Coiled coils are a major motif in proteins and orchestrate multimerization of various complexes important for biological processes. Inhibition of coiled coil-mediated interactions has significant biomedical potential. However, general approaches that afford short peptides with defined coiled coil conformation remain elusive. We evaluated several strategies to stabilize minimal helical bundles, with the dimer motif as the initial focus. A stable dimeric scaffold was realized in a synthetic sequence by replacing an interhelical ionic bond with a covalent bond. Application of this strategy to a more challenging native protein–protein interaction (PPI) suggested that an additional constraint, a disulfide bond at the internal a/d′ position along with a linker at the e/e′ position, is required for enhanced conformational stability. We anticipate the coiled coil stabilization methodology described herein to yield new classes of modulators for PPIs.

The modulation of protein–protein interactions (PPIs) by means of creating or stabilizing secondary structure conformations is a rapidly growing area of research. Recent success in the inhibition of difficult PPIs by secondary structure mimetics also points to potential limitations, because often, specific cases require tertiary structure mimetics. To streamline protein structure-based inhibitor design, we have previously described the examination of protein complexes in the Protein Data Bank where α-helices or β-strands form critical contacts. Here, we examined coiled coils and helix bundles that mediate complex formation to create a platform for the discovery of potential tertiary structure mimetics. Though there has been extensive analysis of coiled coil motifs, the interactions between pre-formed coiled coils and globular proteins have not been systematically analyzed. This article identifies critical features of these helical interfaces with respect to coiled coil and other helical PPIs. We expect the analysis to prove useful for the rational design of modulators of this fundamental class of protein assemblies.

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.

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.

Rationally designed protein-protein interaction inhibitors mimic interfacial binding epitopes, specifically residues that contribute significantly to binding. However, direct mimicry often does not lead to high affinity ligands because the natural complexes themselves are functionally transient and of low affinity. The mimics typically need to be optimized for potency. Engineered proteins displaying conformationally-defined epitopes may serve as attractive alternatives to natural protein partners as they can be strictly screened for tight binding. The advantage of focused screens with conformationally-defined protein scaffolds is that conservation of the geometry of the natural binding epitopes may preserve binding site specificity while allowing direct mimicry by various synthetic secondary structure scaffolds. Here we review different classes of engineered proteins for their binding epitope geometry and as leads for synthetic secondary and tertiary structure mimics.