Development of a 3rd generation HIV-fusion inhibitor
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.
Optimization of a bicycle-CLIPS inhibitor of the uPA
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).