The use of peptidomimetic scaffolds to target protein–protein interfaces is a promising strategy for inhibitor design. The strategy relies on mimicry of protein motifs that exhibit a concentration of native hot spot residues. To address this constraint, we present a pocket-centric computational design strategy guided by AlphaSpace to identify high-quality pockets near the peptidomimetic motif that are both targetable and unoccupied. Alpha-clusters serve as a spatial representation of pocket space and are used to guide the selection of natural and non-natural amino acid mutations to design inhibitors that optimize pocket occupation across the interface. We tested the strategy against a challenging protein–protein interaction target, KIX/MLL, by optimizing a single helical motif within MLL to compete against the full-length wild-type MLL sequence. Molecular dynamics simulation and experimental fluorescence polarization assays are used to verify the efficacy of the optimized peptide sequence.

Substitution of a main chain i → i + 4 hydrogen bond with a covalent bond can nucleate and stabilize the α-helical conformation in peptides. Herein we describe the potential of different alkene isosteres to mimic intramolecular hydrogen bonds and stabilize α-helices in diverse peptide sequences.

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.

Chemoselective reactions for amide bond formation have transformed the ability to access synthetic proteins and other bioconjugates through ligation of fragments. In these ligations, amide bond formation is accelerated by transient enforcement of an intramolecular reaction between the carboxyl and the amine termini of two fragments. Building on this principle, we introduce an aldehyde capture ligation that parlays the high chemoselective reactivity of aldehydes and amines to enforce amide bond formation between amino acid residues and peptides that are difficult to ligate by existing technologies.

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.

•Protein–protein interactions are fertile and challenging targets for inhibition.•Protein–protein interaction analysis and classification aids in inhibitor selection.•Mimicry of critical protein domains offers an attractive strategy for inhibitor design.•Computational tools support every phase of inhibitor design.Protein–protein interactions (PPIs) are emerging as attractive targets for drug design because of their central role in directing normal and aberrant cellular functions. These interactions were once considered “undruggable” because their large and dynamic interfaces make small molecule inhibitor design challenging. However, landmark advances in computational analysis, fragment screening and molecular design have enabled development of a host of promising strategies to address the fundamental molecular recognition challenge. An attractive approach for targeting PPIs involves mimicry of protein domains that are critical for complex formation. This approach recognizes that protein subdomains or protein secondary structures are often present at interfaces and serve as organized scaffolds for the presentation of side chain groups that engage the partner protein(s). Design of protein domain mimetics is in principle rather straightforward but is enabled by a host of computational strategies that provide predictions of important residues that should be mimicked. Herein we describe a workflow proceeding from interaction network analysis, to modeling a complex structure, to identifying a high-affinity sub-structure, to developing interaction inhibitors. We apply the design procedure to peptidomimetic inhibitors of Ras-mediated signaling.

Protein–protein interactions encompass large surface areas, but often a handful of key residues dominate the binding energy landscape. Rationally designed small molecule scaffolds that reproduce the relative positioning and disposition of important binding residues, termed “hotspot residues”, have been shown to successfully inhibit specific protein complexes. Although this strategy has led to development of novel synthetic inhibitors of protein complexes, often direct mimicry of natural amino acid residues does not lead to potent inhibitors. Experimental screening of focused compound libraries is used to further optimize inhibitors but the number of possible designs that can be efficiently synthesized and experimentally tested in academic settings is limited. We have applied the principles of computational protein design to optimization of nonpeptidic helix mimics as ligands for protein complexes. We describe the development of computational tools to design helix mimetics from canonical and noncanonical residue libraries and their application to two therapeutically important protein–protein interactions: p53-MDM2 and p300-HIF1α. The overall study provides a streamlined approach for discovering potent peptidomimetic inhibitors of protein–protein interactions.

