Design and synthesis of a new orthogonally protected glutamic acid analog and its use in the preparation of high affinity polo-like kinase 1 polo-box domain - binding peptide macrocycles

Abstract

Targeting protein - protein interactions (PPIs) has emerged as an important area of discovery for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the polo-like kinase 1 (Plk1) polo-box domain (PBD), a phosphorylated protein residue can provide high-affinity recognition and binding to target protein hot spots. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden "cryptic" pockets that may be revealed on ligand binding. In our current paper, we describe the design and synthesis of macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a phosphothreonine (pT) residue for optimal interaction in the signature phospho-binding pocket. Macrocycles prepared with this new amino acid analog introduce additional hydrogen-bonding interactions not found in the open-chain linear parent peptide. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets.

Fig. 1. Structures of peptides discussed in the text (*ref. 34).

Scheme 1. Synthesis of key macrocycle junction – forming residue 6.

Scheme 2. Synthesis of chain-to-chain cyclic PBD hexapeptides of type 7.

Fig. 2. Comparison of crystal structures of 2 and 7a bound the Plk1 PBD. (A) Crystal structure of PBD-bound 2 (PDB: 3RQ7; carbons purple) superimposed onto the crystal structure of PBD-bound 7a (carbons cyan). The protein surface is rendered as electrostatic potential (blue = positive; red = negative; white = neutral), with key binding regions color-highlighted (phosphate-binding pocket = lime; cryptic pocket = cyan; pyrrolidine-binding region = orange); (B) closeup of the critical region where the alkylphenyl groups join the peptides; (C) closeup of the C-terminal residues; (D) closeup of N-terminal interactions.

Fig. 3. Electron density for 7a macrocycle. The final 2F_o − F_c electron density map surrounding the macrocycle is shown at a contour level of 1.25σ. The hydrogens in the final refined model have been omitted for clarity.

Fig. 4. Structures of pT, Pmab analogs and Pmab-containing macrocycle 13.

Fig. 5. X-ray crystal structure of PDB – 7a highlighting hydrogen bonding interactions in the phosphate – binding pocket and the lack of interactions with the phosphoryl ether oxygen (shown in yellow circle).