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 proofof-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.Ena | VASP | protein-protein interaction | actin cytoskeleton | cell migration
A practical and scalable synthesis of a Fmoc-protected tricyclic dipeptide mimetic (6), that is, a 1,4-diaza-tricyclo-[8.3.0(3,7)]-tridec-8-ene derivative resembling a rigidified di-L-proline in a polyproline type II (PPII) helix conformation, was developed. The strategy is based on a Ru-catalyzed ring-closing metathesis of a dipeptide (4) prepared by PyBOP coupling of cis-5-vinylproline tert-butylester (2) and trans-N-Boc-3-vinylproline (rac-3) followed by chromatographic diastereomer separation. Building block 2 was prepared from L-proline in six steps via electrochemical C5-methoxylation, cyanation and conversion of the nitrile into a vinyl substituent. Building block rac-3 was prepared in five steps exploiting a Cu-catalyzed 1,4-addition of vinyl-MgBr to a 2,3-dehydroproline derivative in the key step. In the course of the investigation subtle dependencies of protecting groups on the reactivity of the 2,3- and 2,5-disubstituted pyrrolidine derivatives were observed. The configuration and conformational preference of several intermediates were determined by X-ray crystallography. The developed synthesis allows the preparation of substantial amounts of 6, which will be used in the search for new small molecules for the modulation of protein-protein interactions involving proline-rich motifs (PRDs).
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.
Polycyclic proline‐derived scaffolds (ProMs) have recently demonstrated their value as conformationally defined dipeptide analogs for the modular construction of secondary structure mimetics, specifically interfering with PPII helix‐mediated protein–protein interactions. We disclose the stereoselective synthesis of two new tricyclic amino acid scaffolds (ProM‐4 and ProM‐8) that differ from the first generation scaffold ProM‐1 by the size of ring A. Conformational preferences and subtle structural differences of the three homologous scaffolds were analyzed by X‐ray crystallography, computational calculations, and NMR spectroscopy. N‐tert‐butoxycarbonyl(Boc)‐3‐(1‐propenyl)azetidine‐2‐carboxylic acid was prepared from L‐aspartic acid through β‐lactam intermediates. The corresponding piperidine‐based building block rac‐N‐Boc‐3‐vinylpipecolic acid was synthesized by Cu‐catalyzed 1,4‐addition of vinyl‐MgBr to methyl N‐Boc‐2,3‐dehydropipecolate. Target molecules were prepared through peptide coupling of the respective ring A building blocks with cis‐5‐vinylproline tert‐butyl ester and subsequent ring‐closing metathesis. Selective deprotection of a tert‐butyl carbamate (N‐Boc protecting group) in the presence of a tert‐butyl ester was achieved with trifluoroacetic acid at 0 °C.
Das X markiert den Punkt, an dem die Aminosäure X (siehe Struktur; grau C, cyan H, blau N, rot O, gelb Doppelbindung) als Pro‐Pro‐Ersatz in zwei Peptide eingebaut werden konnte, die an die prolinreiche Motive erkennende Domäne Fyn‐SH3 binden, ohne ihre Bindungsfähigkeit zu gefährden. Das Dipeptidanalogon X, das in einer Polyprolin‐Typ‐II‐Helixkonformation fixiert ist, wurde durch stereoselektive Einführung einer Vinylidenbrücke in eine Diprolineinheit erzeugt.
Helixinduktion: Die synthetische tricyclische Aminosäure ProM‐5, die formal durch stereoselektive Einführung einer Vinylidenbrücke in eine Diprolineinheit entsteht, ist ein leistungsstarkes Gerüst für die Nukleierung der α‐Helix‐Bildung in einer linearen Peptidkette. Dieses Verhalten könnte bei der Entwicklung neuer Proteomimetika zur Modulation von Proteinwechselwirkungen genutzt werden.
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