While they account for a large portion of drug targets, membrane proteins present a unique challenge for drug discovery. Peripheral membrane proteins (PMPs), a class of water-soluble proteins that bind to membranes, are also difficult targets, particularly those that function only when bound to membranes. The protein–membrane interface in PMPs is often where functional interactions and catalysis occur, making it a logical target for inhibition. However, protein–membrane interfaces are underexplored spaces in inhibitor design, and there is a need for enhanced methods for small-molecule ligand discovery. In an effort to better initiate drug discovery efforts for PMPs, this study presents a screening methodology using membrane-mimicking reverse micelles (mmRM) and NMR-based fragment screening to assess ligandability at the protein–membrane interface. The proof-of-principle target, glutathione peroxidase 4 (GPx4), is a lipid hydroperoxidase that is essential for the oxidative protection of membranes and thereby the prevention of ferroptosis. GPx4 inhibition is promising for therapy-resistant cancer therapy, but current inhibitors are generally covalent ligands with limited clinical utility. Presented here is the discovery of noncovalent small-molecule ligands for membrane-bound GPx4 revealed through the mmRM fragment screening methodology. The fragments were tested against GPx4 under bulk aqueous conditions and displayed little to no binding to the protein without embedment into the membrane. The 9 hits had varying affinities and partitioning coefficients and revealed properties of fragments that bind within the protein–membrane interface. Additionally, a secondary screen confirmed the potential to progress the fragments by enhancing the affinity from >200 to ∼15 μM with the addition of certain hydrophobic groups. This study presents an advancement of screening capabilities for membrane-associated proteins, reveals ligandability within the GPx4 protein–membrane interface, and may serve as a starting point for developing noncovalent inhibitors of GPx4.
{"title":"Ligandability at the Membrane Interface of GPx4 Revealed through a Reverse Micelle Fragment Screening Platform","authors":"Courtney L. Labrecque, Brian Fuglestad","doi":"10.1021/jacsau.4c00427","DOIUrl":"https://doi.org/10.1021/jacsau.4c00427","url":null,"abstract":"While they account for a large portion of drug targets, membrane proteins present a unique challenge for drug discovery. Peripheral membrane proteins (PMPs), a class of water-soluble proteins that bind to membranes, are also difficult targets, particularly those that function only when bound to membranes. The protein–membrane interface in PMPs is often where functional interactions and catalysis occur, making it a logical target for inhibition. However, protein–membrane interfaces are underexplored spaces in inhibitor design, and there is a need for enhanced methods for small-molecule ligand discovery. In an effort to better initiate drug discovery efforts for PMPs, this study presents a screening methodology using membrane-mimicking reverse micelles (mmRM) and NMR-based fragment screening to assess ligandability at the protein–membrane interface. The proof-of-principle target, glutathione peroxidase 4 (GPx4), is a lipid hydroperoxidase that is essential for the oxidative protection of membranes and thereby the prevention of ferroptosis. GPx4 inhibition is promising for therapy-resistant cancer therapy, but current inhibitors are generally covalent ligands with limited clinical utility. Presented here is the discovery of noncovalent small-molecule ligands for membrane-bound GPx4 revealed through the mmRM fragment screening methodology. The fragments were tested against GPx4 under bulk aqueous conditions and displayed little to no binding to the protein without embedment into the membrane. The 9 hits had varying affinities and partitioning coefficients and revealed properties of fragments that bind within the protein–membrane interface. Additionally, a secondary screen confirmed the potential to progress the fragments by enhancing the affinity from >200 to ∼15 μM with the addition of certain hydrophobic groups. This study presents an advancement of screening capabilities for membrane-associated proteins, reveals ligandability within the GPx4 protein–membrane interface, and may serve as a starting point for developing noncovalent inhibitors of GPx4.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xueqian Chen, Jiatian Liu, Yong Zhang, Xueyun Gao, Dongdong Su
The precise localization of metastatic tumors with subtle growth is crucial for timely intervention and improvement of tumor prognosis but remains a paramount challenging. To date, site-specific activation of fluorogenic probes for single-stimulus-based diagnosis typically targets an occult molecular event in a complex biosystem with limited specificity. Herein, we propose a highly specific site-specific cascade-activated strategy to enhance detection accuracy, aiming to achieve the accurate detection of breast cancer (BC) lung metastasis in a cascade manner. Specifically, cascade-activatable NIR fluorogenic nanomicelles HPNs were constructed using ultra-pH-sensitive (UPS) block copolymers as carriers and nitroreductase (NTR)-activated fluorogenic reporters. HPNs exhibit programmable cascade response characteristics by first instantaneous dissociating under in situ tumor acidity, facilitating deep tumor penetration followed by selective fluorescence activation through NTR-mediated enzymatic reaction resulting in high fluorescence ON/OFF contrast. Notably, this unique feature of HPNs enables high-precision diagnosis of orthotopic BC as well as its lung metastases with a remarkable signal-to-background ratio (SBR). This proposed site-specific cascade activation strategy will offer opportunities for a specific diagnosis with high signal fidelity of various insidious metastatic lesions in situ across different diseases.
