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Antagonists (e.g., Ziconotide, Gabapentin) of the CaV2.2 (N-type) calcium channels are used clinically as analgesics for chronic pain. However, their use is limited by narrow therapeutic windows, difficult dosing routes (Ziconotide), misuse and overdoses (Gabapentin), as well as a litany of adverse effects. Expansion of novel pain therapeutics may emerge from mechanism-based interrogation of CaV2.2. Here we report the identification of C2230, an aryloxy-hydroxypropylamine, as a CaV2.2 blocker. C2230 trapped and stabilized inactivated CaV2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited CaV2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited CaV2.2 in dorsal root and trigeminal ganglia neurons from rats, marmosets, and humans in a G-protein-coupled receptor-independent manner. Further, C2230 reduced evoked excitatory postsynaptic currents and excitatory neurotransmitter release in the spinal cord, leading to relief of neuropathic, orofacial, and osteoarthritic pain-like behaviors via three different routes of administration. C2230 also decreased fiber photometry-based calcium responses in the parabrachial nucleus, mitigated aversive behavioral responses to mechanical stimuli after neuropathic injury, and preserved protective pain responses, all without affecting motor or cardiovascular function. Finally, site-directed mutation analysis demonstrated that C2230 binds differently than other known CaV2.2 blockers, making it a promising lead compound for analgesic development.

Transmembrane Ca v 2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Ca v 2.2 (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 Ca v 2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Ca v 2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A 1 R 2 dipeptide in CBD3 as the anchoring Ca v 2.2 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 Ca v 2.2 from CRMP2, reduced membrane Ca v 2.2 expression and Ca 2+ 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 Ca v 2.2 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.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. β-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-β-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic β-sheets without the requirement for natural or synthetic β-turns, thereby providing a minimal mimic of a protein β-sheet. To access turn-less antiparallel β-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein β-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in β-sheets. In correspondence with natural β-sheets, we find that minimal HBS mimics show robust β-sheet formation when specific amino acid residue pairings are incorporated. In isolated β-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., (© 2023 Wiley-VCH GmbH.)

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., (© 2023 Wiley-VCH GmbH.)

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., (© 2022 Wiley-VCH GmbH.)

Minimal protein mimics have yielded novel classes of protein-protein interaction inhibitors; however, this success has not been extended to targeting intrinsically disordered proteins, which represent a significant proportion of important therapeutic targets. We sought to determine the requirements for binding an intrinsically disordered region (IDR) by its native binding partner as a prelude to developing minimal protein mimics that regulate IDR interactions. Our analysis reinforces the hypothesis that IDRs reside on a fulcrum between unfolded and folded states and that a handful of key binding residues on partner protein surfaces dictate their folding. Our studies also suggest that minimal mimics of protein surfaces may not offer specific ligands for IDRs and that it would be more judicious to target the globular protein partners of IDRs.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Metabolic (dysfunction)-associated fatty liver disease (MAFLD) affects a third of the population and is a leading cause of liver-related death. Since no effective treatments exist, novel approaches to drug development are required. Unfortunately, outdated terminology and definitions of the disease are hampering efforts to develop new drugs and treatments. An international consensus panel has put forth an influential proposal for the disease to be renamed from nonalcoholic fatty liver disease (NAFLD) to MAFLD, including a proposal for how the disease should be diagnosed. As allies with the many stakeholders in MAFLD care-including patients, patients' advocates, clinicians, researchers, nurse and allied health groups, regional societies, and others-we are aware of the negative consequences of the NAFLD term and definition. We share the sense of urgency for change and will act in new ways to achieve our goals. Although there is much work to be done to overcome clinical inertia and reverse worrisome recent trends, the MAFLD initiative provides a firm foundation to build on. It provides a roadmap for moving forward toward more efficient care and affordable, sustainable drug and device innovation in MAFLD care. We hope it will bring promising new opportunities for a brighter future for MAFLD care and improve care and outcomes for patients of one of the globe's largest and costliest public health burdens. From this viewpoint, we have revisited this initiative through the perspectives of drug development and regulatory science., Competing Interests: YF has been an editorial board member of Journal of Clinical and Translational Hepatology since 2021. The other authors have no conflict of interests related to this publication. The views presented in this article do not necessarily reflect those of the USA Food and Drug Administration. Any mention of commercial products is for clarification and is not intended as an endorsement., (© 2022 Authors.)

