Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1

Abstract

Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.

Figure 1. Sequences and structures of cystine-knot trypsin inhibitors.

(A) Knottin oMCoTI-II (pdb: 1ha9). (B) SOTI-III (pdb: 4aor). Secondary structure is shown as cartoon with surface, and cysteine residues are depicted as yellow sticks; protease-binding regions (or inhibitor loops) are depicted in red, β-sheets - in blue, and α-helices - in green. Cystine-forming residues are marked bold, and the numbering of respective cysteines is according to their appearance in the sequence.

Figure 2. Yeast surface display of SOTI-III wild type and screening against matriptase-1.

(A) Schematic illustration of Aga1p/Aga2p surface-displayed inhibitor (red) flanked by the amino terminal HA (Human influenza hemagglutinin) epitope (green) and the carboxy terminal cMyc epitope (purple). Functional display of the inhibitor is monitored by incubation with biotinylated trypsin followed by fluorescence labeling with streptavidin, R-phycoreythrin conjugate (SPE). (B) FACS histogram overlay of yeast surface presented SOTI-III wild type labeled with anti-cMyc antibody (yellow), trypsin (blue), matriptase-1 (green) and chymotrypsin (brown). (C) FACS overlays of matriptase-1 binder enrichment. The sorting round (R) and the matriptase-1 concentration used in each round (µM) is given in the figures. Dark grey: FACS histogram during sorting. Light grey: FACS histogram during resort (only rounds 2 and 4). (D) Sequence alignment of SOTI-III wild type and matriptase-1-binding SOTI variant 1. Randomized residues are colored in red. Cysteines are depicted in bold letters, while cystine connections are omitted for clarity.

Figure 3. Summary of MCoTI-II-based library design and screening against matriptase-1.

(A) Sequence of open-chain MCoTI-II wild type. Cysteines are depicted in bold letters. R¹ represents the amino-terminal flanking sequence, including the HA-epitope. R² represents the carboxy-terminal flanking sequence, including the cMyc-epitope. Codon randomization for (A), (B), and (D) as indicated by color (at pos. 6 only Lys or Arg was allowed, grey). (B) Secondary structure of MCoTI-II is shown as cartoon with surface, cysteine residues are shown as orange sticks. (C) FACS histograms showing four rounds of sorting with decreasing target concentration for enrichment of matriptase-1 binders. R denotes the sort round with the concentration of matriptase-1 indicated. Actual sort gates are shown. (D) Sequence alignment of matriptase-1-binding MCoTI variants. Cysteines are numbered according to the appearance in the sequence and depicted in bold letters, while cystine connections are omitted for clarity.

Figure 4. Inhibition assay of uPA activation by matriptase-1 on the surface of PC-3 cells.

Depicted is the logarithmic inhibitor concentration against the absorption at 405