{"title":"Site-Specific Cascade-Activatable Fluorogenic Nanomicelles Enable Precision and Accuracy Imaging of Pulmonary Metastatic Tumor","authors":"Xueqian Chen, Jiatian Liu, Yong Zhang, Xueyun Gao, Dongdong Su","doi":"10.1021/jacsau.4c00356","DOIUrl":"https://doi.org/10.1021/jacsau.4c00356","url":null,"abstract":"The precise localization of metastatic tumors with subtle growth is crucial for timely intervention and improvement of tumor prognosis but remains a paramount challenging. To date, site-specific activation of fluorogenic probes for single-stimulus-based diagnosis typically targets an occult molecular event in a complex biosystem with limited specificity. Herein, we propose a highly specific site-specific cascade-activated strategy to enhance detection accuracy, aiming to achieve the accurate detection of breast cancer (BC) lung metastasis in a cascade manner. Specifically, cascade-activatable NIR fluorogenic nanomicelles <b>HPNs</b> were constructed using ultra-pH-sensitive (UPS) block copolymers as carriers and nitroreductase (NTR)-activated fluorogenic reporters. <b>HPNs</b> exhibit programmable cascade response characteristics by first instantaneous dissociating under <i>in situ</i> tumor acidity, facilitating deep tumor penetration followed by selective fluorescence activation through NTR-mediated enzymatic reaction resulting in high fluorescence ON/OFF contrast. Notably, this unique feature of <b>HPNs</b> enables high-precision diagnosis of orthotopic BC as well as its lung metastases with a remarkable signal-to-background ratio (SBR). This proposed site-specific cascade activation strategy will offer opportunities for a specific diagnosis with high signal fidelity of various insidious metastatic lesions <i>in situ</i> across different diseases.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"176 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Soraya Learte-Aymamí, Laura Martínez-Castro, Carmen González-González, Miriam Condeminas, Pau Martin-Malpartida, María Tomás-Gamasa, Sandra Baúlde, José R. Couceiro, Jean-Didier Maréchal, Maria J. Macias, José L. Mascareñas, M. Eugenio Vázquez
The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered β-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain β-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.