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.

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.

β-Strands are a fundamental component of protein structure, and these extended peptide regions serve as binding epitopes for numerous protein-protein complexes. However, synthetic mimics that capture the conformation of these epitopes and inhibit selected protein-protein interactions are rare. Here we describe covalent and noncovalent β-hairpin mimics of an extended strand region mediating the Tcf4/β-catenin interaction. Our efforts afford a rationally designed lead for an underexplored region of β-catenin, which has been the subject of numerous ligand discovery campaigns.

Minimal mimics of protein conformations provide rationally designed ligands to modulate protein function. The advantage of minimal mimics is that they can be chemically synthesized and coaxed to be proteolytically resistant; a key disadvantage is that minimization of the protein binding epitope may be associated with loss of affinity and specificity. Several approaches to overcome this challenge may be envisioned, including deployment of covalent warheads and use of nonnatural residues to improve contacts with the binding surface. Herein, we describe our computational and experimental efforts to enhance the minimal protein mimics with fragments that can contact undiscovered binding pockets on Mdm2 and MdmX-two well-studied protein partners of p53.

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 β-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 β-sheet structure. It was found that the conformational shift only occurs upon introduction of Zn 2+ ; other metal ions (Cu 2+ , Fe 3+ , Co 2+ , Mg 2 , and Ni 2+ ) 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 Zn 2+ supporting the role of this metal in stabilizing the β-sheet conformation in a defined manner., (© 2021 Wiley-VCH GmbH.)

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., (© 2021 Wiley Periodicals LLC.)

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., Competing Interests: The authors declare no competing interest.

The coiled coil is a common protein tertiary structure intimately involved in mediating protein recognition and function. Due to their structural simplicity, coiled coils have served as attractive scaffolds for the development of functional biomaterials. Herein we describe the design of conformationally-defined coiled coil photoswitches as potential environmentally-sensitive biomaterials.

Examination of complexes of proteins with biomolecular ligands reveals that proteins tend to interact with partners via folded sub-domains, in which the backbone possesses secondary structure. α-Helices comprising the largest class of protein secondary structures, play fundamental roles in a multitude of highly specific protein-protein and protein-nucleic acid interactions. We have demonstrated a unique strategy for stabilization of the α-helical conformation that involves replacement of one of the main chain i and i+4 hydrogen bonds in the target α-helix with a covalent bond. We termed this synthetic strategy a hydrogen bond surrogate (HBS) approach. Two salient features of this approach are: (1) the internal placement of the crosslink allows development of helices such that none of the solvent-exposed surfaces are blocked by the constraining element, i.e., all side chains of the constrained helices remain available for molecular recognition. (2) This approach can be deployed to constrain very short peptides (<10 amino acid residues) into highly stable α-helices. This chapter presents the biophysical basis for the development of the hydrogen bond surrogate approach, as well as methods for the synthesis and conformational analysis of the artificial helices., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoβ (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects., Competing Interests: Competing interest statement: All authors, except for L.M., were employees of Genentech, Inc., a member of the Roche group, at the time this work was performed and own/owned Roche stock and/or options at the time of their employment. L.M. is an employee and shareholder of ProSciento, Inc.; ProSciento, Inc. received research funding from Genentech, Inc. A.B. is currently an employee of Calico Life Sciences. J.S. is currently an employee of Regeneron Pharmaceuticals. R.B. is currently an employee of IgGenix. P.S.A. and L.W.C. are currently employees of Principia Biopharma., (Copyright © 2020 the Author(s). Published by PNAS.)

