A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

A Sos proteomimetic as a pan-Ras inhibitor.

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

Macrocyclic ß-Sheets Stabilized by Hydrogen Bond Surrogates.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. ß-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-ß-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic ß-sheets without the requirement for natural or synthetic ß-turns, thereby providing a minimal mimic of a protein ß-sheet. To access turn-less antiparallel ß-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein ß-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in ß-sheets. In correspondence with natural ß-sheets, we find that minimal HBS mimics show robust ß-sheet formation when specific amino acid residue pairings are incorporated. In isolated ß-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.

Two-Component Redox Organocatalyst for Peptide Bond Formation.

Peptides are fundamental therapeutic modalities whose sequence-specific synthesis can be automated. Yet, modern peptide synthesis remains atom uneconomical and requires an excess of coupling agents and protected amino acids for efficient amide bond formation. We recently described the rational design of an organocatalyst that can operate on Fmoc amino acids─the standard monomers in automated peptide synthesis (J. Am. Chem. Soc. 2019, 141, 15977). The catalytic cycle centered on the conversion of the carboxylic acid to selenoester, which was activated by a hydrogen bonding scaffold for amine coupling. The selenoester was generated in situ from a diselenide catalyst and stoichiometric amounts of phosphine. Although the prior system catalyzed oligopeptide synthesis on solid phase, it had two significant requirements that limited its utility as an alternative to coupling agents─it depended on stoichiometric amounts of phosphine and required molecular sieves as dehydrating agent. Here, we address these limitations with an optimized method that requires only catalytic amounts of phosphine and no dehydrating agent. The new method utilizes a two-component organoreductant/organooxidant-recycling strategy to catalyze amide bond formation.

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

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.

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

Radical addition to dehydroalanine (Dha) represents an appealing, modular strategy to access non-canonical peptide analogues for drug discovery. Prior studies on radical addition to the Dha residue of peptides and proteins have demonstrated outstanding functional group compatibility, but the lack of stereoselectivity has limited the synthetic utility of this approach. Herein, we address this challenge by employing chiral nickel catalysts to control the stereoselectivity of radical addition to Dha on oligopeptides. The conditions accommodate a variety of primary and secondary electrophiles to introduce polyethylene glycol, biotin, halo-tag, and hydrophobic and hydrophilic side chains to the peptide. The reaction features catalyst control to largely override substrate-based control of stereochemical outcome for modification of short peptides. We anticipate that the discovery of chiral nickel complexes that confer catalyst control will allow rapid, late-stage modification of peptides featuring nonnatural sidechains.

Dual Control of Peptide Conformation with Light and Metal Coordination.

The design of a stimuli-responsive peptide whose conformation is controlled by wavelength-specific light and metal coordination is described. The peptide adopts a defined tertiary structure and its conformation can be modulated between an α-helical coiled coil and ß-sheet. The peptide is designed with a hydrophobic interface to induce coiled coil formation and is based on a recently described strategy to obtain switchable helix dimers. Herein, we endowed the helix dimer with 8-hydroxyquinoline (HQ) groups to achieve metal coordination and shift to a ß-sheet structure. It was found that the conformational shift only occurs upon introduction of Zn2+ ; other metal ions (Cu2+ , Fe3+ , Co2+ , Mg2 , and Ni2+ ) do not offer switching likely due to non-specific metal-peptide coordination. A control peptide lacking the metal-coordinating residues does not show conformational switching with Zn2+ supporting the role of this metal in stabilizing the ß-sheet conformation in a defined manner.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 2 million fatalities since it first emerged in late 2019. As we write, infection rates are at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary target of SARS-CoV-2 is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results while finding additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

Macropinocytosis as a Key Determinant of Peptidomimetic Uptake in Cancer Cells.

Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, ß-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.

Rational Design of an Organocatalyst for Peptide Bond Formation.

Amide bonds are ubiquitous in peptides, proteins, pharmaceuticals, and polymers. The formation of amide bonds is a straightforward process: amide bonds can be synthesized with relative ease because of the availability of efficient coupling agents. However, there is a substantive need for methods that do not require excess reagents. A catalyst that condenses amino acids could have an important impact by reducing the significant waste generated during peptide synthesis. We describe the rational design of a biomimetic catalyst that can efficiently couple amino acids featuring standard protecting groups. The catalyst design combines lessons learned from enzymes, peptide biosynthesis, and organocatalysts. Under optimized conditions, 5 mol % catalyst efficiently couples Fmoc amino acids without notable racemization. Importantly, we demonstrate that the catalyst is functional for the synthesis of oligopeptides on solid phase. This result is significant because it illustrates the potential of the catalyst to function on a substrate with a multitude of amide bonds, which may be expected to inhibit a hydrogen-bonding catalyst.