开发能促进活细胞内生物正交反应的过渡金属催化平台仍然是化学生物学领域的一大挑战。钯基催化剂尤其如此，它们在有机合成中非常强大，但在细胞环境中却表现不佳，主要原因是它们会迅速失活。我们现在证明，将 Pd(II) 复合物接枝到模型 WW 结构域的工程化 β 片上，就能产生与细胞兼容的钯多聚蛋白，在 HeLa 细胞内有效催化去丙炔化反应。事实证明，WW结构域β片的凹面形状特别适合容纳金属中心，并保护其在细胞环境中不会迅速失活。通过全面的核磁共振和计算研究，我们证实了金属叠肽的形成，并提出了这种新型金属蛋白图案的三维结构。
{"title":"De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts","authors":"Soraya Learte-Aymamí, Laura Martínez-Castro, Carmen González-González, Miriam Condeminas, Pau Martin-Malpartida, María Tomás-Gamasa, Sandra Baúlde, José R. Couceiro, Jean-Didier Maréchal, Maria J. Macias, José L. Mascareñas, M. Eugenio Vázquez","doi":"10.1021/jacsau.4c00379","DOIUrl":"https://doi.org/10.1021/jacsau.4c00379","url":null,"abstract":"The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered β-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain β-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CO2 electroreduction (CO2RR) to generate valuable chemicals in acidic electrolytes can improve the carbon utilization rate in comparison to that under alkaline conditions. However, the thermodynamically more favorable hydrogen evolution reaction under an acidic electrolyte makes the CO2RR a big challenge. Herein, robust metal phthalocyanine(Pc)-based (M = Ni, Co) conductive metal-covalent organic frameworks (MCOFs) connected by strong metal tetraaza[14]annulene (TAA) linkage, named NiPc–NiTAA and NiPc–CoTAA, are designed and synthesized to apply in the CO2RR in acidic electrolytes for the first time. The optimal NiPc–NiTAA exhibited an excellent Faradaic efficiency (FECO) of 95.1% and a CO partial current density of 143.0 mA cm–2 at −1.5 V versus the reversible hydrogen electrode in an acidic electrolyte, which is 3.1 times that of the corresponding metal–organic framework NiPc–NiN4. The comparison tests and theoretical calculations reveal that in-plane full π–d conjugation MCOF with a good conductivity of 3.01 × 10–4 S m–1 accelerates migration of the electrons. The NiTAA linkage can tune the electron distribution in the d orbit of metal centers, making the d-band center close to the Fermi level and then activating CO2. Thus, the active sites of NiPc and NiTAA collaborate to reduce the *COOH formation energy barrier, favoring CO production in an acid electrolyte. It is a helpful route for designing outstanding conductive MCOF materials to enhance CO2 electrocatalysis under an acidic electrolyte.
与碱性条件下的碳利用率相比，在酸性电解质中利用二氧化碳电还原（CO2RR）生成有价值的化学物质可以提高碳利用率。然而，酸性电解质下热力学上更有利的氢进化反应使得 CO2RR 成为一大挑战。本文设计并合成了基于强金属四氮杂[14]环烯（TAA）连接的强金属酞菁（Pc）（M = Ni、Co）导电金属共价有机框架（MCOFs），命名为 NiPc-NiTAA 和 NiPc-CoTAA，首次应用于酸性电解质中的 CO2RR。最佳的 NiPc-NiTAA 在酸性电解质中与可逆氢电极相比，法拉第效率（FECO）为 95.1%，二氧化碳部分电流密度为 143.0 mA cm-2（-1.5 V），是相应金属有机框架 NiPc-NiN4 的 3.1 倍。对比试验和理论计算显示，面内全 π-d 共轭 MCOF 具有 3.01 × 10-4 S m-1 的良好导电性，可加速电子的迁移。NiTAA 连接可以调整金属中心 d 轨道上的电子分布，使 d 带中心接近费米级，进而激活 CO2。因此，NiPc 和 NiTAA 的活性位点共同降低了 *COOH 形成的能障，有利于在酸性电解质中生成 CO。这为设计优异的导电 MCOF 材料以增强酸性电解质下的 CO2 电催化提供了一条有益的途径。
{"title":"Electrocatalytic Reduction of Carbon Dioxide in Acidic Electrolyte with Superior Performance of a Metal–Covalent Organic Framework over Metal–Organic Framework","authors":"Chang-Pu Wan, Hui Guo, Duan-Hui Si, Shui-Ying Gao, Rong Cao, Yuan-Biao Huang","doi":"10.1021/jacsau.4c00246","DOIUrl":"https://doi.org/10.1021/jacsau.4c00246","url":null,"abstract":"CO<sub>2</sub> electroreduction (CO<sub>2</sub>RR) to generate valuable chemicals in acidic electrolytes can improve the carbon utilization rate in comparison to that under alkaline conditions. However, the thermodynamically more favorable hydrogen evolution reaction under an acidic electrolyte makes the CO<sub>2</sub>RR a big challenge. Herein, robust metal phthalocyanine(Pc)-based (M = Ni, Co) conductive metal-covalent organic frameworks (MCOFs) connected by strong metal tetraaza[14]annulene (TAA) linkage, named NiPc–NiTAA and NiPc–CoTAA, are designed and synthesized to apply in the CO<sub>2</sub>RR in acidic electrolytes for the first time. The optimal NiPc–NiTAA exhibited an excellent Faradaic efficiency (FE<sub>CO</sub>) of 95.1% and a CO partial current density of 143.0 mA cm<sup>–2</sup> at −1.5 V versus the reversible hydrogen