title: Clustering of disulfide-rich peptides provides scaffolds for hit discovery by phage display: application to interleukin-23.
abstract: BACKGROUND
  Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity.
  
  
  RESULTS
  We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC50of 3.3 μM.
  
  
  CONCLUSIONS
  Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Barkan
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Cheng
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA.
authors.forename: Herodion
authors.initials: H
authors.lastname: Celino
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA.
authors.forename: Tran T
authors.initials: TT
authors.lastname: Tran
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA.
authors.forename: Ashok
authors.initials: A
authors.lastname: Bhandari
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 94158, USA.|California Institute for Quantitative Biosciences (QB3), University of California, San Francisco, San Francisco, CA, 94158, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, 94158, USA.|Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 94158, USA.|California Institute for Quantitative Biosciences (QB3), University of California, San Francisco, San Francisco, CA, 94158, USA.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
authors.affiliation: Protagonist Therapeutics, Inc., 521 Cottonwood Drive, Suite 100, Milpitas, CA, 95035-74521, USA. m.smythe@protagonist-inc.com.|Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, 4072, Australia. m.smythe@protagonist-inc.com.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Smythe
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D016000:Cluster Analysis; D004220:Disulfides / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D053759:Interleukin-23 / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D004220:Disulfides; D053759:Interleukin-23; D019151:Peptide Library; D010455:Peptides
keywords: Clustering; Disulfide-rich peptides; Drug discovery; Interleukin-23; Knottins; Phage display; Structure conservation