The development of inhibitors for protein–protein interactions frequently involves the mimicry of secondary structure motifs. While helical protein–protein interactions have been heavily targeted, a similar level of success for the inhibition of β-strand and β-sheet rich interfaces has been elusive. We describe an assessment of the full range of β-strand interfaces whose high-resolution structures are available in the Protein Data Bank. This analysis identifies complexes where a β-stand or β-sheet contributes significantly to binding. The results highlight the molecular recognition complexity in strand-mediated interactions relative to helical interfaces and offer guidelines for the construction of β-strand and β-sheet mimics as ligands for protein receptors. The online data set will potentially serve as an entry-point to new classes of protein–protein interaction inhibitors.

Helix–coil transition theory connects observable properties of the α-helix to an ensemble of microstates and provides a foundation for analyzing secondary structure formation in proteins. Classical models account for cooperative helix formation in terms of an energetically demanding nucleation event (described by the σ constant) followed by a more facile propagation reaction, with corresponding s constants that are sequence dependent. Extensive studies of folding and unfolding in model peptides have led to the determination of the propagation constants for amino acids. However, the role of individual side chains in helix nucleation has not been separately accessible, so the σ constant is treated as independent of sequence. We describe here a synthetic model that allows the assessment of the role of individual amino acids in helix nucleation. Studies with this model lead to the surprising conclusion that widely accepted scales of helical propensity are not predictive of helix nucleation. Residues known to be helix stabilizers or breakers in propagation have only a tenuous relationship to residues that favor or disfavor helix nucleation.

Development of small-molecule inhibitors of protein–protein interactions is a fundamental challenge at the interface of chemistry and cancer biology. Successful methods for design of protein–protein interaction inhibitors include computational and experimental high-throughput and fragment-based screening strategies to locate small-molecule fragments that bind protein surfaces. An alternative rational design approach seeks to mimic the orientation and disposition of critical binding residues at protein interfaces. We describe the design, synthesis, biochemical, and in vivo evaluation of a small-molecule scaffold that captures the topography of α-helices. We designed mimics of a key α-helical domain at the interface of hypoxia-inducible factor 1α and p300 to develop inhibitors of hypoxia-inducible signaling. The hypoxia-inducible factor/p300 interaction regulates the transcription of key genes, whose expression contributes to angiogenesis, metastasis, and altered energy metabolism in cancer. The designed compounds target the desired protein with high affinity and in a predetermined manner, with the optimal ligand providing effective reduction of tumor burden in experimental animal models.

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for bioorganic and medicinal chemists. This Perspective discusses emerging principles for the rational design of PPI inhibitors. Fundamentally, the approach seeks to adapt nature’s protein recognition principles for the design of suitable secondary structure mimetics.

Selective blockade of gene expression by designed small molecules is a fundamental challenge at the interface of chemistry, biology, and medicine. Transcription factors have been among the most elusive targets in genetics and drug discovery, but the fields of chemical biology and genetics have evolved to a point where this task can be addressed. Herein we report the design, synthesis, and in vivo efficacy evaluation of a protein domain mimetic targeting the interaction of the p300/CBP coactivator with the transcription factor hypoxia-inducible factor-1α. Our results indicate that disrupting this interaction results in a rapid down-regulation of hypoxia-inducible genes critical for cancer progression. The observed effects were compound-specific and dose-dependent. Gene expression profiling with oligonucleotide microarrays revealed effective inhibition of hypoxia-inducible genes with relatively minimal perturbation of nontargeted signaling pathways. We observed remarkable efficacy of the compound HBS 1 in suppressing tumor growth in the fully established murine xenograft models of renal cell carcinoma of the clear cell type. Our results suggest that rationally designed synthetic mimics of protein subdomains that target the transcription factor–coactivator interfaces represent a unique approach for in vivo modulation of oncogenic signaling and arresting tumor growth.

Oligomers composed of β3-amino acid residues and a mixture of α- and β3-residues have emerged as proteolytically stable structural mimics of α-helices. An attractive feature of these oligomers is that they adopt defined conformations in short sequences. In this manuscript, we evaluate the impact of β3-residues as compared to their α-amino acid analogs in prenucleated helices. Our hydrogen–deuterium exchange results suggest that heterogeneous sequences composed of “αααβ” repeats are conformationally more rigid than the corresponding homogeneous α-peptide helices, with the macrocycle templating the helical conformation having a significant influence.

