Ligand Design for PRDs

A first strategy for PRM:PRD inhibition was based on the introduction of non-natural amino acids into the known "wild-type" peptides (Nguyen et al. 1998, 2000). They demonstrated that the second proline residue of the PxxP motif recognized by SH3 domains could be replaced with ^-alkylated glycine derivatives to create ligands exhibiting enhanced affinities. Their strategy for inhibitor design can be outlined as follows: maintain the required hybrid Ca- and N-substituted scaffold, but vary side-chain identity along this scaffold to optimize complementarity. Using this strategy, they could not only enhance the affinity of the peptoid structures compared to wild type peptides, but also improve the selectivity within the family of SH3 domains. The replacement of prolines in the conserved

PxxP motif by N-substituted glycines was also successfully applied to peptides recognized by the class-1 EVH1 domain of human VASP (Zimmermann et al. 2003). It could be shown that the first proline of the PxxP motif of the ActA-derived peptide SFEFPPPPTEDEL can be replaced by an N-propylphenyl-substi-tuted glycine without loss of binding. However, substitution of the last proline with this approach was not possible, indicating that successful replacement of prolines within a PPII helix is position-dependent. The ability of these structures to replace prolines depends mostly on their ability to reach additional binding sites on the domain surface. The relevance of epitope recognition was exemplified for the EVH1 domain (Zimmermann et al. 2003). It was found that the mutated peptide SFEAPPPPTEDEL is not able to bind to the class-1 EVH1 domain due to the F to A exchange at position 4. However, the binding could be restored by replacement of the first proline (position 5) with N-proylphenyl-gly-cine. In that case, the phenyl ring of the non-natural amino acid is able to reach the F4 binding epitope of the wild type peptide.

For short proline-rich peptides, little or no ordered secondary structure is observed before binding takes place. The association of an unfolded peptide with PRD involves unfavorable binding entropy due to the loss of rotational freedom on forming a PPII helix. Despite the slight preference of unbound PRM peptide structures for forming a PPII helix, only a small fraction of peptides is available in a preformed PPII conformation. Thus, concepts were developed to stabilize the PPII helix by replacing residues within PRM by conformationally restricted PPII mimetics. However, as yet only few examples for isosteric PPII helix modules have been published. Several of those are shown in Fig. 2.

Compound A was synthesized recently as a potential PPII helix isostere (Bandur et al. 2005). The stereo-selective construction of a tri-substituted double bond in an

Fig. 2 Selection of known PPII mimetics

¿■-Pro-Pro isostere induces an anti-periplanar conformational lock that constrains the two rings in a PPII-typical orientation. However, this compound lacks the "central" carbonyl group that seems to be important for the binding to PRDs. Tremmel and Geyer (2002) described the synthesis and the structural analysis of a hexapep-tide surrogate based on a trimer of scaffold B displaying all characteristic features of the PPII helix. The sugar-derived peptide mimetics have the amphiphilic character of Ser-Pro dipeptide units. Castelhano et al. (Witter et al. 1998) (WO 98/54208) developed a highly constrained spirolactam mimetic (compound C) of two prolines within the PLPPLP sequence of SH3-binding peptides. They also showed that incorporating a tricylic spirolactam scaffold into a p85-derived peptide (see compound D) instead of a PV-motif maintained the affinity of the wild-type peptide. The other diastereoisomers (3R,6S; 3S,6S; and 3S,6R) showed lower affinity. Lack of enhanced binding of compound D reflects the absence of entropy gain from constraining the structure. This is in agreement with a recently published study (Ruzza et al. 2006), in which specific proline positions of a PRM peptide corresponding to a sequence 394-403 of hematopoietic progenitor kinase (HPK-1) were replaced by 4(R)- or 4(S)-4-fluoro-L-proline. 4-Fluoro-substituted prolines are known to induce and stabilize the PPII helix in solution (Holmgren et al. 1999; Doi et al. 2005; Nishi et al. 2005). The propensity for a PPII helix was determined using a CD thermal analysis, and the affinity of the HPK-1 peptide to HS1-SH3 was measured. They found that none of the peptides with induced PPII helix was able to bind with higher affinity than the parent peptide. These results clearly show that the induction of a PPII helix in short peptides is not sufficient to increase the affinity towards an SH3 domain. However, the situation is different when peptides are incorporated into a protein scaffold (Golemi-Kotra et al. 2004). Schaepartz et al. used a miniature protein design strategy based on the avian pancreatic polypeptide (aPP). aPP consists of an eight-residue PPII helix linked through a type-I P-turn to a 20-residue helix characterized by stabilizing interactions between the PPII helix and the a-helix. The architecture of aPP enables the protein to display a solventexposed binding epitope at the a-helical or the PPII helical element of the structure. Using this strategy, it was possible to design aPP-based mini-proteins with nanomo-lar affinity to different proteins (Rutledge et al. 2003; Montclare and Schepartz 2003). The application of the "grafting" strategy on the PPII-part of aPP results in a mini-protein that binds with ten-fold higher affinity to Mena than the ActAu ligand from Listeria monocytogenes, the best previously known Mena ligand. The peptides forming the PPII helix in the mini-protein alone showed very weak affinity to Mena. Thus, stabilization of the PPII conformation within the context of a scaffold can improve binding affinity.

