l-Carboranylalanine (LCba) is a unique artificial amino acid containing a cluster of 10 boron atoms. Since the three-dimensional aromaticity and charge distributions of the carborane side chain are ...quite different from any side chains of proteinogenic amino acids, there is no report whether LCba can be a substrate for the translation machinery. Here, we report studies on the ribosomal incorporation of LCba into peptide via initiation and elongation using the flexizyme-assisted translation system. Our results indicate that only the initiation step could tolerate LCba incorporation, but the elongation steps could not, very likely due to its steric bulkiness of the side chain. Based on this knowledge, we have designed a library of macrocyclic peptides initiated by α-N-(2-choloroacetyl)-l-carboranylalanine (ClAc-LCba) and selected molecules capable of binding to human epidermal growth factor receptor (hEGFR). Two peptides that were forwarded to deeper studies exhibited affinities with K D values at 16 and 20 nM against hEGFR. Computational modeling of one of the peptides suggested that the carborane side chain might be directly involved in the interaction with the hydrophobic β-sheet core in the EGF binding site of hEGFR, which is consistent with the mutational data where replacing LCba residue with LPhe completely eliminated the binding activity. Cell lines that stably express hEGFR could be stained by incubation with the C-terminal fluorescein-labeled peptides, whereas hEGFR-negative cells could not be stained. This study provides a general strategy for the de novo discovery of carborane-containing macrocyclic peptides targeting various tumor biomarker proteins, potentially applicable to boron neutron capture therapy.
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned ...by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, ...we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC
values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.
New chemical inhibitors of protein-protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein ...interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the β-catenin:TCF interaction. The β-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on β-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited.
Peptoids are a family of synthetic oligomers composed of N-substituted glycine units. Along with other “foldamer” systems, peptoid oligomer sequences can be predictably designed to form a variety of ...stable secondary structures. It is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions. Computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs. We introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design. We find that peptoids can be described by a “rotamer” treatment, similar to that established for proteins, in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape. Because of the insufficient number of solved peptoid structures, we have calculated the relative energies of side-chain conformational states to provide a backbone-dependent (BBD) rotamer library for a set of 54 different peptoid side chains. We evaluated two rotamer library development methods that employ quantum mechanics (QM) and/or molecular mechanics (MM) energy calculations to identify side-chain rotamers. We show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces. We have incorporated our peptoid rotamer libraries into ROSETTA, a molecular design package previously validated in the context of protein design and structure prediction.
A major role for the intracellular post-translational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area focused on O-GlcNAc ...modification of the amyloid-forming proteins themselves. Here we used synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we found that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we found that, although O-GlcNAc levels are globally reduced in Alzheimer's disease brains, the modification of relevant sHSPs is either maintained or increased, which suggests a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.
Synthetic proteins bearing site-specific posttranslational modifications have revolutionized our understanding of their biological functions in vitro and in vivo. One such modification, ...O-GlcNAcylation, is the dynamic addition of β-N-acetyl glucosamine to the side chains of serine and threonine residues of proteins, yet our understanding of the site-specific impact of O-GlcNAcylation remains difficult to evaluate in vivo because of the potential for enzymatic removal by endogenous O-GlcNAcase (OGA). Thioglycosides are generally perceived to be enzymatically stable structural mimics of O-GlcNAc; however, in vitro experiments with small-molecule GlcNAc thioglycosides have demonstrated that OGA can hydrolyze these linkages, indicating that S-linked β-N-acetyl glucosamine (S-GlcNAc) on peptides or proteins may not be completely stable. Here, we first develop a robust synthetic route to access an S-GlcNAcylated cysteine building block for peptide and protein synthesis. Using this modified amino acid, we establish that S-GlcNAc is an enzymatically stable surrogate for O-GlcNAcylation in its native protein setting. We also applied nuclear magnetic resonance spectroscopy and computational modeling to find that S-GlcNAc is an good structural mimic of O-GlcNAc. Finally, we demonstrate that site-specific S-GlcNAcylation results in biophysical characteristics that are the same as those of O-GlcNAc within the context of the protein α-synuclein. While this study is limited in focus to two model systems, these data suggest that S-GlcNAc broadly resembles O-GlcNAc and that it is indeed a stable analogue in the context of peptides and proteins.
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single ...GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed ...184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.
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•Computational design of diverse permeable macrocycles beyond the “rule-of-five” space•X-ray and NMR structures of designed macrocycles match their computational models•Designed macrocycles are permeable in vitro and orally bioavailable in vivo•Designed chameleonic peptides show solvent-dependent conformational switching
An investigation of the design principles of macrocyclic peptide membrane permeability and oral bioavailability enables the generation of synthetic macrocycles that fold into the predicted conformation, can cross membranes, and even adopt different conformations depending on polar versus nonpolar contexts.
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