electrode in an acidic electrolyte, which is 3.1 times that of the corresponding metal–organic framework NiPc–NiN<sub>4</sub>. The comparison tests and theoretical calculations reveal that in-plane full π–d conjugation MCOF with a good conductivity of 3.01 × 10<sup>–4</sup> S m<sup>–1</sup> accelerates migration of the electrons. The NiTAA linkage can tune the electron distribution in the d orbit of metal centers, making the d-band center close to the Fermi level and then activating CO<sub>2</sub>. Thus, the active sites of NiPc and NiTAA collaborate to reduce the *COOH formation energy barrier, favoring CO production in an acid electrolyte. It is a helpful route for designing outstanding conductive MCOF materials to enhance CO<sub>2</sub> electrocatalysis under an acidic electrolyte.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"53 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Soochan Lee, Sungmin Lee, Yuna Kwak, Masood Yousaf, Eunchan Cho, Hoi Ri Moon, Sung June Cho, Noejung Park, Wonyoung Choe
A new topology previously unknown in metal–organic frameworks (MOFs) provides an important clue to uncovering a new series of polyhedral MOFs. We report a novel MOF crystallized in a parsimonious mep topology based on Frank–Kasper (FK) polyhedra. The distribution of angles in a tetrahedral arrangement (T-O-T) is crucial for the formation of FK polyhedra in mep topology. This finding led us to investigate the T-O-T angle distribution in related zeolites and zeolitic imidazolate frameworks (ZIFs). Unlike zeolites, it is extremely difficult to achieve high T-O-T angles in ZIFs, which prevents the formation of some FK topologies. Density functional theory (DFT) total energy calculations support a correlation between T-O-T angles and the feasibility of new tetrahedron-based FK frameworks. This result may lead to innovative ways of accessing new cellular topologies by simple chemical tweaking of T-O-T angles.
金属有机框架（MOFs）中以前未知的一种新拓扑结构为发现一系列新的多面体 MOFs 提供了重要线索。我们报告了一种基于弗兰克-卡斯帕（Frank-Kasper，FK）多面体的简约 mep 拓扑结晶出的新型 MOF。四面体排列（T-O-T）中的角度分布对于在 mep 拓扑中形成 FK 多面体至关重要。这一发现促使我们研究了相关沸石和沸石咪唑啉框架（ZIFs）中的 T-O-T 角分布。与沸石不同，ZIFs 中的 T-O-T 角很难达到很高，这阻碍了一些 FK 拓扑的形成。密度泛函理论（DFT）总能量计算支持 T-O-T 角与基于四面体的新型 FK 框架的可行性之间的相关性。这一结果可能会带来创新的方法，通过对 T-O-T 角进行简单的化学调整就能获得新的细胞拓扑结构。
{"title":"Parsimonious Topology Based on Frank-Kasper Polyhedra in Metal–Organic Frameworks","authors":"Soochan Lee, Sungmin Lee, Yuna Kwak, Masood Yousaf, Eunchan Cho, Hoi Ri Moon, Sung June Cho, Noejung Park, Wonyoung Choe","doi":"10.1021/jacsau.4c00285","DOIUrl":"https://doi.org/10.1021/jacsau.4c00285","url":null,"abstract":"A new topology previously unknown in metal–organic frameworks (MOFs) provides an important clue to uncovering a new series of polyhedral MOFs. We report a novel MOF crystallized in a parsimonious <b>mep</b> topology based on Frank–Kasper (FK) polyhedra. The distribution of angles in a tetrahedral arrangement (T-O-T) is crucial for the formation of FK polyhedra in <b>mep</b> topology. This finding led us to investigate the T-O-T angle distribution in related zeolites and zeolitic imidazolate frameworks (ZIFs). Unlike zeolites, it is extremely difficult to achieve high T-O-T angles in ZIFs, which prevents the formation of some FK topologies. Density functional theory (DFT) total energy calculations support a correlation between T-O-T angles and the feasibility of new tetrahedron-based FK frameworks. This result may lead to innovative ways of accessing new cellular topologies by simple chemical tweaking of T-O-T angles.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Saer Samanipour, Leon Patrick Barron, Denice van Herwerden, Antonia Praetorius, Kevin V. Thomas, Jake William O’Brien
