Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

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 (Kd=120 nM,MW=734Da). 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.

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

“…Zyxin, which contains four clustered FP4 motifs, has been shown to bind to the VASP EVH1 domain by contacting both the canonical FP4 site and a noncanonical site on the opposite side of the EVH1 domain (Acevedo et al, 2017). Interestingly, a crystal structure of the ENAH EVH1 domain bound to a single-FP4 motif peptide at the canonical site also contains a second single-FP4 peptide bound to the region corresponding to the noncanonical binding site in VASP (PDB 5NC7, Barone, 2020). To test whether multi-FP4 peptides engage this noncanonical site, we designed ENAH EVH1 R47A.…”

“…Barone, et al have shown that small-molecule Ena/VASP EVH1 domain inhibitors reduce breast cancer-mediated invasion in zebrafish models, and in in vitro invasion assays, establishing Ena/VASP EVH1 domains as promising therapeutic targets (21). Yet Ena/VASP paralogs have distinct functions in cancer.…”

“…For example, reduced expression of ENAH or VASP decreases invasive potential in migratory breast cancer cells (17, 18), whereas EVL suppresses breast cancer cell invasion (19). Previous work has explored the development of ENAH EVH1 inhibitors (20, 21), but previously reported molecules lack high paralog selectivity. This is not surprising, given that the three proteins are 100% identical in sequence in the core FP4 binding groove and share 62-72% sequence similarity throughout the EVH1 domain.…”

Fe65: A Scaffolding Protein of Actin Regulators

“…Hence, it is not surprising that Fe65, APP, Mena, cortactin, and ELMO/DOCK180/Rac are involved in suppressing or supporting different kinds of cancerous signaling cascades, for example, in breast, thyroid, colon, lung, and pancreas cancer [ 56 , 128 , 185 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 ]. It was astonishing that especially breast cancer was highly investigated in association with Fe65 interaction partners (APP, Mena, cortactin, ELMO/DOCK180/Rac) separately but not in a common pathway [ 56 , 187 , 188 , 189 , 190 , 191 , 196 , 197 , 198 , 199 , 200 , 201 , 202 , 203 , 204 ].…”

Improved Synthesis of MediPhos Ligands and Their Use in the Pd‐Catalyzed Enantioselective N‐Allylation of Glycine Esters

“…Aiming at the synthesis of the dipeptide mimetic ProM‐17 ( 9 ) [5c] we recognized that the developed amination protocol afforded the required amine building block, i. e. the N‐allylated glycine ester 10 a , only with unsatisfying enantioselectivity (80 % ee ) under the established conditions using L3* (Scheme 3; Table 1).…”