The emergence of SARS-CoV-2 has caused over a million human deaths and massive global disruption. The viral infection may also represent a threat to our closest living relatives, nonhuman primates. The contact surface of the host cell receptor, ACE2, displays amino acid residues that are critical for virus recognition, and variations at these critical residues modulate infection susceptibility. Infection studies have shown that some primate species develop COVID-19-like symptoms; however, the susceptibility of most primates is unknown. Here, we show that all apes and African and Asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ACE2. Monkeys in the Americas, and some tarsiers, lemurs and lorisoids, differ at critical contact residues, and protein modeling predicts that these differences should greatly reduce SARS-CoV-2 binding affinity. Other lemurs are predicted to be closer to catarrhines in their susceptibility. Our study suggests that apes and African and Asian monkeys, and some lemurs, are likely to be highly susceptible to SARS-CoV-2. Urgent actions have been undertaken to limit the exposure of great apes to humans, and similar efforts may be necessary for many other primate species.

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, β-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.

We report a diaryldiselenide catalyst for cross-dehydrogenative nucleophilic functionalization of hydrophosphoryl compounds. The proposed organocatalytic cycle closely resembles the mechanism of the Atherton-Todd reaction, with the catalyst serving as a recyclable analogue of the halogenating agent employed in the named reaction. Phosphorus and selenium NMR studies reveal the existence of a P-Se bond intermediate, and structural analyses indicate a stereospecific reaction.

Protein-protein interactions (PPIs) play a critical role in fundamental biological processes. Competitive inhibition of these interfaces requires compounds that can access discontinuous binding epitopes along a large, shallow binding surface area. Conformationally defined protein surface mimics present a viable route to target these interactions. However, the development of minimal protein mimics that engage intracellular targets with high affinity remains a major challenge because mimicry of a portion of the binding interface is often associated with the loss of critical binding interactions. Covalent targeting provides an attractive approach to overcome the loss of noncovalent contacts but have the inherent risk of dominating noncovalent contacts and increasing the likelihood of nonselective binding. Here, we report the iterative design of a proteolytically stable α 3 β chimeric helix mimic that covalently targets oncogenic Ras G12C as a model system. We explored several electrophiles to optimize preferential alkylation with the desired C12 on Ras. The designed lead peptide modulates nucleotide exchange, inhibits activation of the Ras-mediated signaling cascade, and is selectively toxic toward mutant Ras G12C cancer cells. The relatively high frequency of acquired cysteines as missense mutations in cancer and other diseases suggests that covalent peptides may offer an untapped therapeutic approach for targeting aberrant protein interactions.

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.

Protein secondary and tertiary structure mimics have served as model systems to probe biophysical parameters that guide protein folding and as attractive reagents to modulate protein interactions. Here we review contemporary methods to reproduce loop, helix, sheet and coiled-coil conformations in short peptides.

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.

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.

Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein-protein interaction (PPI) inhibitors. While a wide variety of strategies have been used to stabilize or imitate α-helices, similar strategies for β-sheet stabilization are more limited. Synthetic scaffolds that stabilize reverse turns and cross-strand interactions have provided important insights into β-sheet stability and folding. However, these templates occupy regions of the β-sheet that might impact the β-sheet's ability to bind at a PPI interface. Here, we present the hydrogen bond surrogate (HBS) approach for stabilization of β-hairpin peptides. The HBS linkage replaces a cross-strand hydrogen bond with a covalent linkage, conferring significant conformational and proteolytic resistance. Importantly, this approach introduces the stabilizing linkage in the buried β-sheet interior, retains all side chains for further functionalization, and allows efficient solid-phase macrocyclization. We anticipate that HBS stabilization of PPI β-sheets will enhance the development of β-sheet PPI inhibitors and expand the repertoire of druggable PPIs.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