Synthetic Control of Tertiary Helical Structures in Short Peptides.

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 Domain Mimics as Modulators of Protein-Protein Interactions.

Protein-protein interactions (PPIs) are ubiquitous in biological systems and often misregulated in disease. As such, specific PPI modulators are desirable to unravel complex PPI pathways and expand the number of druggable targets available for therapeutic intervention. However, the large size and relative flatness of PPI interfaces make them challenging molecular targets. This Account describes our systematic approach using secondary and tertiary protein domain mimics (PDMs) to specifically modulate PPIs. Our strategy focuses on mimicry of regular secondary and tertiary structure elements from one of the PPI partners to inspire rational PDM design. We have compiled three databases (HIPPDB, SIPPDB, and DIPPDB) of secondary and tertiary structures at PPI interfaces to guide our designs and better understand the energetics of PPI secondary and tertiary structures. Our efforts have focused on three of the most common secondary and tertiary structures: α-helices, ß-strands, and helix dimers (e.g., coiled coils). To mimic α-helices, we designed the hydrogen bond surrogate (HBS) as an isosteric PDM and the oligooxopiperazine helix mimetic (OHM) as a topographical PDM. The nucleus of the HBS approach is a peptide macrocycle in which the N-terminal i, i + 4 main-chain hydrogen bond is replaced with a covalent carbon-carbon bond. In mimicking a main-chain hydrogen bond, the HBS approach stabilizes the α-helical conformation while leaving all helical faces available for functionalization to tune binding affinity and specificity. The OHM approach, in contrast, envisions a tetrapeptide to mimic one face of a two-turn helix. We anticipated that placement of ethylene bridges between adjacent amides constrains the tetrapeptide backbone to mimic the i, i + 4, and i + 7 side chains on one face of an α-helix. For ß-strands, we developed triazolamers, a topographical PDM where the peptide bonds are replaced by triazoles. The triazoles simultaneously stabilize the extended, zigzag conformation of ß-strands and transform an otherwise ideal protease substrate into a stable molecule by replacement of the peptide bonds. We turned to a salt bridge surrogate (SBS) approach as a means for stabilizing very short helix dimers. As with the HBS approach, the SBS strategy replaces a noncovalent interaction with a covalent bond. Specifically, we used a bis-triazole linkage that mimics a salt bridge interaction to drive helix association and folding. Using this approach, we were able to stabilize helix dimers that are less than half of the length required to form a coiled coil from two independent strands. In addition to demonstrating the stabilization of desired structures, we have also shown that our designed PDMs specifically modulate target PPIs in vitro and in vivo. Examples of PPIs successfully targeted include HIF1α/p300, p53/MDM2, Bcl-xL/Bak, Ras/Sos, and HIV gp41. The PPI databases and designed PDMs created in these studies will aid development of a versatile set of molecules to probe complex PPI functions and, potentially, PPI-based therapeutics.

Targeting Unoccupied Surfaces on Protein-Protein Interfaces.

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.

Effects of side chains in helix nucleation differ from helix propagation.

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.

In vivo modulation of hypoxia-inducible signaling by topographical helix mimetics.

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.

Side-Chain Conformational Preferences Govern Protein-Protein Interactions.

Protein secondary structures serve as geometrically constrained scaffolds for the display of key interacting residues at protein interfaces. Given the critical role of secondary structures in protein folding and the dependence of folding propensities on backbone dihedrals, secondary structure is expected to influence the identity of residues that are important for complex formation. Counter to this expectation, we find that a narrow set of residues dominates the binding energy in protein-protein complexes independent of backbone conformation. This finding suggests that the binding epitope may instead be substantially influenced by the side-chain conformations adopted. We analyzed side-chain conformational preferences in residues that contribute significantly to binding. This analysis suggests that preferred rotamers contribute directly to specificity in protein complex formation and provides guidelines for peptidomimetic inhibitor design.

Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling.

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.

An Effective Strategy for Stabilizing Minimal Coiled Coil Mimetics.

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.