Appropriately-placed hydrogen bond surrogates have been demonstrated to efficiently nucleate helical conformations. Herein we describe an efficient method for the synthesis of thioether-based hydrogen bond surrogate (teHBS) helices. A teHBS helix is shown to adopt a stable conformation and target its cognate protein receptor with high affinity.

Structure-based design of synthetic inhibitors of protein–protein interactions (PPIs) requires adept molecular design and synthesis strategies as well as knowledge of targetable complexes. To address the significant gap between the elegant design of helix mimetics and their sporadic use in biology, we analyzed the full set of helical protein interfaces in the Protein Data Bank to obtain a snapshot of how helices that are critical for complex formation interact with the partner proteins. The results of this study are expected to guide the systematic design of synthetic inhibitors of PPIs. We have experimentally evaluated new classes of protein complexes that emerged from this data set, highlighting the significance of the results described herein.

A new class of nonpeptidic α-helix mimetics derived from α-amino acids and featuring chiral backbones is described. NMR and circular dichroism spectroscopies, in combination with molecular modeling studies, provide compelling evidence that oligooxopiperazine dimers adopt stable conformations that reproduce the arrangement of i, i+4, and i+7 residues on an α-helix.

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein–protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.

Synthetic inhibitors of protein−protein interactions are being discovered despite the inherent challenge in targeting large contact surfaces with small molecules. An analysis of available examples identifies common features of complexes that make them tractable for small molecules. We deduced that relative disposition and energetic contributions of “hot spot” residues provide a predictive scale for the potential of protein−protein interactions to be inhibited by small molecules. On the basis of this model, we analyzed the full set of helical protein interfaces in the Protein Data Bank to identify those that are potentially suitable candidates for synthetic ligands.

Solid phase synthesis of HBS helices involving the Fukuyama–Mitsunobu reaction and triphosgene coupling is described.

Designed ligands that inhibit hypoxia-inducible gene expression could offer new tools for genomic research and, potentially, drug discovery efforts for the treatment of neovascularization in cancers. We report a stabilized α-helix designed to target the binding interface between the C-terminal transactivation domain (C-TAD) of hypoxia-inducible factor 1α (HIF-1α) and cysteine-histidine rich region (CH1) of transcriptional coactivator CBP/p300. The synthetic helix disrupts the structure and function of this complex, resulting in a rapid downregulation of two hypoxia-inducible genes (VEGF and GLUT1) in cell culture.

In a recent report published in PNAS, Gellman and coworkers describe the design, characterization, and potent activity of α/β-peptides that mimic a long α helix involved in HIV viral entry.

Herein we identify and analyze helical protein interfaces as potential targets for synthetic modulators of protein–protein interactions.

Proteases typically recognize their peptide substrates in extended conformations. General approaches for designing protease inhibitors often consist of peptidomimetics that feature this conformation. Herein we discuss a combination of computational and experimental studies to evaluate the potential of triazole-linked β-strand mimetics as inhibitors of HIV-1 protease activity.Design of nonpeptidic β-strand mimetics as protease inhibitors is reported.

