ABSTRACT
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.
Subject(s)
Chronic Pain , Peptidomimetics , Rats , Animals , Chronic Pain/drug therapy , Chronic Pain/metabolism , Rats, Sprague-Dawley , Peptidomimetics/pharmacology , Calcium/metabolism , Calcium Channels, N-Type/genetics , Calcium Channels, N-Type/metabolism , Sensory Receptor Cells/metabolism , Ganglia, Spinal/metabolismABSTRACT
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.
Subject(s)
Leucine Zippers , Transcription Factors , Amino Acid Sequence , Ligands , Transcription Factors/chemistry , DNA/chemistry , RNA, Double-Stranded , Binding SitesABSTRACT
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.
Subject(s)
Proteins , Protein Conformation, beta-Strand , Hydrogen Bonding , Models, Molecular , Protein Structure, Secondary , Proteins/chemistryABSTRACT
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.
Subject(s)
Intrinsically Disordered Proteins , Intrinsically Disordered Proteins/chemistry , Membrane ProteinsABSTRACT
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.
Subject(s)
Nickel , Peptides , Nickel/chemistry , Catalysis , Peptides/chemistry , OligopeptidesABSTRACT
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.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcal Infections , Anti-Bacterial Agents/chemistry , Bacterial Proteins/metabolism , Cryoelectron Microscopy , Humans , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/pharmacology , Staphylococcus aureus/metabolismABSTRACT
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 acidsâthe 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 agentsâit 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.
Subject(s)
Amino Acids , Peptides , Amides , Amines , Amino Acids/chemistry , Oxidation-Reduction , Peptide Biosynthesis , Peptides/chemistryABSTRACT
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.
Subject(s)
PeptidomimeticsABSTRACT
ß-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 ß-hairpin mimics of an extended strand region mediating the Tcf4/ß-catenin interaction. Our efforts afford a rationally designed lead for an underexplored region of ß-catenin, which has been the subject of numerous ligand discovery campaigns.
Subject(s)
Peptides, Cyclic/metabolism , Protein Binding/drug effects , Transcription Factor 4/metabolism , beta Catenin/metabolism , Amino Acid Sequence , Escherichia coli/chemistry , Escherichia coli Proteins/metabolism , Peptides, Cyclic/chemistry , Protein ConformationABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Peptides/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Humans , Ligands , Molecular Docking Simulation , Peptides/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-mdm2/chemistryABSTRACT
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.
Subject(s)
Peptides , Proteins , Hydrogen Bonding , Protein Conformation , Protein Conformation, alpha-Helical , Protein Structure, SecondaryABSTRACT
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.
Subject(s)
Metals , Peptides , Amino Acid Sequence , Circular Dichroism , Protein Conformation, beta-Strand , Protein Structure, SecondaryABSTRACT
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.
Subject(s)
Multiprotein Complexes/ultrastructure , Protein Conformation , Son of Sevenless Protein, Drosophila/ultrastructure , ras Proteins/ultrastructure , Animals , Biomimetics , Crystallography, X-Ray , Drug Discovery , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/ultrastructure , HCT116 Cells , Helix-Loop-Helix Motifs/genetics , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Proteome/genetics , Signal Transduction/genetics , Son of Sevenless Protein, Drosophila/chemistry , Son of Sevenless Protein, Drosophila/genetics , ras Proteins/chemistry , ras Proteins/geneticsABSTRACT
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.
Subject(s)
COVID-19/veterinary , Lemur , Lorisidae , Primate Diseases/epidemiology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/epidemiology , Lemur/genetics , Lorisidae/genetics , Primate Diseases/virology , Risk FactorsABSTRACT
The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts 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 and finds 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.
ABSTRACT
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.
Subject(s)
Photochemical Processes , Proteins/chemistry , Models, Molecular , Protein Engineering/methods , Protein Structure, SecondaryABSTRACT
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.
Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/veterinary , Host Specificity/genetics , Pandemics/veterinary , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/veterinary , Primate Diseases/enzymology , Primates/genetics , Receptors, Virus/genetics , Amino Acid Sequence , Amino Acid Substitution , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/physiology , Biological Evolution , COVID-19 , Chiroptera/genetics , Conserved Sequence , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Genetic Predisposition to Disease , Mammals/genetics , Models, Molecular , Mutation, Missense , Peptidyl-Dipeptidase A/chemistry , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Point Mutation , Primate Diseases/virology , Protein Binding , Protein Conformation , Risk , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
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.
Subject(s)
Neoplasms/chemistry , Peptides/chemistry , Peptidomimetics/chemistry , Pinocytosis , Cell Line , Flow Cytometry , Humans , Microscopy, Fluorescence , Neoplasms/pathology , Protein Binding , Protein ConformationABSTRACT
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.