Recently, Lawrence and colleagues described a strategy to develop peptide/non-peptide chimeras that bind the SH3 domain of Fyn, a Src kinase family member, in a selective manner (Lawrence 2005; Li and Lawrence 2005). Starting with the consensus peptide RALPPLP coupled to a disulfide-linked Tentagel resin, certain positions flanking the PxxP motif were modified by introducing (L)-2,3,-diami-nopropionic acid (DAP). The strategy behind this is based on the idea that modified residues adjacent to the core motif may contribute significantly to the binding energy by engaging in non-covalent interactions with sub-sites of the domain that cannot be reached by natural amino acids. Using this approach, it was possible to develop a highly selective Fyn-SH3 inhibitor with an affinity in the nanomolar range. Utilizing the recognition of non-conserved sites of individual adaptor domains is a promising way of obtaining specificity. This can only be achieved by recruitment of additional hot spot areas for binding located on the domain surface and related to the non-conserved sequence segments within the domain family.

Another interesting case is the discovery of UCS15A (Sharma et al. 2001; Oneyama et al. 2002; Oneyama et al. 2003). This compound was selected from a screen for Src kinase signaling antagonists using a yeast-based high-throughput assay identifying compounds that are able to rescue the growth arrest of v-Src overexpressing strains. Surprisingly, it was found that although UCS15A was able to inhibit the tyrosine phosphorylation of two Src substrates in a dose-dependent manner, no in vitro inhibition of Src-kinase activity was observed. Follow-up experiments indicated that UCS15A was able to block the SH3-mediated proteinprotein interaction for a number of typical SH3-mediated protein-protein interactions, such as cortactin-Zo1 and Grb2-Sos1. Further, it was shown that the compound binds to the PRM directly and not to SH3 domains. It is interesting to note that UCS15A contains many H-donating groups destined for interaction with exposed main-chain carbonyl oxygens of a PPII helix.

Using the mouse Tec kinase SH3 domain as a model system for structure-based ligand design, Inglis and co-workers (2004, 2006) have identified several simple heterocyclic compounds that selectively bind to the Tec-SH3 domain. They reported that 6-substituted 2-aminochinolines interact with the solvent-exposed Trp of the Tec-SH3 domain core motif. NMR chemical shift perturbation from ['H,15N]-HSQC experiments using 15N-labeled Tec SH3 protein were analyzed to reveal A"D-values within the 125-300 |M range.

In summary, there are several strategies emerging to design peptide-based high-affinity ligands for PRDs. Key building blocks have been identified that might serve as potent leads for the design of second-generation compounds with improved affinities and selectivities.

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