ABSTRACT
Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.
Subject(s)
Breast Neoplasms/drug therapy , Cell Adhesion Molecules/metabolism , DNA-Binding Proteins/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Protein Interaction Domains and Motifs/drug effects , Small Molecule Libraries/pharmacology , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Female , Humans , Jurkat Cells , Proline/metabolism , Protein Binding/drug effects , ZebrafishABSTRACT
With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.
Subject(s)
Peptides/chemistry , Proteins/chemistry , Dipeptides/chemistry , Peptidomimetics , Protein Conformation , StereoisomerismABSTRACT
Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.
Subject(s)
Proline-Rich Protein Domains , Protein Interaction Mapping , Animals , Cell Adhesion Molecules/chemistry , Cell Line, Tumor , Cell Membrane Permeability , Crystallography, X-Ray , Drosophila melanogaster/metabolism , Esterification , Fluorescent Antibody Technique , Humans , Kinetics , Ligands , Microfilament Proteins/chemistry , Models, Molecular , Molecular Weight , Peptides/chemistry , Phosphoproteins/chemistry , Protein Binding , Protein Structure, Tertiary , Pseudopodia , Stress Fibers/metabolism , Zyxin/chemistryABSTRACT
A one-pot protocol for the cyanomethylation of aryl halides through a palladium-catalyzed reaction with isoxazole-4-boronic acid pinacol ester was developed. Mechanistically, the reaction proceeds through (1) Suzuki coupling, (2) base-induced fragmentation, and (3) deformylation as shown by characterization of all postulated intermediates. Under optimized conditions (PdCl(2)dppf, KF, DMSO/H(2)O, 130 °C) a broad spectrum of aryl bromides could be converted into arylacetonitriles with up to 88% yield.