Around two-thirds of chronic human disease can not be explained by genetics alone. The Lancet Commission on Pollution and Health estimates that 16% of global premature deaths are linked to pollution. Additionally, it is now thought that humankind has surpassed the safe planetary operating space for introducing human-made chemicals into the Earth System. Direct and indirect exposure to a myriad of chemicals, known and unknown, poses a significant threat to biodiversity and human health, from vaccine efficacy to the rise of antimicrobial resistance as well as autoimmune diseases and mental health disorders. The exposome chemical space remains largely uncharted due to the sheer number of possible chemical structures, estimated at over 1060 unique forms. Conventional methods have cataloged only a fraction of the exposome, overlooking transformation products and often yielding uncertain results. In this Perspective, we have reviewed the latest efforts in mapping the exposome chemical space and its subspaces. We also provide our view on how the integration of data-driven approaches might be able to bridge the identified gaps.
{"title":"Exploring the Chemical Space of the Exposome: How Far Have We Gone?","authors":"Saer Samanipour, Leon Patrick Barron, Denice van Herwerden, Antonia Praetorius, Kevin V. Thomas, Jake William O’Brien","doi":"10.1021/jacsau.4c00220","DOIUrl":"https://doi.org/10.1021/jacsau.4c00220","url":null,"abstract":"Around two-thirds of chronic human disease can not be explained by genetics alone. The Lancet Commission on Pollution and Health estimates that 16% of global premature deaths are linked to pollution. Additionally, it is now thought that humankind has surpassed the safe planetary operating space for introducing human-made chemicals into the Earth System. Direct and indirect exposure to a myriad of chemicals, known and unknown, poses a significant threat to biodiversity and human health, from vaccine efficacy to the rise of antimicrobial resistance as well as autoimmune diseases and mental health disorders. The exposome chemical space remains largely uncharted due to the sheer number of possible chemical structures, estimated at over 10<sup>60</sup> unique forms. Conventional methods have cataloged only a fraction of the exposome, overlooking transformation products and often yielding uncertain results. In this Perspective, we have reviewed the latest efforts in mapping the exposome chemical space and its subspaces. We also provide our view on how the integration of data-driven approaches might be able to bridge the identified gaps.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"111 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jin-Da Zhuang, Jin-Min Shi, Chen-Cheng Hong, Ting-Ting Wu, Li Liu, Josef Voglmeir
The biotechnological production of uridine diphosphate-d-xylose (UDP-d-xylose), the glycosyl donor in enzymatic for d-xylose, is an important precursor for advancing glycoengineering research on biopharmaceuticals such as heparin and glycosaminoglycans. Leveraging a recently discovered UDP-xylose salvage pathway, we have engineered a series of bifunctional chimeric biocatalysts derived from Solitalea canadensis galactokinase/uridyltransferase, facilitating the conversion of d-xylose to UDP-d-xylose. This study elucidates the novel assembly of eight fusion protein constructs, differing in domain orientations and linker peptide lengths, to investigate their functional expression in Escherichia coli, resulting in the synthesis of the first bifunctional enzyme that orchestrates a direct transformation from d-xylose to UDP-d-xylose. Fusion constructs with a NH2-GSGGGSGHM-COOH peptide linker demonstrated the highest expression and catalytic tenacity. For the highest catalytic conversion from d-xylose to UDP-d-xylose, we established an optimum pH of 7.0 and a temperature optimum of 30 °C, with an optimal fusion enzyme concentration of 3.3 mg/mL for large-scale UDP-d-xylose production. Insights into ATP and ADP inhibition further helped to optimize the reaction conditions. Testing various ratios of unfused galactokinase and uridyltransferase biocatalysts for UDP-xylose synthesis from d-xylose revealed that a 1:1 ratio was optimal. The Kcat/Km value for the NH2-GSGGGSGHM-COOH peptide linker showed a 10% improvement compared with the unfused counterparts. The strategic design of these fusion enzymes efficiently routes for the convenient and efficient biocatalytic synthesis of xylosides in biotechnological and pharmaceutical applications.
