Peptide Lead Optimization

Increasing peptide drug potency and improving pharmacokinetic properties through lead optimization of linear and CLIPS-constrained peptides


Schematic visualization of the lead optimization proces. Based on an initial lead (left), full replacement analysis using both natural & non-natural amino acids identifies those amino acids in the lead sequence that are essential to binding, as well as the amino acids that contribute to affinity improvement. Iterative synthesis & screening cycles with affinity-optimized sequences result in an optimized candidate peptide.

Lead optimization is a critical step in the design of novel peptide-based drugs. Peptide leads can be optimized in three ways:

1. through amino acid replacement analysis,
2. by constraining peptides using our in-house CLIPS technology, or
3. by improving the peptide length/size.

By combining these modifications in iterative screening rounds, we have demonstrated that we can improve the affinity of peptide-based lead drug candidates up to 1000 times.

We have developed a diverse library of enhanced CLIPS molecules, which after incorporation can lead to improved peptide conformation as well as better pharmacokinetic properties. This addition directly affects the potency of lead drug candidates, for example through:

  • improved specificity,
  • complex stability, and
  • decreased elimination/clearance.

Pepscan’s scientists build on over 25 years of experience. Our highly skilled project teams combine chemistry and biology expertise and are well-equipped to bring a peptide lead drug candidate to the next level.

Key benefits of Pepscan’s lead optimization

  • Detailed insights into the critical amino acids of the core epitope of a lead peptide
  • Possibility to further improve binding affinity by introducing a large variety of non-natural amino acids
  • Additional insights through mutational analysis and alanine scanning (ala scan)
  • Development of an enhanced molecule by constraining it (e.g. using CLIPS) for better pharmacokinetic properties, such as improved specificity, complex stability and decreased elimination/clearance, which directly affects the potency of a lead drug candidate
  • Possibility to include various in-house functional assays to determine proteolytic and/or enzymatic stability profiles of a lead peptide candidate

Lead optimization using Pepscan’s peptide-array platform

Multiple peptide modifications can be evaluated simultaneously using our peptide arrays, where peptides undergo high-throughput synthesis on a solid support (455 wells in polypropylene mini-cards). The format of the lead peptide may be either linear or conformationally constrained, e.g. a CLIPS-constrained peptide. Moreover, our CLIPS technology is often used to improve the binding potential of linear peptides by fixing them in a certain conformation. Binding of the modified peptides to their target of interest (e.g. a protein, (monoclonal) antibody, enzyme, etc.) is determined via read-out in an ELISA-type assay to ultimately identify those peptides that show a sufficient increase in binding.

Non-natural amino acids (both L and D) are also frequently used in our peptide arrays to further extend the horizon for discovering new chemical entities (NCEs) beyond the reach of phage-display-type library optimizations that solely rely on the use of the 20 natural L-amino acids. This not only leads to new NCEs but also opens up novel options for the IP protection of existing peptide drugs.

Our peptide platform has helped identify small peptide binders designed or derived from whole protein sequences, and create constrained (e.g. CLIPS) derivatives of lead sequences with significantly improved binding activity.

Case report 1 of 2: Development of a 3rd generation HIV-fusion inhibitor


X-ray of the 26-mer peptide sequence in complex with the 5 helix bundle that mimicks the gp41 target protein

In a recent collaboration with Janssen Pharmaceuticals the combined HiSense/CLIPS-technology platform was successfully used for the development of a 3rd generation HIV-fusion inhibitor. Known inhibitors either have a too low antiviral activity, or are too long (38-mer) to be synthesized in a cost-effective manner. Pepscan succeeded to identify a truncated version of T2635, a known HIV fusion-inhibitor with antiviral activity in the low nanomolar range, and similar activity against >10 Fuuzeon-resistant HIV-mutants. The ~50 nM antiviral activity of the lead T2635-trunc peptide (26-mer CLIPS-peptide) was successfully optimized in several iterative screening rounds to ~50 pM, simply by combining several individual amino acid mutation that were identified using the Hisense-platfrom technology. Moreover, slight variations in the type of CLIPS-scaffold and the anchor positions in the sequences also resulted in impressive activity improvement of ≥20. For the optimized 26-mer lead peptide, a X-ray crystal structure was obtained (collaboration with W. Weissenhorn, Univ. of Grenoble/France), which identified the new peptide inhibitor to be the first that does neither bind to the Lipid Binding Domain (LBD) nor to the Protein Binding Domain (PBD), but just in between the two. A patent application has meanwhile been filed.

Case report 2 of 2: Optimization of a bicycle-CLIPS inhibitor of the uPA

Figure 3. Results of a full replacement analysis of the 6×6 bicycle-CLIPS uPA inhibitor (Heinis et al., ACS Chem Biol. 2012)

In collaboration with Professor Christian Heinis, who applied Pepscan’s CLIPS technology in phage display libraries for the first time and is also the co-founder of Bicycle Therapeutics, we worked on the optimization of UK18 (ACT3SRYEVDCT3RGRGSACT3AGAA), a lead inhibitor identified by Heinis and Winter in a phage-display screening campaign with a 6×6 bicycle-CLIPS library against the target protein uPA (uPA; ACS Chem. Biol. 2012). Previous attempts to optimize the activity of this inhibitor using computational modelling had failed (Angew. Chem. 2013).

Using Pepscan’s peptide array, we identified four different amino acid replacements that showed a small but decent improvement in both uPA binding as well as inhibitory activity towards uPA. The individual mutations (L-A-1/L-Ile, L-Val-6/L-Thr, Gly-11/D-Asp, L-Ala-13/L-Leu) showed increases of the IC50 values ranging from 1.17-1.51 (fold-change, FC). When combined in the fully optimized bicycle-CLIPS inhibitor, these four mutations together improved the activity almost 2-fold compared to the original lead compound (i.e. IC50 from 68.0 nM to 34.9 nM, Ki from 48.3 to 24.8 nM).

Figure 4. Structure of lead and fully optimized bicycle-CLIPS uPA inhibitors

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