Bioorganic & Medicinal Chemistry，Volume 110, August 2024, 117793
The pathogenic role of anti-phospholipase A2 receptor (PLA2R) antibodies in primary membranous nephropathy (MN) has been well-established. This study aimed to identify potential small-molecule inhibitors against the PLA2R-antibody interaction, offering potential therapeutic benefits. A comprehensive screening of over 4000 small-molecule compounds was conducted by ELISA to assess their inhibitory effects on the binding between the immobilized full-length extracellular PLA2R and its antibodies. The affinity of anti-PLA2R IgG from MN patients and the inhibitory efficacy of each compound were evaluated via surface plasmon resonance (SPR). Human podocyte injuries were analyzed using CCK-8 assay, wound healing assay, western blot analysis, and immunofluorescence, after exposure to MN plasma +/- blocking compound. Fifteen compounds were identified as potential inhibitors, demonstrating inhibition rates >20 % for the PLA2R-antibody interaction. Anti-PLA2R IgG exhibited a consistent affinity among patients (KD = 10−8 M). Macrocarpal B emerged as the most potent inhibitor, reducing the antigen–antibody interaction by nearly 30 % in a dose-dependent manner, comparable to the performance of the 31-mer peptide from the CysR domain. Macrocarpal B bound to the immobilized PLA2R with an affinity of 1.47 × 10−6 M, while showing no binding to anti-PLA2R IgG. Human podocytes exposed to MN plasma showed decreased podocin expression, impaired migration function, and reduced cell viability. Macrocarpal B inhibited the binding of anti-PLA2R IgG to podocytes and reduced the cellular injuries.
New adjuvants that trigger cellular immune responses are urgently needed for the effective development of cancer and virus vaccines. Motivated by recent discoveries that show activation of type I interferon (IFN-I) signaling boosts T cell immunity, this study proposes that targeting this pathway can be a strategic approach to identify novel vaccine adjuvants. Consequently, a comprehensive chemical screening of 6,800 small molecules is performed, which results in the discovery of the natural compound picrasidine S (PS) as an IFN-I inducer. Further analysis reveals that PS acts as a powerful adjuvant, significantly enhancing both humoral and cellular immune responses. At the molecular level, PS initiates the activation of the cGAS-IFN-I pathway, leading to an enhanced T cell response. PS vaccination notably increases the population of CD8+ central memory (TCM)-like cells and boosts the CD8+ T cell-mediated anti-tumor immune response. Thus, this study identifies PS as a promising candidate for developing vaccine adjuvants in cancer prevention.
Haowen Zhang, Shuhan Xie, Jun Yang, Ning Ye, Feng Gao, Fabrice Gallou, Lei Gao, Xiaoguang Lei
Angewandte Chemie International Edition Pub Date : 2024-05-15 , DOI: 10.1002/anie.202405833
Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.
Qi Ding, Nianxin Guo, Lei Gao, Michelle McKee, Dongshan Wu, Jun Yang, Junping Fan, Jing-Ke Weng & Xiaoguang Lei Nature Communications ，volume 15, Article number: 2492 (2024) Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.
Bin Jiang†, Lei Gao†, Haijun Wang†, Yaping Sun†, Xiaolin Zhang, Han Ke, Shengchao Liu,Pengchen Ma, Qinggang Liao, Yue Wang, Huan Wang, Yugeng Liu, Ran Du, Torben Rogge, Wei Li, Yi Shang, K. N. Houk, Xingyao Xiong, Daoxin Xie, Sanwen Huang, Xiaoguang Lei*, Jianbin Yan* Science, 2024, 383(6681), 622-629. Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.
