Pub Date : 2024-03-18DOI: 10.1021/acs.bioconjchem.4c00013
Amissi Sadiki*, Shanshan Liu, Shefali R. Vaidya, Eric M. Kercher, Ryan T. Lang, James McIsaac, Bryan Q. Spring, Jared R. Auclair and Zhaohui Sunny Zhou*,
A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.
使用转谷氨酰胺酶(TGase,E.C. 2.3.2.13)是对原生(非工程)抗体进行位点特异性修饰的一种多功能化学酶工具。这种酶以各种胺为共底物,将肽和蛋白质中谷氨酰胺(Gln 或 Q)的未取代侧链酰胺转化为取代的酰胺(即共轭物)。令人惊喜的是,微生物 TG 酶(mTGase,EC 2.3.2.13)只修饰 IgG Fc 区域中的一个保守谷氨酰胺(Gln295),从而提供了一种高度特异且普遍适用的共轭方法。然而,在转氨基(mTGase 进入谷氨酰胺残基)之前,必须减少来自附近保守 N-糖(IgG1 中为 Asn297)的立体阻碍。在以前的方法中,酰胺酶(PNGase F,EC 3.5.1.52)被用来完全去除 N-聚糖。然而,PNGase F 还会将净中性天冬酰胺(Asn297)转化为带负电荷的天冬氨酸(Asp297)。这种电荷变化可能会显著改变 IgG 抗体的结构、功能和免疫原性。与此相反,在我们介绍的新方法中,N-聚糖被内切糖苷酶(EndoS2,EC 3.2.1.96)修剪,从而保留了核心的 N-乙酰葡糖胺(GlcNAc)分子和中性天冬酰胺。修剪后的聚糖还减少或取消了 Fc 受体介导的功能,从而减少了与其他细胞(如免疫细胞)的非特异性结合,从而获得更好的成像剂。此外,剩余的核心聚糖还可以进一步衍生,如聚糖重塑和双重共轭。我们的方法既实用又稳健,能生成接近定量的共轭物,而且这两种酶都可以在市场上买到。
{"title":"Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase","authors":"Amissi Sadiki*, Shanshan Liu, Shefali R. Vaidya, Eric M. Kercher, Ryan T. Lang, James McIsaac, Bryan Q. Spring, Jared R. Auclair and Zhaohui Sunny Zhou*, ","doi":"10.1021/acs.bioconjchem.4c00013","DOIUrl":"10.1021/acs.bioconjchem.4c00013","url":null,"abstract":"<p >A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.bioconjchem.4c00013","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140157032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-16DOI: 10.1021/acs.bioconjchem.4c00038
Jiaxing Di, Pei Huang* and Xiaoyuan Chen*,
mRNA therapeutics hold great promise for disease treatment, yet a key challenge lies in achieving site-specific mRNA delivery to maximize therapeutic efficacy while minimizing off-target side effects. This viewpoint delves into multiple complementary targeting strategies to achieve precise site-specific mRNA delivery, covering topics of administration routes, passive targeting, and active targeting. It highlights the critical importance of rationally designed nanocarriers for obtaining desired therapeutic effects and accelerating the clinical translation of mRNA therapeutics.
{"title":"Targeting Strategies for Site-Specific mRNA Delivery","authors":"Jiaxing Di, Pei Huang* and Xiaoyuan Chen*, ","doi":"10.1021/acs.bioconjchem.4c00038","DOIUrl":"10.1021/acs.bioconjchem.4c00038","url":null,"abstract":"<p >mRNA therapeutics hold great promise for disease treatment, yet a key challenge lies in achieving site-specific mRNA delivery to maximize therapeutic efficacy while minimizing off-target side effects. This viewpoint delves into multiple complementary targeting strategies to achieve precise site-specific mRNA delivery, covering topics of administration routes, passive targeting, and active targeting. It highlights the critical importance of rationally designed nanocarriers for obtaining desired therapeutic effects and accelerating the clinical translation of mRNA therapeutics.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140139580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-14DOI: 10.1021/acs.bioconjchem.4c00024
Aryn A. Alanizi, Alexandre M. Sorlin, Matthew F. L. Parker, Marina López-Álvarez, Hecong Qin, Sang Hee Lee, Joseph Blecha, Oren S. Rosenberg, Joanne Engel, Michael A. Ohliger, Robert R. Flavell and David M. Wilson*,
Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent “click” cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide–alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.