α-Helices constitute the largest class of protein secondary structures and play a major role in mediating protein−protein interactions. Development of stable mimics of short α-helices would be invaluable for inhibition of protein−protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in α-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS α-helices feature a carbon−carbon bond derived from a ring-closing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix−coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino acids into the helical orientation inherently limits the stability of short α-helices. The HBS method affords preorganized α-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an attractive strategy for generation of ligands for protein receptors because placement of the cross-link on the inside of the helix does not block solvent-exposed molecular recognition surfaces of the molecule. Our metathesis-based synthetic strategy utilizes standard Fmoc solid phase peptide synthesis methodology, resins, and reagents and provides HBS helices in sufficient amounts for subsequent biophysical and biological analyses. Extensive conformational analysis of HBS α-helices with 2D NMR, circular dichroism spectroscopies and X-ray crystallography confirms the α-helical structure in these compounds. The crystal structure indicates that all i and i + 4 C═O and NH hydrogen-bonding partners fall within distances and angles expected for a fully hydrogen-bonded α-helix. The backbone conformation of HBS α-helix in the crystal structure superimposes with an rms difference of 0.75 Å onto the backbone conformation of a model α-helix. Significantly, the backbone torsion angles for the HBS helix residues fall within the range expected for a canonical α-helix. Thermal and chemical denaturation studies suggest that the HBS approach provides exceptionally stable α-helices from a variety of short sequences, which retain their helical conformation in aqueous buffers at exceptionally high temperatures. The high degree of thermal stability observed for HBS helices is consistent with the theoretical predictions for a nucleated helix. The HBS approach was devised to afford internally constrained helices so that the molecular recognition surface of the helix and its protein binding properties are not compromised by the constraining moiety. Notably, our preliminary studies illustrate that HBS helices can target their expected protein receptors with high affinity.

We report the design, synthesis, and characterization of a short peptide trapped in a π-helix configuration. This high-energy conformation was nucleated by a preorganized π-turn, which was obtained by replacing an N-terminal intramolecular main chain i and i + 5 hydrogen bond with a carbon−carbon bond. Our studies highlight the nucleation parameter as a key factor contributing to the relative instability of the π-helix and allow us to estimate fundamental helix−coil transition parameters for this conformation.

We have reported a new class of artificial α-helices in which a pre-organized α-turn nucleates the helical conformation [R. N. Chapman, G. Dimartino, and P. S. Arora, J. Am. Chem. Soc., 2004, 126, 12252 and D. Wang, K. Chen, J. L. Kulp, III, and P. S. Arora, J. Am. Chem. Soc., 2006, 128, 9248]. This manuscript describes the effect of the core nucleation template on the overall helicity of the peptides and demonstrates that the macrocycle which most closely mimics the 13-membered hydrogen-bonded α-turn in canonical α-helices also affords the most stable artificial α-helix. We also investigate the stability of these synthetic helices through classical helix–coil parameters and find that the denaturation behavior of HBS α-helices agrees with the theoretical properties of a peptide with a well-defined and stable helix nucleus.

Examination of complexes of proteins with other biomolecules reveals that proteins tend to interact with partners via folded subdomains, in which the backbone possesses secondary structure. α-Helices, the largest class of protein secondary structures, play fundamental roles in a multitude of highly specific protein–protein and protein–nucleic acids interactions. Herein, we describe the potential of a helix nucleation strategy to afford modulators of protein–protein interactions.

Over the past decade, protein–protein interactions (PPIs) have gone from being neglected as ‘undruggable’ to being considered attractive targets for the development of therapeutics. Recent advances in computational analysis, fragment-based screening, and molecular design have revealed promising strategies to address the basic molecular recognition challenge: how to target large protein surfaces with specificity. Several systematic and complementary workflows have been developed to yield successful inhibitors of PPIs. Here we review the major contemporary approaches utilized for the discovery of inhibitors and focus on a structure-based workflow, from the selection of a biological target to design.

Solid phase synthesis of HBS helices involving the Fukuyama–Mitsunobu reaction and triphosgene coupling is described.

Appropriately-placed hydrogen bond surrogates have been demonstrated to efficiently nucleate helical conformations. Herein we describe an efficient method for the synthesis of thioether-based hydrogen bond surrogate (teHBS) helices. A teHBS helix is shown to adopt a stable conformation and target its cognate protein receptor with high affinity.

Substitution of a main chain i → i + 4 hydrogen bond with a covalent bond can nucleate and stabilize the α-helical conformation in peptides. Herein we describe the potential of different alkene isosteres to mimic intramolecular hydrogen bonds and stabilize α-helices in diverse peptide sequences.

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein–protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.