Jun Yang, Kai Liang, Han Ke, Yuebin Zhang, Qian Meng, Lei Gao, Junping Fan, Guohui Li, Hu Zhou, Junyu Xiao,* and Xiaoguang Lei* JACS Au. 2024. In the era of global climate change, the increasingly severe Fusarium head blight (FHB) and deoxynivalenol (DON) contamination have caused economic losses and brought food and feed safety concerns. Recently, an FHB resistance gene Fhb7 coding a glutathione-S transferase (GST) to degrade DON by opening the critical toxic epoxide moiety was identified and opened a new window for wheat breeding and DON detoxification. However, the poor stability of Fhb7 and the elusiveness of the catalytic mechanism hinder its practical application. Herein, we report the first structure of Fhb7 at 2.41 Å and reveal a unique catalytic mechanism of epoxide opening transformation in GST family proteins. Furthermore, variants V29P and M10 showed that 5.5-fold and 266.7-fold longer half-life time than wild-type, respectively, were identified. These variants offer broad substrate scope, and the engineered biosafe Bacillus subtilis overexpressing the variants shows excellent DON degradation performance, exhibiting potential at bacterium engineering to achieve DON detoxification in the feed and biomedicine industry. This work provides a profound mechanistic insight into the enzymatic activities of Fhb7 and paves the way for further utilizing Fhb7-related enzymes in crop breeding and DON detoxification by synthetic biology.
Fan Xiao, Mengze Sun, Liyun Zhang, and Xiaoguang Lei* he Journal of Organic Chemistry. 2023. ortho-Phthalaldehyde (OPA) with high reactivity to the amine group has been widely used to modify proteins. We discovered new modifications of OPA and 2-acylbenzaldehyde and proposed the reaction mechanism. Using isotope labeling mass spectrometry-based experiment, we identified new cross-linking properties of OPA and 2-acylbenzaldehyde. This reactivity revealed that OPA has the potential to probe proximal amino acids in biological systems.
Yihui Gao, Mingzhe Ma, Wenyang Li,* and Xiaoguang Lei* Advanced Science, 2023, 2305608. As a vital project of forward chemical genetic research, target deconvolution aims to identify the molecular targets of an active hit compound. Chemoproteomics, either with chemical probe-facilitated target enrichment or probe-free, provides a straightforward and effective approach to profile the target landscape and unravel the mechanisms of action. Canonical methods rely on chemical probes to enable target engagement, enrichment, and identification, whereas click chemistry and photoaffinity labeling techniques improve the efficiency, sensitivity, and spatial accuracy of target recognition. In comparison, recently developed probe-free methods detect protein-ligand interactions without the need to modify the ligand molecule. This review provides a comprehensive overview of different approaches and recent advancements for target identification and highlights the significance of chemoproteomics in investigating biological processes and advancing drug discovery processes.
Qixuan Wang, Fusheng Guo, Jin Wang，Xiaoguang Lei Chemical Science, 2023, 14(37), 10353-10359. We herein report the first total syntheses of several bis-b-carboline alkaloids, picrasidines G, S, R, and T, and natural product-like derivatives in a divergent manner. Picrasidines G, S, and T feature an indolotetrahydroquinolizinium (ITHQ) skeleton, while picrasidine R possesses a 1,4-diketone linker between two b-carboline fragments. The synthesis of ITHQ-type bis-b-carboline alkaloids could be directly achieved by a late-stage regio-selective aza-[4 + 2] cycloaddition of vinyl b-carboline alkaloids, suggesting that this remarkable aza-[4 + 2] cycloaddition might be involved in the biosynthetic pathway. Computational studies revealed that such aza-[4 + 2] cycloaddition is a stepwise process and explained the unique regioselectivity (DDG = 3.77 kcal mol−1). Moreover, the successful application of iridiumcatalyzed C–H borylation on b-carboline substrates enabled the site-selective C-8 functionalization for efficient synthesis and structural diversification of this family of natural products. Finally, concise synthesis of picrasidine R by the thiazolium-catalyzed Stetter reaction was also accomplished.