{"title":"Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO","authors":"Aryn A. Alanizi, Alexandre M. Sorlin, Matthew F. L. Parker, Marina López-Álvarez, Hecong Qin, Sang Hee Lee, Joseph Blecha, Oren S. Rosenberg, Joanne Engel, Michael A. Ohliger, Robert R. Flavell and David M. Wilson*, ","doi":"10.1021/acs.bioconjchem.4c00024","DOIUrl":"10.1021/acs.bioconjchem.4c00024","url":null,"abstract":"<p ><i>Purpose:</i> This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled <span>d</span>-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label <span>d</span>-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N<sub>3</sub>) bearing <span>d</span>-amino acid is incorporated into peptidoglycan muropeptides, with subsequent “click” cycloaddition with an <sup>18</sup>F-labeled strained cyclooctyne partner. <i>Procedures:</i> A water-soluble, <sup>18</sup>F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([<sup>18</sup>F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing <span>d</span>-amino acids, and incorporated <sup>18</sup>F was determined via gamma counting. <i>In vitro</i> uptake in bacteria previously treated with azide-modified <span>d</span>-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [<sup>18</sup>F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future <i>in vivo</i> imaging. <i>Results:</i> The new strain-promoted azide–alkyne cycloaddition (SPAAC) radiotracer [<sup>18</sup>F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via <i>N</i>-succinimidyl 4-[<sup>18</sup>F]fluorobenzoate ([<sup>18</sup>F]SFB). Accumulation of [<sup>18</sup>F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified <span>d</span>-amino acids than in controls; for example, we observed 7 times greater [<sup>18</sup>F]FB-sulfo-DBCO ligation in <i>Staphylococcus aureus</i> cultures incubated with 3-azido-<span>d</span>-alanine versus those incubated with <span>d</span>-alanine. <i>Conclusions:</i> The SPAAC radiotracer [<sup>18</sup>F]FB-sulfo-DBCO was validated <i>in vitro</i> via metabolic labeling of azide-bearing peptidoglycan muropeptides. <span>d</span>-Amino acid-derived PET radiotracers may be more efficiently screened via [<sup>18</sup>F]FB-sulfo-DBCO modification.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.bioconjchem.4c00024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140118117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-12DOI: 10.1021/acs.bioconjchem.4c00019
Natalie Y. Luo, Rachel L. Minne, Joseph P. Gallant, Gihan S. Gunaratne, Jayden L. West, Saahil Javeri, Austin J. Robertson, Eric W. Lake, Jonathan W. Engle, Jason C. Mixdorf, Eduardo Aluicio-Sarduy, Kwang P. Nickel, Reinier Hernandez, Randall J. Kimple, Andrew M. Baschnagel* and Aaron M. LeBeau*,
The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.
