Robert Opitz

Addressing Protein–Protein Interactions with Small Molecules: A Pro-Pro Dipeptide Mimic with a PPII Helix Conformation as a Module for the Synthesis of PRD-Binding Ligands†

Interactions of so-called proline-rich motif-recognizing domains (PRDs) with proteins containing proline-rich motifs (PRMs) are widely utilized by nature and are involved in several relevant processes, such as tyrosine kinase receptor signaling, endocytosis, cytoskeletal rearrangements, 6] transcription, and splicing. 9] In recent years, some PRDs were identified as putative therapeutical targets that can possibly be addressed by synthetic small molecules. An example is the Fyn-SH3 domain, which is involved in the regulation of enzymatic activity and the assembly of signaling complexes. A common property of all PRMs is that they preferentially form a left-handed polyproline type II (PPII) helix with an overall shape resembling a triangular prism (Figure 1). This structural element has a helical pitch of 9.3 , three residues per turn, and typical torsion angles F of 758 and Y of 1458. Of note, the PPII helix is characterized by a complete lack of main-chain hydrogen bonding patterns. Structural studies have shown that the recognition of PRMs by the respective domains (PRDs) requires the polyproline region to adopt a PPII conformation. Moreover, the interaction is mostly stabilized by formation of a hydrogen bond between a conserved tryptophane (located at the domain surface) and a carbonyl oxygen at the backbone of the central PRM of the peptide ligand. In the course of our research into the development of small molecules that specifically interfere with intracellular protein–protein interactions, we searched for a conformationally defined diproline mimic in a PPII helix conformation that could be incorporated into peptide chains or related modular constructs. To the best of our knowledge, related attempts in other laboratories have not led to the development of systems with satisfying properties to date. By investigating molecular models, we envisioned that the PPII helix could possibly be stabilized by an appropriate C2 bridge between two adjacent proline residues. Computeraided modeling then suggested that the diproline analogue X, generated by formal introduction of a “rigid” vinylidene bridge, adopts a single preferred conformation that almost perfectly mimics a Pro-Pro unit of a PPII helix (Figure 2). Herein we describe the stereoselective synthesis of this novel compound (as a Fmoc-protected derivative) and its successful incorporation as a Pro-Pro substitute in the core motif of SH3 domain-binding peptides. Figure 1. An ideal PPII helix formed by four l-proline units as seen from the side (left) and along the helix axis (right). C cyan, N blue, O red.

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

Significance Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. Protein domains specialized in recognition of these motifs expose a flat and relatively rigid binding site that preferentially interacts with sequences adopting a left-handed polyproline helix II. Here, we present a toolkit of new chemical entities that enables rational construction of selective small-molecule inhibitors for these protein domains. As proof of principle, we developed a selective, cell-permeable inhibitor of Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains of the Ena/VASP protein family. Invasive breast-cancer cells treated with our EVH1 inhibitor showed strongly reduced cell invasion. 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.

Design and Stereoselective Synthesis of ProM‐2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation

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.

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.