Oral Presentation 14th Australian Peptide Conference 2022

Three ways to take down cholesterol with peptides (#19)

Benjamin J Tombling 1 , David J Craik 1 , Conan K Wang 1
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

BACKGROUND:

Protein-protein interfaces that are large, flat, and featureless are notoriously difficult to target by drugs. This is the case for the interaction interface between the serum protein PCSK9 and the receptor LDLR, which is a validated target for lowering cholesterol levels. Here, we developed new strategies for design of PCSK9-targeted peptide inhibitors with potent activities.

 

METHODS: 

We explored the use of three different design strategies:

  1. Loop expansion – reengineering of a LDLR microdomain by loop elongation [1]
  2. Bioactive cyclisation – functionalisation of a linker sequence [2]
  3. Increasing valency – increasing avidity effects on a multivalent scaffold [3]

Peptides were designed using a combination of computational, phage library screening and chemical synthesis. Subsequently, structures were characterized by NMR and MD, binding characterized by SPR and ITC, inhibition of PCSK9:LDLR interaction by competition ELISAs and on-cell Westerns, effect on cellular metabolism of cholesterol using dye-labelled lipids.

 

RESULTS:

All three design strategies were successful in generating potent lead peptides with affinities in the pico- to nanomolar range. We looked beyond the current results and performed a computational analysis of microdomains and peptide:protein interactions, and identified a vast range of lead peptides amenable to the approaches described herein.

 

SIGNIFICANCE:

The success of the strategies in generating potent peptide inhibitors suggest they could be of broad interest to the peptide research community, as many protein-protein interactions remain underexplored due to their challenging topological nature.

 

REFERENCES:

  1. Tombling BJ, Lammi C, Lawrence N, Li J, Arnoldi A, Craik DJ, Wang CK. Engineered EGF-A peptides with improved affinity for proprotein convertase subtilisin/kexin type 9 (PCSK9). ACS Chem Biol 2020. 16:429-439.
  2. Tombling BJ, Lammi C, Lawrence N, Gilding EK, Grazioso G, Craik DJ, Wang CK. Bioactive Cyclization Optimizes the Affinity of a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Peptide Inhibitor. J Med Chem 2020. 64:2523-33.
  3. Tombling BJ, Lammi C, Bollati C, Anoldi A, Craik DJ, Wang CK. Increased valency improves inhibitory activity of peptides targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). Chembiochem 2021. 22:2154-60.