Kai Wang, Zhiwei Zhang, Jing Hang, Jia Liu, Fusheng Guo, Yong Ding, Meng Li, Qixing Nie, Jun Lin, Yingying Zhuo, Lulu Sun, Xi Luo, Qihang Zhong, Chuan Ye, Chuyu Yun, Yi Zhang, Jue Wang, Rui Bao, Yanli Pang, Guang Wang, Frank J. Gonzalez, Xiaoguang Lei, Jie Qiao, and Changtao Jiang. Science. 2023, 381, eadd5787.
A mechanistic understanding of how microbial proteins affect the host could yield deeper insights into gut microbiota–host cross-talk. We developed an enzyme activity–screening platform to investigate how gut microbiota–derived enzymes might influence host physiology. We discovered that dipeptidyl peptidase 4 (DPP4) is expressed by specific bacterial taxa of the microbiota. Microbial DPP4 was able to decrease the active glucagon like peptide-1 (GLP-1) and disrupt glucose metabolism in mice with a leaky gut. Furthermore, the current drugs targeting human DPP4, including sitagliptin, had little effect on microbial DPP4. Using high-throughput screening, we identified daurisoline-d4 (Dau-d4) as a selective microbial DPP4 inhibitor that improves glucose tolerance in diabetic mice.
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Macrocarpal B blocks the binding between the phospholipase A2 receptor and its antibodies
Zixin Feng, Fu-sheng Guo, Qian Wang, Miao Wang, Ming-Hui Zhao, Zhao Cui, Xiaoguang Lei
Bioorganic & Medicinal Chemistry，Volume 110, August 2024, 117793
The pathogenic role of anti-phospholipase A2 receptor (PLA2R) antibodies in primary membranous nephropathy (MN) has been well-established. This study aimed to identify potential small-molecule inhibitors against the PLA2R-antibody interaction, offering potential therapeutic benefits. A comprehensive screening of over 4000 small-molecule compounds was conducted by ELISA to assess their inhibitory effects on the binding between the immobilized full-length extracellular PLA2R and its antibodies. The affinity of anti-PLA2R IgG from MN patients and the inhibitory efficacy of each compound were evaluated via surface plasmon resonance (SPR). Human podocyte injuries were analyzed using CCK-8 assay, wound healing assay, western blot analysis, and immunofluorescence, after exposure to MN plasma +/- blocking compound. Fifteen compounds were identified as potential inhibitors, demonstrating inhibition rates >20 % for the PLA2R-antibody interaction. Anti-PLA2R IgG exhibited a consistent affinity among patients (KD = 10−8 M). Macrocarpal B emerged as the most potent inhibitor, reducing the antigen–antibody interaction by nearly 30 % in a dose-dependent manner, comparable to the performance of the 31-mer peptide from the CysR domain. Macrocarpal B bound to the immobilized PLA2R with an affinity of 1.47 × 10−6 M, while showing no binding to anti-PLA2R IgG. Human podocytes exposed to MN plasma showed decreased podocin expression, impaired migration function, and reduced cell viability. Macrocarpal B inhibited the binding of anti-PLA2R IgG to podocytes and reduced the cellular injuries.
Picrasidine S Induces cGAS-Mediated Cellular Immune Response as a Novel Vaccine Adjuvant
Xiaofan Ding, Mengxue Sun, Fusheng Guo, Xinmin Qian, Haoyu Yuan, Wenjiao Lou, Qixuan Wang, Xiaoguang Lei, Wenwen Zeng
Advanced science (Weinheim, Baden-Wurttemberg, Germany), e2310108. Advance online publication. https://doi.org/10.1002/advs.202310108
New adjuvants that trigger cellular immune responses are urgently needed for the effective development of cancer and virus vaccines. Motivated by recent discoveries that show activation of type I interferon (IFN-I) signaling boosts T cell immunity, this study proposes that targeting this pathway can be a strategic approach to identify novel vaccine adjuvants. Consequently, a comprehensive chemical screening of 6,800 small molecules is performed, which results in the discovery of the natural compound picrasidine S (PS) as an IFN-I inducer. Further analysis reveals that PS acts as a powerful adjuvant, significantly enhancing both humoral and cellular immune responses. At the molecular level, PS initiates the activation of the cGAS-IFN-I pathway, leading to an enhanced T cell response. PS vaccination notably increases the population of CD8+ central memory (TCM)-like cells and boosts the CD8+ T cell-mediated anti-tumor immune response. Thus, this study identifies PS as a promising candidate for developing vaccine adjuvants in cancer prevention.
