Unconventional Secondary Structure Mimics: Ladder-Rungs

Abstract

Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well-defined patterns. Bioinformatics studies to match side-chain orientations of a novel hydantoin triazole chemotype (1) to protein-protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein-protein interaction uPA⋅uPAR. Consequently, chemotype 1 was made with appropriate side-chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.

Keywords: cancer; peptide; peptidomimetics; protein-protein interactions; uPAR.

Figure 1.

a RMSD (Å) of the best overlays of 1aaa (all stereochemistries) on each of the ideal secondary structures. b Distribution of secondary structures at PPI interfaces where an overlay of <0.3 Å RMSD was deduced (516 interfaces) as derived by DSSP^[18-20] calculations (these do not differentiate strand-turn-strand, parallel- and antiparallel-sheets). c Overlay of 1aaa on uPA in uPA•uPAR (PDB 3bt1, uPA is urokinase plasminogen activator, RMSD 0.14 Å), on Ku70 in Ku70•Ku80 (PDB 1jeq, RMSD 0.19 Å), and on complement C4-A in C4-A•C4-B (PDB 4fxk; RMSD 0.21 Å).

Figure 2.

a Mimic DLL-1aaa overlaid on uPA at ²²Asn, ²¹Ser, and ²⁸Ile without unfavorable interactions of the backbone with uPAR, implying DLL-1nsi should be prepared and tested. b Overlay of a preferred conformer of DDD-1aaa on an ideal strand-turn-strand illustrating how the mimic projects two side-chains above and one below the plane of equator of the secondary structure.

Figure 3.

a Competitive ELISA of all compounds at 50 μM. b Dose response of three (lead) compounds to obtain IC₅₀ values. c Competitive fluorescence polarization to determine the binding, K_i, of the lead compounds 1.

Figure 3.

a Competitive ELISA of all compounds at 50 μM. b Dose response of three (lead) compounds to obtain IC₅₀ values. c Competitive fluorescence polarization to determine the binding, K_i, of the lead compounds 1.

Figure 4.

a Dose response wound healing assay (peptide AE105 at 0.5 μM as a positive control). Data processed using ibidi software (available online).^[36] b Normalized migration and c invasion assays using MDA-MB-231 compared with a DMSO control.

Figure 4.

a Dose response wound healing assay (peptide AE105 at 0.5 μM as a positive control). Data processed using ibidi software (available online).^[36] b Normalized migration and c invasion assays using MDA-MB-231 compared with a DMSO control.

Scheme 1.

Solid phase syntheses of the featured compounds 1.