β-Amino acids offer attractive opportunities to develop biologically active peptidomimetics, either employed alone or in conjunction with natural α-amino acids. Owing to their potential for unique conformational preferences that deviate considerably from α-peptide geometries, β-amino acids greatly expand the possible chemistries and physical properties available to polyamide foldamers. Complete in silico support for designing new molecules incorporating non-natural amino acids typically requires representing their side-chain conformations as sets of discrete rotamers for model refinement and sequence optimization. Such rotamer libraries are key components of several state-of-the-art design frameworks. Here we report the development, incorporation in to the Rosetta macromolecular modeling suite, and validation of rotamer libraries for β 3 -amino acids., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Herein, the design of a catalyst that combines lessons learned from peptide biosynthesis, enzymes, and organocatalysts is described. The catalyst features a urea scaffold for carbonyl recognition and elements of nucleophilic catalysis. In the presence of 10 mol % of the organocatalyst, the rate of peptide bond formation is accelerated by 10000-fold over the uncatalyzed reaction between Fmoc-amino acid thioesters and amino acid methyl esters.

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, β-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 β-strands, we developed triazolamers, a topographical PDM where the peptide bonds are replaced by triazoles. The triazoles simultaneously stabilize the extended, zigzag conformation of β-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.

Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest-host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest-host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest-host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We describe a modular approach to identify and inhibit protein-protein interactions (PPIs) that are mediated by protein secondary and tertiary structures with rationally designed peptidomimetics. Our analysis begins with entries of high-resolution complexes in the Protein Data Bank and utilizes conformational sampling, scoring, and design capabilities of advanced biomolecular modeling software to develop peptidomimetics.

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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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.

Inhibition of protein-protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy--that builds on the principles of fragment-based drug discovery (FBDD)--is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein's Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features--alpha-atom and alpha-space--and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors.

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.

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., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Involvement of superficial temporal space secondary to odontogenic infections of the maxillary and mandibular teeth is not uncommon. Usually, infections of the temporal space are drained via temporal approach. Authors propose a new approach for decompression and drainage of superficial temporal space which offers an advantage of dependent drainage and hidden scar.

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.

Amyloid fibrils are ordered, insoluble protein aggregates that are associated with neurodegenerative conditions such as Alzheimer's disease. The fibrils have a common rod-like core structure, formed from an elongated stack of β-strands, and have a rigidity similar to that of silk (Young's modulus of 0.2-14 GPa). They also exhibit high thermal and chemical stability and can be assembled in vitro from short synthetic non-disease-related peptides. As a result, they are of significant interest in the development of self-assembled materials for bionanotechnology applications. Synthetic DNA molecules have previously been used to form intricate structures and organize other materials such as metal nanoparticles and could in principle be used to nucleate and organize amyloid fibrils. Here, we show that DNA origami nanotubes can sheathe amyloid fibrils formed within them. The fibrils are built by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin and a DNA origami construct is used to form 20-helix DNA nanotubes with sufficient space for the fibrils inside. Once formed, the fibril-filled nanotubes can be organized onto predefined two-dimensional platforms via DNA-DNA hybridization interactions.

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 α-helix is a prevalent secondary structure in proteins and is critical in mediating protein-protein interactions (PPIs). Peptide mimetics that adopt stable helices have become powerful tools for the modulation of PPIs in vitro and in vivo. Hydrogen-bond surrogate (HBS) α-helices utilize a covalent bond in place of an N-terminal i to i+4 hydrogen bond and have been used to target and disrupt PPIs that become dysregulated in disease states. These compounds have improved conformational stability and cellular uptake as compared to their linear peptide counterparts. The protocol presented here describes current methodology for the synthesis of HBS α-helical mimetics. The solid-phase synthesis of HBS helices involves solid-phase peptide synthesis with three key steps involving incorporation of N-allyl functionality within the backbone of the peptide, coupling of a secondary amine, and a ring-closing metathesis step., (Copyright © 2014 John Wiley & Sons, Inc.)