Chemoenzymatic Synthesis of 2-Aryl Thiazolines from 4- Hydroxybenzaldehydes Using Vanillyl Alcohol Oxidases
Haowen Zhang, Shuhan Xie, Jun Yang, Ning Ye, Feng Gao, Fabrice Gallou, Lei Gao, Xiaoguang Lei
Angewandte Chemie International Edition Pub Date : 2024-05-15 , DOI: 10.1002/anie.202405833
Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.
The evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba
Qi Ding, Nianxin Guo, Lei Gao, Michelle McKee, Dongshan Wu, Jun Yang, Junping Fan, Jing-Ke Weng & Xiaoguang Lei
Nature Communications ，volume 15, Article number: 2492 (2024)
Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.
Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III
Bin Jiang†, Lei Gao†, Haijun Wang†, Yaping Sun†, Xiaolin Zhang, Han Ke, Shengchao Liu,Pengchen Ma, Qinggang Liao, Yue Wang, Huan Wang, Yugeng Liu, Ran Du, Torben Rogge, Wei Li, Yi Shang, K. N. Houk, Xingyao Xiong, Daoxin Xie, Sanwen Huang, Xiaoguang Lei*, Jianbin Yan*
Science, 2024, 383(6681), 622-629.
Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.
Enzymatic Degradation of Deoxynivalenol with the Engineered Detoxification Enzyme Fhb7
Jun Yang, Kai Liang, Han Ke, Yuebin Zhang, Qian Meng, Lei Gao, Junping Fan, Guohui Li, Hu Zhou, Junyu Xiao,* and Xiaoguang Lei*
JACS Au. 2024.
In the era of global climate change, the increasingly severe Fusarium head blight (FHB) and deoxynivalenol (DON) contamination have caused economic losses and brought food and feed safety concerns. Recently, an FHB resistance gene Fhb7 coding a glutathione-S transferase (GST) to degrade DON by opening the critical toxic epoxide moiety was identified and opened a new window for wheat breeding and DON detoxification. However, the poor stability of Fhb7 and the elusiveness of the catalytic mechanism hinder its practical application. Herein, we report the first structure of Fhb7 at 2.41 Å and reveal a unique catalytic mechanism of epoxide opening transformation in GST family proteins. Furthermore, variants V29P and M10 showed that 5.5-fold and 266.7-fold longer half-life time than wild-type, respectively, were identified. These variants offer broad substrate scope, and the engineered biosafe Bacillus subtilis overexpressing the variants shows excellent DON degradation performance, exhibiting potential at bacterium engineering to achieve DON detoxification in the feed and biomedicine industry. This work provides a profound mechanistic insight into the enzymatic activities of Fhb7 and paves the way for further utilizing Fhb7-related enzymes in crop breeding and DON detoxification by synthetic biology.
Investigation of Peptide Labeling with ortho-Phthalaldehyde and 2‑Acylbenzaldehyde
Fan Xiao, Mengze Sun, Liyun Zhang, and Xiaoguang Lei*
he Journal of Organic Chemistry. 2023.
ortho-Phthalaldehyde (OPA) with high reactivity to the amine group has been widely used to modify proteins. We discovered new modifications of OPA and 2-acylbenzaldehyde and proposed the reaction mechanism. Using isotope labeling mass spectrometry-based experiment, we identified new cross-linking properties of OPA and 2-acylbenzaldehyde. This reactivity revealed that OPA has the potential to probe proximal amino acids in biological systems.
