Phage display identification of nanomolar ligands for human NEDD4-WW3: Energetic and dynamic implications for the development of broad-spectrum antivirals Castillo, Francisco Corbi Verge, Carles Murciano Calles, Javier Candel, Adela M. Iglesias Bexiga, Manuel Ruiz Sanz, Javier Martínez Herrerías, José Cristóbal Luque Fernández, Irene NEDD4 WW domains NEDD4-WW3 domain Drug discovery Polyproline recognition Broad-spectrum antivirals Phage display Molecular dynamics simulations Calorimetry Nuclear magnetic resonance This research has been financed by grants BIO2016-78746-C2-1-R and PID2020-112895RB-100 from the Spanish Ministry of Education and Science (I.L.) including AEI/FEDER EU funds. R.N.H. was funded in part by National Institutes of Health grants AI138052 and AI138630. M. I.B. and J.M.C. were recipients of a research contract from the Spanish Ministry of Education and Science. F.C. was funded by a predoctoral fellowship from the Andalusian Government P10-CVI-5915. J.M.C. ac-knowledges a reincorporation research contract from the University of Granada. We thank Dr. Sachdev Sidhu for his invaluable assistance setting up the phage display techniques in our laboratory. We also thank the support of the C.I.C. of the University of Granada. The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions. 2022-05-06T11:51:14Z 2022-05-06T11:51:14Z 2022-03-05 journal article Francisco Castillo... [et al.]. Phage display identification of nanomolar ligands for human NEDD4-WW3: Energetic and dynamic implications for the development of broad-spectrum antivirals, International Journal of Biological Macromolecules, Volume 207, 2022, Pages 308-323, ISSN 0141-8130, [https://doi.org/10.1016/j.ijbiomac.2022.03.010] http://hdl.handle.net/10481/74732 10.1016/j.ijbiomac.2022.03.010 eng http://creativecommons.org/licenses/by-nc-nd/3.0/es/ open access Atribución-NoComercial-SinDerivadas 3.0 España Elsevier