Helical secondary and tertiary motifs are commonly observed as binding epitopes in natural and engineered protein scaffolds. While several strategies have been described to constrain α-helices or reproduce their binding attributes in synthetic mimics, general strategies to mimic tertiary helical motifs remain in their infancy. We recently described a synthetic strategy to develop helical dimers (J. Am. Chem. Soc. 2015, 137, 11618−11621). We found that replacement of an interhelical salt bridge with a covalent bond can stabilize antiparallel motifs in short sequences. Here we show that the approach can be generalized to obtain antiparallel and parallel dimers as well as trimer motifs. Helical stabilization requires judiciously designed cross-linkers as well as optimal interhelical hydrophobic packing. We anticipate that these mimics would afford new classes of modulators of biological function.

We describe the use of thioester exchange equilibria to measure the propensities of amino acid residues to participate in helical secondary structure at room temperature in the absence of denaturants. Thermally or chemically induced unfolding has previously been employed to measure α-helix propensities among proteinogenic α-amino acid residues, and quantitative comparison with precedents indicates that the thioester exchange system is reliable for residues that lack side chain charge. This system allows the measurement of α-helix propensities for D-α-amino acid residues and propensities of residues with non-proteinogenic backbones, such as those derived from a β-amino acid, to participate in an α-helix-like secondary structure.

Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.

Bacterial DnaK is an ATP-dependent molecular chaperone important for maintaining cellular proteostasis in concert with cofactor proteins. The cofactor DnaJ delivers nonnative client proteins to DnaK and activates its ATPase activity, which is required for protein folding. In the bacterial pathogen Mycobacterium tuberculosis, DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein−protein interactions (PPIs) between DnaK and at least one DnaJ are essential for survival of mycobacteria; hence, these PPIs represent untapped antibacterial targets. Here, we synthesize peptide-based mimetics of DnaJ1 and DnaJ2 N-terminal domains as rational inhibitors of DnaK− cofactor interactions. We find that covalently stabilized DnaJ mimetics are capable of disrupting DnaK−cofactor activity in vitro and prevent mycobacterial recovery from proteotoxic stress in vivo, leading to cell death. Since chaperones and cofactors are highly conserved, we anticipate these results will inform the design of other mimetics to modulate chaperone function across cell types.

A thiol-thioester exchange system has been used to measure the propensities of diverse β-amino acid residues to participate in an α-helix-like conformation. These measurements depend on formation of a parallel coiled-coil tertiary structure when two peptide segments become linked by thioester formation. One peptide segment contains a "guest" site that accommodates diverse β residues and is distal to the coiled-coil interface. We find that helix propensity is influenced by side chain placement within the β residue [β (side chain adjacent to nitrogen) slightly favored relative to β (side chain adjacent to carbonyl)]. The previously recognized helix stabilization resulting from five-membered ring incorporation is quantified. These results are significant because so few quantitative thermodynamic measurements have been reported for α/β-peptide folding.