间质上皮转化(MET)受体酪氨酸激酶在 5% 的非小细胞肺癌(NSCLC)患者中上调或突变,并在其他多种癌症中过表达。我们试图开发一种对 MET 具有高亲和力的新型单域驼科抗体,用于向表达 MET 的癌症递送共轭有效载荷。我们从幼稚的驼科可变重型(VHH)结构域噬菌体展示文库中发现了一种名为 1E7 的 VHH 克隆,它对人类 MET 具有高亲和力,并与多种物种的 MET 具有交叉反应性。当表达为二价人类 Fc 融合蛋白时,通过流式细胞术和免疫荧光成像,我们发现 1E7-Fc 能选择性地与 EBC-1(MET 扩增)和 UW-Lung 21(MET 第 14 外显子突变)细胞系结合。接下来,我们通过 PET/CT 成像研究了[89Zr]Zr-1E7-Fc 在体内检测 MET 表达的能力。[89Zr]Zr-1E7-Fc在两种异种移植物中均显示出快速定位和高肿瘤摄取率,在24小时内,EBC-1和华大肺21的%ID/g分别为6.4和5.8。在 24 小时的时间点上,还观察到次要和非目标组织的清除情况。总之,我们的数据表明,1E7-Fc代表了一种平台技术,可用于对MET改变的NSCLC进行成像和治疗。
{"title":"Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer","authors":"Natalie Y. Luo, Rachel L. Minne, Joseph P. Gallant, Gihan S. Gunaratne, Jayden L. West, Saahil Javeri, Austin J. Robertson, Eric W. Lake, Jonathan W. Engle, Jason C. Mixdorf, Eduardo Aluicio-Sarduy, Kwang P. Nickel, Reinier Hernandez, Randall J. Kimple, Andrew M. Baschnagel* and Aaron M. LeBeau*, ","doi":"10.1021/acs.bioconjchem.4c00019","DOIUrl":"10.1021/acs.bioconjchem.4c00019","url":null,"abstract":"<p >The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [<sup>89</sup>Zr]Zr-1E7-Fc to detect MET expression <i>in vivo</i> by PET/CT imaging. [<sup>89</sup>Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140108361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-08DOI: 10.1021/acs.bioconjchem.3c00549
Liang Fang, Simian Cai, Patrick McMullen, Yi-Chen Hsu, Michelle Yi Qin Chen and Shaoyi Jiang*,
Living microbial therapies have been proposed as a course of action for a variety of diseases. However, problematic interactions between the host immune system and the microbial organism present significant clinical concerns. Previously, we developed a genetically encoded superhydrophilic zwitterionic peptide, termed EKP, to mimic low-immunogenic zwitterionic materials, which have been used for the chemical modification of biologics such as protein and nucleic acid drugs to increase their in vivo circulation time and reduce their immunogenicity. Herein, we demonstrate the protective effects of the EKP polypeptide genetically cloaking the surface of Saccharomyces cerevisiae as a model microbe in both in vitro and in vivo systems. First, we show that EKP peptide cloaking suppresses the interactions between yeast cells and their specific antibodies, thereby illustrating its cloaking behavior. Then, we examine the in vitro interactions between EKP peptide surface cloaked yeast cells and murine macrophage cells, which exhibit phagocytotic behavior in the presence of foreign microbes. Our results indicate that EKP cloaking suppresses macrophage interactions and thus reduces phagocytosis. Furthermore, EKP cloaked yeast cells demonstrate a prolonged circulation time in mice in vivo.
