Stevens, Andrej Sali, Liping Sun,* and Wenqing Shui* ACS Cent. Sci. 2023, 9, 992−1007
Despite advances in characterizing the structures and functions of G protein-coupled receptors (GPCRs), our understanding of GPCR activation and signaling is still limited by the lack of information on conformational dynamics. It is particularly challenging to study the dynamics of GPCR complexes with their signaling partners because of their transient nature and low stability. Here, by combining cross-linking mass spectrometry (CLMS) with integrative structure modeling, we map the conformational ensemble of an activated GPCR-G protein complex at near-atomic resolution. The integrative structures describe heterogeneous conformations for a high number of potential alternative active states of the GLP-1 receptor−Gs complex. These structures show marked differences from the previously determined cryo-EM structure, especially at the receptor−Gs interface and in the interior of the Gs heterotrimer. Alaninescanning mutagenesis coupled with pharmacological assays validates the functional significance of 24 interface residue contacts only observed in the integrative structures, yet absent in the cryo-EM structure. Through the integration of spatial connectivity data from CLMS with structure modeling, our study provides a new approach that is generalizable to characterizing the conformational dynamics of GPCR signaling complexes.
Diversity-oriented synthesis of cyclohexenes by combining enzymatic intermolecular DielsAlder reactions and decarboxylative functionalizations
Diversity-oriented synthesis of cyclohexenes by combining enzymatic intermolecular DielsAlder reactions and decarboxylative functionalizations，
Jin Wang, Han Ke, Jun Yang, Nianxin Guo, Kangdelong Hu, Ruyao Tang,Qi Ding, Lei Gao*and Xiaoguang Lei*
Chem Catalysis, 2023, 3(1), 100451
Substituted cyclohexanes are common scaffolds found in both natural products and drug molecules. Diels-Alderases that can efficiently catalyze intermolecular Diels-Alder reactions to generate cyclohexene ring systems have received considerable interest. However, the synthetic power of Diels-Alderases is incomparable with chemo-catalysts due to their limited substrate scopes. Here, we report a new chemo-enzymatic strategy for the diversity-oriented syntheses of functionalized cyclohexenes. We first applied focused rational iterative site-specific mutagenesis to generate a natural Diels-Alderase variant M3, which shows a 34-fold increase in catalytic efficiency, broad substrate scope, and good to perfect stereoselectivity. Then, we used diverse transition-metal-catalyzed decarboxylative coupling reactions to functionalize the enzymatic Diels-Alder products. This work offers an efficient synthetic route to structurally diverse cyclohexenes that are not accessible by solely using biocatalysis or chemo-catalysis and illustrates how chemo-catalysis can cooperate with biocatalysis to expand the synthetic application of biocatalysts.
C−H Glycosylation of Native Carboxylic Acids: Discovery of Antidiabetic SGLT‑2 Inhibitors
C−H Glycosylation of Native Carboxylic Acids: Discovery of Antidiabetic SGLT‑2 Inhibitors，
Sanshan Wang, Kaiqi Chen, Fusheng Guo, Wenneng Zhu, Chendi Liu, Haoran Dong, Jin-Quan Yu,*and Xiaoguang Lei*
https://doi.org/10.1021/acscentsci.3c00201
Publication Date:June 9, 2023
C-Glycosides are critical motifs embedded in many bioactive natural products. The inert C-glycosides are privileged structures for developing therapeutic agents owing to their high chemical and metabolic stability. Despite the comprehensive strategies and tactics established in the past few decades, highly efficient C-glycoside syntheses via C−C coupling with excellent regio-, chemo-, and stereoselectivity are still needed. Here, we report the efficient Pd-catalyzed glycosylation of C−H bonds promoted by weak coordination with native carboxylic acids without external directing groups to install various glycals to the structurally diverse aglycon parts. Mechanistic evidence points to the participation of a glycal radical donor in the C−H coupling reaction. The method has been applied to a wide range of substrates (over 60 examples), including many marketed drug molecules. Natural product- or drug-like scaffolds with compelling bioactivities have been constructed using a late-stage diversification strategy. Remarkably, a new potent sodium-glucose cotransporter-2 inhibitor with antidiabetic potential has been discovered, and the pharmacokinetic/ pharmacodynamic profiles of drug molecules have been changed using our C−H glycosylation approach. The method developed here provides a powerful tool for efficiently synthesizing C-glycosides to facilitate drug discovery.
Total Synthesis of Diverse Tetramic Acid Bearing cis-Decalin Natural Products
Total Synthesis of Diverse Tetramic Acid Bearing cis-Decalin Natural Products
Haoran Dong, Dachao Hu, Benke Hong, Jin Wang, and Xiaoguang Lei*
Angew. Chem. Int. Ed. 2023, e202301872
We here report the first total syntheses of four natural antibiotics vermisporin (1), PF1052/AB4015-A (2), AB4015-L (3), AB4015-B (4) and one hydrogenated natural product derivative AB4015-A2 (5) that all feature a tetramic acid bearing cis-decalin ring. The construction of the functionalized cis-decalin ring was achieved by a diastereoselective intramolecular Diels-Alder (IMDA) reaction which underwent a rare endo-boat transition state. Through an intramolecular neighboring-group-oriented strategy, the sterically hindered epoxy group in vermisporin (1), PF1052/AB4015-A (2) and AB4015-L (3) was installed efficiently. A one-pot aminolysis/Dieckmann-condensation cascade using L-amino acid derivatives afforded the desired tetramic acid structure. The total synthesis led to the unambiguous verification of the absolute configuration of these natural products
Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites
Xueping Wang, Victor Tomilin, Merve Ertem, Abigail McKernan, Xiaoguang Lei, Oleh Pochynyuk, Ossama B Kashlan*
J. Physiol. 2022, 600, 4695-4711.
Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel.
