Sunhee Hwang, Aaron T. Balana, Bryan Martin, Michael Clarkson, Paola Di Lello, Hao Wu, Yanjie Li, Jakob Fuhrmann, Yavuz Dagdas, Patrick Holder, Christina I. Schroeder, Stephen E. Miller* and Xinxin Gao*,
{"title":"生成功能化二硫约束肽类似物的生物生产平台","authors":"Sunhee Hwang, Aaron T. Balana, Bryan Martin, Michael Clarkson, Paola Di Lello, Hao Wu, Yanjie Li, Jakob Fuhrmann, Yavuz Dagdas, Patrick Holder, Christina I. Schroeder, Stephen E. Miller* and Xinxin Gao*, ","doi":"10.1021/acsbiomedchemau.4c0002610.1021/acsbiomedchemau.4c00026","DOIUrl":null,"url":null,"abstract":"<p >Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced <sup>15</sup>N/<sup>13</sup>C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost, and expedite the timeline for DCP discovery.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.4c00026","citationCount":"0","resultStr":"{\"title\":\"Bioproduction Platform to Generate Functionalized Disulfide-Constrained Peptide Analogues\",\"authors\":\"Sunhee Hwang, Aaron T. Balana, Bryan Martin, Michael Clarkson, Paola Di Lello, Hao Wu, Yanjie Li, Jakob Fuhrmann, Yavuz Dagdas, Patrick Holder, Christina I. Schroeder, Stephen E. Miller* and Xinxin Gao*, \",\"doi\":\"10.1021/acsbiomedchemau.4c0002610.1021/acsbiomedchemau.4c00026\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced <sup>15</sup>N/<sup>13</sup>C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. 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Bioproduction Platform to Generate Functionalized Disulfide-Constrained Peptide Analogues
Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced 15N/13C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost, and expedite the timeline for DCP discovery.
期刊介绍:
ACS Bio & Med Chem Au is a broad scope open access journal which publishes short letters comprehensive articles reviews and perspectives in all aspects of biological and medicinal chemistry. Studies providing fundamental insights or describing novel syntheses as well as clinical or other applications-based work are welcomed.This broad scope includes experimental and theoretical studies on the chemical physical mechanistic and/or structural basis of biological or cell function in all domains of life. It encompasses the fields of chemical biology synthetic biology disease biology cell biology agriculture and food natural products research nucleic acid biology neuroscience structural biology and biophysics.The journal publishes studies that pertain to a broad range of medicinal chemistry including compound design and optimization biological evaluation molecular mechanistic understanding of drug delivery and drug delivery systems imaging agents and pharmacology and translational science of both small and large bioactive molecules. Novel computational cheminformatics and structural studies for the identification (or structure-activity relationship analysis) of bioactive molecules ligands and their targets are also welcome. The journal will consider computational studies applying established computational methods but only in combination with novel and original experimental data (e.g. in cases where new compounds have been designed and tested).Also included in the scope of the journal are articles relating to infectious diseases research on pathogens host-pathogen interactions therapeutics diagnostics vaccines drug-delivery systems and other biomedical technology development pertaining to infectious diseases.