活微生物疗法已被提议作为治疗多种疾病的一种方法。然而,宿主免疫系统与微生物有机体之间的相互作用问题给临床带来了极大的困扰。此前,我们开发了一种基因编码的超亲水性齐聚物肽(称为 EKP)来模拟低免疫原性齐聚物,这种齐聚物已被用于对蛋白质和核酸药物等生物制剂进行化学修饰,以延长其体内循环时间并降低其免疫原性。在本文中,我们在体外和体内系统中展示了将 EKP 多肽基因包覆在作为模式微生物的酿酒酵母表面的保护作用。首先,我们发现 EKP 多肽隐形后会抑制酵母细胞与其特异性抗体之间的相互作用,从而说明了它的隐形行为。然后,我们研究了 EKP 肽表面隐形的酵母细胞与小鼠巨噬细胞之间的体外相互作用。我们的研究结果表明,EKP 斗篷会抑制巨噬细胞之间的相互作用,从而减少吞噬作用。此外,EKP 斗篷酵母细胞在小鼠体内的循环时间延长。
{"title":"Passivating the Background of Living Microbes with a Zwitterionic Peptide for Therapies","authors":"Liang Fang, Simian Cai, Patrick McMullen, Yi-Chen Hsu, Michelle Yi Qin Chen and Shaoyi Jiang*, ","doi":"10.1021/acs.bioconjchem.3c00549","DOIUrl":"10.1021/acs.bioconjchem.3c00549","url":null,"abstract":"<p >Living microbial therapies have been proposed as a course of action for a variety of diseases. However, problematic interactions between the host immune system and the microbial organism present significant clinical concerns. Previously, we developed a genetically encoded superhydrophilic zwitterionic peptide, termed EKP, to mimic low-immunogenic zwitterionic materials, which have been used for the chemical modification of biologics such as protein and nucleic acid drugs to increase their in vivo circulation time and reduce their immunogenicity. Herein, we demonstrate the protective effects of the EKP polypeptide genetically cloaking the surface of <i>Saccharomyces cerevisiae</i> as a model microbe in both in vitro and in vivo systems. First, we show that EKP peptide cloaking suppresses the interactions between yeast cells and their specific antibodies, thereby illustrating its cloaking behavior. Then, we examine the in vitro interactions between EKP peptide surface cloaked yeast cells and murine macrophage cells, which exhibit phagocytotic behavior in the presence of foreign microbes. Our results indicate that EKP cloaking suppresses macrophage interactions and thus reduces phagocytosis. Furthermore, EKP cloaked yeast cells demonstrate a prolonged circulation time in mice in vivo.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide–alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.
{"title":"3-Acyl-4-Pyranone as a Lysine Residue-Selective Bioconjugation Reagent for Peptide and Protein Modification","authors":"Keyi Nong, Yi-Lu Zhao, Shandong Yi, Xuchun Zhang, Siyuan Wei and Zhu-Jun Yao*, ","doi":"10.1021/acs.bioconjchem.3c00447","DOIUrl":"10.1021/acs.bioconjchem.3c00447","url":null,"abstract":"<p >Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide–alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140048117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-07DOI: 10.1021/acs.bioconjchem.4c00073
Ying Wang, Wei Liu*, Yan Sun and Xiaoyan Dong*,
The design of a potent amyloid-β protein (Aβ) inhibitor plays a pivotal role in the prevention and treatment of Alzheimer’s disease (AD). Despite endogenous transthyretin (TTR) being recognized as an Aβ inhibitor, the weak inhibitory and blood brain barrier (BBB) crossing capabilities hinder it for Aβ aggregation inhibition and transport. Therefore, we have herein designed a recombinant TTR by conjugating a cationic cell penetrating peptide (penetratin, Pen), which not only enabled the fusion protein, TTR-Pen (TP), to present high BBB penetration but also greatly enhanced the potency of Aβ inhibition. Namely, the protein fusion made TP positively charged, leading to a potent suppression of Aβ40 fibrillization at a low concentration (1.5 μM), while a TTR concentration as high as 12.5 μM was required to gain a similar function. Moreover, TP could mitigate Aβ-induced neuronal death, increase cultured cell viability from 72% to 92% at 2.5 μM, and extend the lifespan of AD nematodes from 14 to 18 d. Thermodynamic studies revealed that TP, enriched in positive charges, presented extensive electrostatic interactions with Aβ40. Importantly, TP showed excellent BBB penetration performance, with a 10 times higher BBB permeability than TTR, which would allow TP to enter the brain of AD patients and participate in the transport of Aβ species out of the brain. Thus, it is expected that the fusion protein has great potential for drug development in AD treatment.