{"title":"Engineering Bifunctional Galactokinase/Uridyltransferase Chimera for Enhanced UDP-d-Xylose Production","authors":"Jin-Da Zhuang, Jin-Min Shi, Chen-Cheng Hong, Ting-Ting Wu, Li Liu, Josef Voglmeir","doi":"10.1021/jacsau.4c00288","DOIUrl":"https://doi.org/10.1021/jacsau.4c00288","url":null,"abstract":"The biotechnological production of uridine diphosphate-<span>d</span>-xylose (UDP-<span>d</span>-xylose), the glycosyl donor in enzymatic for <span>d</span>-xylose, is an important precursor for advancing glycoengineering research on biopharmaceuticals such as heparin and glycosaminoglycans. Leveraging a recently discovered UDP-xylose salvage pathway, we have engineered a series of bifunctional chimeric biocatalysts derived from <i>Solitalea canadensis</i> galactokinase/uridyltransferase, facilitating the conversion of <span>d</span>-xylose to UDP-<span>d</span>-xylose. This study elucidates the novel assembly of eight fusion protein constructs, differing in domain orientations and linker peptide lengths, to investigate their functional expression in <i>Escherichia coli</i>, resulting in the synthesis of the first bifunctional enzyme that orchestrates a direct transformation from <span>d</span>-xylose to UDP-<span>d</span>-xylose. Fusion constructs with a NH<sub>2</sub>-GSGGGSGHM-COOH peptide linker demonstrated the highest expression and catalytic tenacity. For the highest catalytic conversion from <span>d</span>-xylose to UDP-<span>d</span>-xylose, we established an optimum pH of 7.0 and a temperature optimum of 30 °C, with an optimal fusion enzyme concentration of 3.3 mg/mL for large-scale UDP-<span>d</span>-xylose production. Insights into ATP and ADP inhibition further helped to optimize the reaction conditions. Testing various ratios of unfused galactokinase and uridyltransferase biocatalysts for UDP-xylose synthesis from <span>d</span>-xylose revealed that a 1:1 ratio was optimal. The <i>K</i><sub>cat</sub>/<i>K</i><sub>m</sub> value for the NH<sub>2</sub>-GSGGGSGHM-COOH peptide linker showed a 10% improvement compared with the unfused counterparts. The strategic design of these fusion enzymes efficiently routes for the convenient and efficient biocatalytic synthesis of xylosides in biotechnological and pharmaceutical applications.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kotaro Sakamoto, Seigo Iwata, Zihao Jin, Lu Chen, Tatsunori Miyaoka, Mei Yamada, Kaiga Katahira, Rei Yokoyama, Ami Ono, Satoshi Asano, Kotaro Tanimoto, Rika Ishimura, Shinsaku Nakagawa, Takatsugu Hirokawa, Yukio Ago, Eijiro Miyako
Establishing drug delivery systems (DDSs) for transporting drugs from peripheral tissues to the brain is crucial for treating central nervous system diseases. We previously reported the interactions of (1) KS-133, a selective antagonist peptide, with vasoactive intestinal peptide receptor 2 (VIPR2), a drug target for schizophrenia, and (2) KS-487, a selective binding peptide, with the cluster IV domain of low-density lipoprotein receptor-related protein 1 (LRP1), which is involved in crossing the blood–brain barrier. We developed a novel DDS-based strategy for treating schizophrenia using KS-487 as a brain-targeting peptide and KS-133 as a drug. Dibenzocyclooctyne-KS-487 was conjugated with N3-indocyanine green (ICG) using a click reaction and administered intravenously into mice. Fluorescence was clearly observed from ICG in the brains of the mice. Nanoparticles (NPs) encapsulating ICG and displaying KS-487 were prepared and subcutaneously administered to mice, resulting in a significant accumulation of ICG in the brain. Pharmacokinetic analysis of NPs containing KS-133 and displaying KS-487 (KS-133/KS-487 NPs) revealed the time-dependent transport of KS-133 into the brain. KS-133/KS-487 NPs were subcutaneously administered to mouse models of schizophrenia, which significantly improved cognitive dysfunction. This is the first study to demonstrate the potential therapeutic efficacy of a multifunctionalized multipeptide NP in inhibiting VIPR2.