Testing the polar auxin transport model with a selective plasma membrane H + -ATPase inhibitor
Yongqing Yang*, Xiaohui Liu, Wei Guo, Wei Liu, Wei Shao, Jun Zhao, Junhong Li, Qing Dong, Liang Ma, Qun He, Yingzhang Li, Jianyong Han, Xiaoguang Lei*
J. Integr. Plant Biol. 2022, 64, 1229-1245
Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H+ gradient across the plasma membrane (PM) established by PM H+-adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H+-ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H+-ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H+-ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H+-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H+-ATPases. Our results support the chemiosmotic model in which PM H+-ATPase itself plays a fundamental role in polar auxin transport
Enzymatic intermolecular Diels-Alder reactions in synthesis: From nature to design
Lei Gao, Jun Yang, Xiaoguang Lei*
Tetrahedron Chem 2022, 2, 100013
The Diels-Alder (D-A) reaction is one of the most critical chemical transformations to construct C–C bonds with predictable regio- and stereo-selectivities. It has been widely used as a retrosynthetic disconnection in synthetic chemistry. Although significant advances have been made, synthetic challenges remain in how to precisely control the stereochemistry of intermolecular Diels-Alder reaction. Enzymes are well known for their remarkable catalytic efficiency and selectivity compared with chemo-catalysts. Therefore, identifying and designing intermolecular Diels-Alderases that can be used in organic synthesis have received considerable attention from the synthetic community. In this review, we review all the enzymes capable of catalyzing formal intermolecular Diels-Alder reactions in natural product biosynthetic pathways, discuss their catalytic mechanisms in detail, and highlight their synthetic potential in the precise and efficient synthesis of enantiopure D-A products. We also discuss the different strategies that can be used to create new artificial Diels-Alderases, especially RNA-based Diels-Alderases.
Perturbation of biological processes with small molecule kinase inhibitors
Wenyang Li, Jue Wang, Ruqi Liang, Xiaoguang Lei*
Current Opinion in Chemical Biology 2022, 70, 102185
The reversible phosphorylation of substrates mediated by kinases and phosphatases affects their subcellular localization, catalytic activity, and/or interaction with other molecules. It is essential for signal transduction and the regulation of nearly all cellular processes, such as proliferation, apoptosis, metabolism, motility, and differentiation. Small molecule kinase inhibitors (SMKIs) have served as critical chemical probes to reveal the biological functions and mechanisms of kinases and their potential as therapeutic targets. In this review, we focused on a few novel SMKIs and their recent application in biological and preclinical studies to showcase how highly selective and potent SMKIs can be developed and utilized to propel the investigations on kinases and the biology behind.
Structural mechanism of SGLT1 inhibitors
Yange Niu, Wenhao Cui, Rui Liu, Sanshan Wang, Han Ke, Xiaoguang Lei, Lei Chen*
Nature Commun. 2022, 13, 6440
Sodium glucose co-transporters (SGLT) harness the electrochemical gradient of sodium to drive the uphill transport of glucose across the plasma membrane. Human SGLT1 (hSGLT1) plays a key role in sugar uptake from food and its inhibitors show promise in the treatment of several diseases. However, the inhibition mechanism for hSGLT1 remains elusive. Here, we present the cryo-EM structure of the hSGLT1-MAP17 hetero-dimeric complex in the presence of the high-affinity inhibitor LX2761. LX2761 locks the transporter in an outward-open conformation by wedging inside the substrate-binding site and the extracellular vestibule of hSGLT1. LX2761 blocks the putative water permeation pathway of hSGLT1. The structure also uncovers the conformational changes of hSGLT1 during transitions from outward-open to inward-open states.
Tiamulin inhibits TNF-α and alleviates psoriasis-like dermatitis
Ruiyu Xiang, Linghan Hu, Siyuan Li, Ziyu Wei, Zhongya Song, Zhiming Chen, Yihe Liu, Juan Liu, Xiaoguang Lei, Yong Yang*
J. Dermatol. Sci. 2022, 107, 32-40
Background
TNF-α elicits a cascade amplification effect in psoriasis. Macromolecule drugs targeting TNF-α are widely used for the clinical treatment of psoriasis. However, there are currently no effective small-molecule inhibitors that can be used in the clinic.
Objective
Novel TNF-α inhibitor was identified via high-throughput screening (HTS) and its anti-inflammatory activity was evaluated.
Methods
Two cell death models were established to identify inhibitors of TNF-α through HTS from a library of 3256 compounds. The effect of the inhibitor of TNF-α was tested by HaCaT cells in vitro and IMQ-induced psoriasis-like mouse model in vivo.
Results
Tiamulin fumarate (TF) was identified as an effective inhibitor of TNF-α. TF significantly blocked the NF-κB and MAPK signaling pathways in TNF-α-stimulated HaCaT cells. Additionally, systemic and topical administration of TF improved IMQ-induced psoriasis-like dermatitis in the mouse model.
Conclusion
Our study established a HTS method to identify TF as an inhibitor of TNF-α. The protective roles of TF in psoriasis-related inflammation reveal the potential therapeutic value of TF for psoriasis.