Cyclic peptides to probe the internal interactome of PDZ domains — ASN Events
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  3. Cyclic peptides to probe the internal interactome of PDZ domains

Cyclic peptides to probe the internal interactome of PDZ domains (#192)

Javier Rodriguez Balboa 1 2 , Louise Strandberg Clemmensen 1 , Søren Østergaard 2 , Kristian Strømgaard 1
  1. Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhaguen, Copenhaguen, Denmark
  2. NovoNordisk A/S, Research Chemistry 3, NovoNordisk A/S, Måløv, Denmark

PDZ domains are part of multi-domain scaffolding proteins that play a key role in organizing and regulating cellular protein complexes1. Mutations of PDZ-domain proteins are linked to cancer or neurological disorders2. Hence, developing inhibitors to compete with PDZ targets is an attractive approach in drug discovery2. PDZ domains typically mediate protein-protein interactions by binding to the C-termini of target proteins1,2. However, increasing evidence suggests that some PDZ domains can also recognize internal binding motifs, a less explored PDZ binding mechanism3. For example, the interaction of the postsynaptic scaffolding protein, PSD-95, with neuronal nitric oxide synthase (nNOS) involves a 30-residue extension within nNOS that adopts an extended β-hairpin fold without a free C-termini3. This interaction is the main target in stroke and ischemia therapies3.

Here, we have mimicked the nNOS β-hairpin motif using cyclic peptides, superior to its linear analogues due to their further increased proteolytic stability and cell penetration capability. To map this interaction, we explored cyclisation strategies on-resin and subsequently transferred the optimized cyclization conditions to the SPOT cellulose membrane to perform peptide arrays. This high-throughput approach has enabled us to obtain key molecular information that has led us to identify which amino acids are relevant in the nNOS β-finger motif and to significantly increase its binding affinity. The SPOT data relevant to the binding mechanism has been validated using fluorescence polarization (FP) and isothermal calorimetry (ITC) assays.

The data gathered in this project will be used to obtain further evidence of this non-canonical mode of PDZ interaction and potentially, provide a cyclic peptide template for drug development.

Acknowledgements

The project receives funding from the European Union´s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No: 675341

  1. 1. Kim, E. & Sheng, M. PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771–81 (2004)
  2. 2. Dev, K. K. Making protein interactions druggable: targeting PDZ domains. Nat. Rev. Drug Discov.3, 1047–1056 (2004)
  3. 3. Haugaard-kedström, L. M., Fernandes, E. F. A. & Strømgaard, K. Neuroprotective Therapy for Stroke and Ischemic Disease. (2017)