{"title":"Cyclic Peptides KS-133 and KS-487 Multifunctionalized Nanoparticles Enable Efficient Brain Targeting for Treating Schizophrenia","authors":"Kotaro Sakamoto, Seigo Iwata, Zihao Jin, Lu Chen, Tatsunori Miyaoka, Mei Yamada, Kaiga Katahira, Rei Yokoyama, Ami Ono, Satoshi Asano, Kotaro Tanimoto, Rika Ishimura, Shinsaku Nakagawa, Takatsugu Hirokawa, Yukio Ago, Eijiro Miyako","doi":"10.1021/jacsau.4c00311","DOIUrl":"https://doi.org/10.1021/jacsau.4c00311","url":null,"abstract":"Establishing drug delivery systems (DDSs) for transporting drugs from peripheral tissues to the brain is crucial for treating central nervous system diseases. We previously reported the interactions of (1) KS-133, a selective antagonist peptide, with vasoactive intestinal peptide receptor 2 (VIPR2), a drug target for schizophrenia, and (2) KS-487, a selective binding peptide, with the cluster IV domain of low-density lipoprotein receptor-related protein 1 (LRP1), which is involved in crossing the blood–brain barrier. We developed a novel DDS-based strategy for treating schizophrenia using KS-487 as a brain-targeting peptide and KS-133 as a drug. Dibenzocyclooctyne-KS-487 was conjugated with N<sub>3</sub>-indocyanine green (ICG) using a click reaction and administered intravenously into mice. Fluorescence was clearly observed from ICG in the brains of the mice. Nanoparticles (NPs) encapsulating ICG and displaying KS-487 were prepared and subcutaneously administered to mice, resulting in a significant accumulation of ICG in the brain. Pharmacokinetic analysis of NPs containing KS-133 and displaying KS-487 (KS-133/KS-487 NPs) revealed the time-dependent transport of KS-133 into the brain. KS-133/KS-487 NPs were subcutaneously administered to mouse models of schizophrenia, which significantly improved cognitive dysfunction. This is the first study to demonstrate the potential therapeutic efficacy of a multifunctionalized multipeptide NP in inhibiting VIPR2.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141506601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yuhan Lyu, Fan Yang, Bharathi Sundaresh, Federico Rosconi, Tim van Opijnen, Jianmin Gao
The ever-expanding antibiotic resistance urgently calls for novel antibacterial therapeutics, especially those with a new mode of action. We report herein our exploration of protein–protein interaction (PPI) inhibition as a new mechanism to thwart bacterial pathogenesis. Specifically, we describe potent and specific inhibitors of the pneumococcal surface protein PspC, an important virulence factor that facilitates the infection of Streptococcus pneumoniae. Specifically, PspC has been documented to recruit human complement factor H (hFH) to suppress host complement activation and/or promote the bacterial attachment to host tissues. The CCP9 domain of hFH was recombinantly expressed to inhibit the PspC–hFH interaction as demonstrated on live pneumococcal cells. The inhibitor allowed for the first pharmacological intervention of the PspC–hFH interaction. This PPI inhibition reduced pneumococci’s attachment to epithelial cells and also resensitized the D39 strain of S. pneumoniae for opsonization. Importantly, we have further devised covalent versions of CCP9, which afforded long-lasting PspC inhibition with low nanomolar potency. Overall, our results showcase the promise of PPI inhibition for combating bacterial infections as well as the power of covalent inhibitors.