Chemoproteomics, a broad avenue to target deconvolution
Yihui Gao, Mingzhe Ma, Wenyang Li,* and Xiaoguang Lei*
Advanced Science, 2023, 2305608.
As a vital project of forward chemical genetic research, target deconvolution aims to identify the molecular targets of an active hit compound. Chemoproteomics, either with chemical probe-facilitated target enrichment or probe-free, provides a straightforward and effective approach to profile the target landscape and unravel the mechanisms of action. Canonical methods rely on chemical probes to enable target engagement, enrichment, and identification, whereas click chemistry and photoaffinity labeling techniques improve the efficiency, sensitivity, and spatial accuracy of target recognition. In comparison, recently developed probe-free methods detect protein-ligand interactions without the need to modify the ligand molecule. This review provides a comprehensive overview of different approaches and recent advancements for target identification and highlights the significance of chemoproteomics in investigating biological processes and advancing drug discovery processes.
Divergent total syntheses of ITHQ-type bis-bcarboline alkaloids by regio-selective formal aza-[4 + 2] cycloaddition and late-stage C–H functionalization
Qixuan Wang, Fusheng Guo, Jin Wang，Xiaoguang Lei
Chemical Science, 2023, 14(37), 10353-10359.
We herein report the first total syntheses of several bis-b-carboline alkaloids, picrasidines G, S, R, and T, and
natural product-like derivatives in a divergent manner. Picrasidines G, S, and T feature an indolotetrahydroquinolizinium (ITHQ) skeleton, while picrasidine R possesses a 1,4-diketone linker
between two b-carboline fragments. The synthesis of ITHQ-type bis-b-carboline alkaloids could be
directly achieved by a late-stage regio-selective aza-[4 + 2] cycloaddition of vinyl b-carboline alkaloids,
suggesting that this remarkable aza-[4 + 2] cycloaddition might be involved in the biosynthetic pathway.
Computational studies revealed that such aza-[4 + 2] cycloaddition is a stepwise process and explained
the unique regioselectivity (DDG = 3.77 kcal mol−1). Moreover, the successful application of iridiumcatalyzed C–H borylation on b-carboline substrates enabled the site-selective C-8 functionalization for
efficient synthesis and structural diversification of this family of natural products. Finally, concise synthesis of picrasidine R by the thiazolium-catalyzed Stetter reaction was also accomplished.
Microbial-host-isozyme analyses reveal microbial DPP4 as a potential antidiabetic target
Kai Wang, Zhiwei Zhang, Jing Hang, Jia Liu, Fusheng Guo, Yong Ding, Meng Li, Qixing Nie, Jun Lin, Yingying Zhuo, Lulu Sun, Xi Luo, Qihang Zhong, Chuan Ye, Chuyu Yun, Yi Zhang, Jue Wang, Rui Bao, Yanli Pang, Guang Wang, Frank J. Gonzalez, Xiaoguang Lei, Jie Qiao, and Changtao Jiang. Science. 2023, 381, eadd5787.
A mechanistic understanding of how microbial proteins affect the host could yield deeper insights into gut microbiota–host cross-talk. We developed an enzyme activity–screening platform to investigate how gut microbiota–derived enzymes might influence host physiology. We discovered that dipeptidyl peptidase 4 (DPP4) is expressed by specific bacterial taxa of the microbiota. Microbial DPP4 was able to decrease the active glucagon like peptide-1 (GLP-1) and disrupt glucose metabolism in mice with a leaky gut. Furthermore, the current drugs targeting human DPP4, including sitagliptin, had little effect on microbial DPP4. Using high-throughput screening, we identified daurisoline-d4 (Dau-d4) as a selective microbial DPP4 inhibitor that improves glucose tolerance in diabetic mice.