Peptide Tethering: Pocket-Directed Fragment Screening for Peptidomimetic Inhibitor Discovery

Abstract

Constrained peptides have proven to be a rich source of ligands for protein surfaces, but are often limited in their binding potency. Deployment of nonnatural side chains that access unoccupied crevices on the receptor surface offers a potential avenue to enhance binding affinity. We recently described a computational approach to create topographic maps of protein surfaces to guide the design of nonnatural side chains [J. Am. Chem. Soc. 2017, 139, 15560]. The computational method, AlphaSpace, was used to predict peptide ligands for the KIX domain of the p300/CBP coactivator. KIX has been the subject of numerous ligand discovery strategies, but potent inhibitors of its interaction with transcription factors remain difficult to access. Although the computational approach provided a significant enhancement in the binding affinity of the peptide, fine-tuning of nonnatural side chains required an experimental screening method. Here we implement a peptide-tethering strategy to screen fragments as nonnatural side chains on conformationally defined peptides. The combined computational–experimental approach offers a general framework for optimizing peptidomimetics as inhibitors of protein–protein interactions.

Rational peptide design for inhibition of the KIX–MLL interaction

“…In contrast, the IC 50 of the T2857W peptide (5.67 ± 0.01 μM) obtained in this study is comparable to the K d for the KIX–MLL interaction (2.02 ± 0.04 μM) and therefore is significantly better than those of the small molecules. Previous studies have also reported peptide-based inhibitors, including a peptide in which a disulfide crosslink was introduced into MLL TAD to improve the peptide stability 33 and a peptide in which non-natural amino acids were introduced into the MLL TAD using the AlphaSpace software 31 , 34 ; however, the KIX-binding affinity of these peptides was lower than that for the wild-type MLL TAD peptide. The K d value obtained for the T2857W peptide in our study (0.95 ± 0.04 μM) is substantially better, thus facilitating tighter KIX binding.…”

Late‐Stage Modification of Oligopeptides by Nickel‐Catalyzed Stereoselective Radical Addition to Dehydroalanine

“…The conformation, proteolytic stability and molecular recognition properties of peptides are intimately tied to their sequences. The introduction of nonnatural sidechains to therapeutic peptides and peptidomimetics can enhance their potency and selectivity for their cognate receptors [1–4] . Oligopeptides comprised of nonnatural building blocks have become increasingly prevalent in the pharmaceutical industry (Scheme 1A) [5] .…”

“…The introduction of nonnatural sidechains to therapeutic peptides and peptidomimetics can enhance their potency and selectivity for their cognate receptors. [1][2][3][4] Oligopeptides comprised of nonnatural building blocks have become increasingly prevalent in the pharmaceutical industry (Scheme 1A). [5] For example, the nonnatural residue L-Hfe (Hfe = homo-phenylalanine) is a constituent of cardiovascular drugs cilazapril, lisinopril, [6] and enalapril 1, [7] and a proteasome inhibitor, carfilzomib, used for treating multiple myeloma.…”

“…[10] Solid-phase peptide synthesis (SPPS) assembles noncanonical peptides from individual building blocks, but the bottom-up approach is not amenable to efficient diversification to prepare analogues for screening of biological activity, as each variant would require a full synthesis-an alternative approach would incorporate side chains as fragments to explore structure-activity relationships at discreet positions. [4] Dha can be introduced into proteins and peptides by genetic [11] and chemical modification of cysteine (Cys), serine (Ser), threonine (Thr), or selenocysteine (Sec). [12] Late-stage modification of dehydroalanine (Dha) residue offers an appealing strategy to rapidly diversify a peptide sequence by introducing a variety of nonnatural coupling partners (Scheme 1B).…”

Synthesis and Studies of Bulky Cycloalkyl α,β-Dehydroamino Acids that Enhance Proteolytic Stability

“…On the basis of the documented correlation between a higher degree of rigidity or folding and greater resistance to degradation by proteases, we posited that cyclic ΔAAs might enhance proteolytic stability more than the previously studied acyclic ΔAAs. We also recognized the value of developing a suite of cycloalkyl ΔAAs of varying ring sizes with the ability to fill different-sized binding pockets on the surface of protein targets of interest . A handful of cycloalkyl ΔAAs are known, but they have primarily been used as substrates for enantioselective hydrogenations .…”