Self-assembled proteomimetic (SAP) with antibody-like binding from short PNA

Article Article scientifique Article Self-assembled proteomimetic (SAP) with antibody-like binding from short PNA–peptide conjugates eng 2025-02-14 2025-02-18 author Brennecke Benjamin Jakob Department of Organic Chemistry, Faculty of Science, University of Geneva, Geneva 1211, Switzerland. 970650 author Civili Beatrice Department of Organic Chemistry, Faculty of Science, University of Geneva, Geneva 1211, Switzerland. 403270 Chimie organique (groupe 5) (CHOR5) author Sabale Pramod Madhavrao Department of Organic Chemistry, Faculty of Science, University of Geneva, Geneva 1211, Switzerland. 909496 author Barluenga Badiola Sofia 0000-0003-3830-1178 Department of Organic Chemistry, Faculty of Science, University of Geneva, Geneva 1211, Switzerland. 810177 Département de chimie organique (CHIOR) author Meyer Benjamin 0000-0003-0601-3550 Center of Vaccinology, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva 1211, Switzerland. 915879 Département de pathologie et immunologie (PATIM) author Winssinger Nicolas 0000-0003-1636-7766 Department of Organic Chemistry, Faculty of Science, University of Geneva, Geneva 1211, Switzerland. 769664 Département de chimie organique (CHIOR) e2412850122 10.1073/pnas.2412850122 39951509 0027-8424 PMC11848287 https://pnas.org/doi/10.1073/pnas.2412850122 Affinity proteins based on a three-helix bundle (affibodies, alphabodies, and computationally de novo designed ones) have been shown to be a general platform to discover binders with properties reminiscent of antibodies, combining high target specificity with affinities reaching well below the nanomolar. Herein, we report a strategy, coined self-assembled proteomimetic (SAP), to mimic such three-helix bundle architecture with a hybridization-enforced two-helix coiled coil that is obtained by templated native chemical ligation (T-NCL) of PNA-peptide conjugates. This SAP strategy stands out by its synthetic accessibility, reducing the length on the longest synthetic peptide to less than 30 amino acids which is readily attainable by standard SPPS methodologies. We show that the T-NCL dramatically accelerates the ligation, enabling this chemistry to proceed in a combinatorial fashion at low micromolar concentrations. We demonstrate that small combinatorial libraries of SAPs can be prepared in one operation and used directly in affinity selections against a target of interest with an LC-MS analysis of the fittest binders. Moreover, we show that the underlying design paradigm is functional for SAPs based on structurally distinct three-helix peptides aimed at different therapeutic targets, namely HER2 and spike's RBD, reaching picomolar affinities. We further illustrate that the affinity of the SAP can be allosterically regulated using a toehold displacement of the hybridizing PNAs to disrupt the coiled coil stabilization. Finally, we show that an RBD-targeting SAP effectively inhibits viral entry of SARS-CoV-2 with an IC<sub>50</sub> of 2.8 nM. Affinity protein Combinatorial libraries Proteomimetics Supramolecular chemistry Dewey 540 Dewey 616.07 Swiss National Science Foundation Encoding and programing chemical biocircuitry 219316 CH Projects 36 Département de chimie organique 141 137 Département de pathologie et immunologie 21F false 762 Groupe Winssinger 1337 Laboratoire d'immunologie vaccinale humaine 1025 https://zenodo.org/records/14224121 Proceedings of the National Academy of Sciences of the United States of America 122 7 98171a26-6454-4b3f-ab8a-8d281df60621 Article (Published version) Public brennecke-et-al-2025-self-assembled.pdf 3999711 application/pdf CC BY-NC-ND-4.0 e9f041845592c390167ffad12c5f5cd97c8cecd92e1e8cb269ba8f4d846975c4 c63e93ea-1553-4cd0-80b2-299057f0df8c Supplemental data Public pnas.2412850122.sapp.pdf 13616479 application/pdf 44e9e3f0cc6b472993fe8c26fe5d6ecc1deef697a27f76fdf2b9c4c1c72cfd00 openAccess Faculté des sciences / Section de chimie et biochimie / Département de chimie organique Faculté de médecine / Section de médecine fondamentale / Département de pathologie et immunologie

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Self-assembled proteomimetic (SAP) with antibody-like binding from short PNA–peptide conjugates	Proceedings of the National Academy of Sciences of the United States of America			2025	102	238

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Brennecke, Benjamin Jakob