{"title":"Covalent Inhibition of a Host–Pathogen Protein–Protein Interaction Reduces the Infectivity of Streptococcus pneumoniae","authors":"Yuhan Lyu, Fan Yang, Bharathi Sundaresh, Federico Rosconi, Tim van Opijnen, Jianmin Gao","doi":"10.1021/jacsau.4c00195","DOIUrl":"https://doi.org/10.1021/jacsau.4c00195","url":null,"abstract":"The ever-expanding antibiotic resistance urgently calls for novel antibacterial therapeutics, especially those with a new mode of action. We report herein our exploration of protein–protein interaction (PPI) inhibition as a new mechanism to thwart bacterial pathogenesis. Specifically, we describe potent and specific inhibitors of the pneumococcal surface protein PspC, an important virulence factor that facilitates the infection of <i>Streptococcus pneumoniae.</i> Specifically, PspC has been documented to recruit human complement factor H (hFH) to suppress host complement activation and/or promote the bacterial attachment to host tissues. The CCP9 domain of hFH was recombinantly expressed to inhibit the PspC–hFH interaction as demonstrated on live pneumococcal cells. The inhibitor allowed for the first pharmacological intervention of the PspC–hFH interaction. This PPI inhibition reduced pneumococci’s attachment to epithelial cells and also resensitized the D39 strain of <i>S. pneumoniae</i> for opsonization. Importantly, we have further devised covalent versions of CCP9, which afforded long-lasting PspC inhibition with low nanomolar potency. Overall, our results showcase the promise of PPI inhibition for combating bacterial infections as well as the power of covalent inhibitors.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"135 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Designing the reactant molecule of an initial reaction, based on quantum chemical pathway exploration, enabled us to access a new reaction, i.e., the tetraborylation reaction of p-benzynes generated from 1,2-diethynylbenzene derivatives, using bis(pinacolato)diborane(4) (B2pin2). Based on the reaction path network generated via the artificial-force-induced reaction (AFIR) method, desired and undesired paths were identified and used to modify the chemical structure of the reactant. After the in silico screening, the optimal structure of the reactant was determined to be a 1,2-diethynylbenzene derivative with a butylene linker. The reaction of the optimized reactant and its derivatives with an excess of B2pin2 gave the tetraborylated products in good yields (up to 58%). It is quite intriguing that the two carbons of p-benzyne behave formally as dicarbenes in this reaction.
{"title":"Tetraborylation of p-Benzynes Generated by the Masamune–Bergman Cyclization through Reaction Design Based on the Reaction Path Network","authors":"Soichiro Nakatsuka, Seiji Akiyama, Yu Harabuchi, Satoshi Maeda, Yuuya Nagata","doi":"10.1021/jacsau.4c00302","DOIUrl":"https://doi.org/10.1021/jacsau.4c00302","url":null,"abstract":"Designing the reactant molecule of an initial reaction, based on quantum chemical pathway exploration, enabled us to access a new reaction, i.e., the tetraborylation reaction of <i>p</i>-benzynes generated from 1,2-diethynylbenzene derivatives, using bis(pinacolato)diborane(4) (B<sub>2</sub>pin<sub>2</sub>). Based on the reaction path network generated via the artificial-force-induced reaction (AFIR) method, desired and undesired paths were identified and used to modify the chemical structure of the reactant. After the in silico screening, the optimal structure of the reactant was determined to be a 1,2-diethynylbenzene derivative with a butylene linker. The reaction of the optimized reactant and its derivatives with an excess of B<sub>2</sub>pin<sub>2</sub> gave the tetraborylated products in good yields (up to 58%). It is quite intriguing that the two carbons of <i>p</i>-benzyne behave formally as dicarbenes in this reaction.","PeriodicalId":14799,"journal":{"name":"JACS Au","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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