{"title":"Transthyretin-Penetratin: A Potent Fusion Protein Inhibitor against Alzheimer’s Amyloid-β Fibrillogenesis with High Blood Brain Barrier Crossing Capability","authors":"Ying Wang, Wei Liu*, Yan Sun and Xiaoyan Dong*, ","doi":"10.1021/acs.bioconjchem.4c00073","DOIUrl":"10.1021/acs.bioconjchem.4c00073","url":null,"abstract":"<p >The design of a potent amyloid-β protein (Aβ) inhibitor plays a pivotal role in the prevention and treatment of Alzheimer’s disease (AD). Despite endogenous transthyretin (TTR) being recognized as an Aβ inhibitor, the weak inhibitory and blood brain barrier (BBB) crossing capabilities hinder it for Aβ aggregation inhibition and transport. Therefore, we have herein designed a recombinant TTR by conjugating a cationic cell penetrating peptide (penetratin, Pen), which not only enabled the fusion protein, TTR-Pen (TP), to present high BBB penetration but also greatly enhanced the potency of Aβ inhibition. Namely, the protein fusion made TP positively charged, leading to a potent suppression of Aβ<sub>40</sub> fibrillization at a low concentration (1.5 μM), while a TTR concentration as high as 12.5 μM was required to gain a similar function. Moreover, TP could mitigate Aβ-induced neuronal death, increase cultured cell viability from 72% to 92% at 2.5 μM, and extend the lifespan of AD nematodes from 14 to 18 d. Thermodynamic studies revealed that TP, enriched in positive charges, presented extensive electrostatic interactions with Aβ<sub>40</sub>. Importantly, TP showed excellent BBB penetration performance, with a 10 times higher BBB permeability than TTR, which would allow TP to enter the brain of AD patients and participate in the transport of Aβ species out of the brain. Thus, it is expected that the fusion protein has great potential for drug development in AD treatment.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140048118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-06DOI: 10.1021/acs.bioconjchem.4c00001
Calin Nicolescu, Jiyoon Kim, Da Sun and Zheng-Rong Lu*,
Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.
{"title":"Assessment of the Efficacy of the Combination of RNAi of lncRNA DANCR with Chemotherapy to Treat Triple Negative Breast Cancer Using Magnetic Resonance Molecular Imaging","authors":"Calin Nicolescu, Jiyoon Kim, Da Sun and Zheng-Rong Lu*, ","doi":"10.1021/acs.bioconjchem.4c00001","DOIUrl":"10.1021/acs.bioconjchem.4c00001","url":null,"abstract":"<p >Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140038191","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-05DOI: 10.1021/acs.bioconjchem.3c00546
Ricarda C. Steffens, Paul Folda, Nikole L. Fendler, Miriam Höhn, Katharina Bücher-Schossau, Susanne Kempter, Nicole L. Snyder, Laura Hartmann, Ernst Wagner and Simone Berger*,
A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.
{"title":"GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery","authors":"Ricarda C. Steffens, Paul Folda, Nikole L. Fendler, Miriam Höhn, Katharina Bücher-Schossau, Susanne Kempter, Nicole L. Snyder, Laura Hartmann, Ernst Wagner and Simone Berger*, ","doi":"10.1021/acs.bioconjchem.3c00546","DOIUrl":"10.1021/acs.bioconjchem.3c00546","url":null,"abstract":"<p >A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and <i>N</i>-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140026807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.
{"title":"Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications","authors":"Ryutaro Ariyoshi, Takashi Matsuzaki, Ryo Sato, Kosuke Minamihata, Kounosuke Hayashi, Taisei Koga, Kensei Orita, Riko Nishioka, Rie Wakabayashi, Masahiro Goto and Noriho Kamiya*, ","doi":"10.1021/acs.bioconjchem.3c00544","DOIUrl":"10.1021/acs.bioconjchem.3c00544","url":null,"abstract":"<p >Microbial transglutaminase (MTG) from <i>Streptomyces mobaraensis</i> is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report <u>e</u>ngineered active <u>z</u>ymogen of MTG (EzMTG) that is expressed in soluble form in the host <i>Escherichia coli</i> cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-02-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988657","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}