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.
Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such ...peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.
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.
Disulfide‐rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a ...stable conformation de novo. Here, we describe a system to produce disulfide‐rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly 13C‐ and 15N‐labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR. The ensemble of NMR structures calculated for both peptides agreed well with the design models, further confirming the accuracy of the design protocol. Collection of NMR data on the peptides under reducing conditions revealed a dependency on disulfide bonds to maintain stability. Furthermore, we performed long‐time molecular dynamics (MD) simulations with tempering to assess the stability of two families of de novo designed peptides. Initial designs which exhibited a stable structure during simulations were more likely to adopt a stable structure in vitro, but attempts to utilize this method to redesign unstable peptides to fold into a stable state were unsuccessful. Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design.
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.
Display omitted
•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.
The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. ...We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC
values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.
Cyclic symmetry is frequent in protein and peptide homo‐oligomers, but extremely rare within a single chain, as it is not compatible with free N‐ and C‐termini. Here we describe the computational ...design of mixed‐chirality peptide macrocycles with rigid structures that feature internal cyclic symmetries or improper rotational symmetries inaccessible to natural proteins. Crystal structures of three C2‐ and C3‐symmetric macrocycles, and of six diverse S2‐symmetric macrocycles, match the computationally‐designed models with backbone heavy‐atom RMSD values of 1 Å or better. Crystal structures of an S4‐symmetric macrocycle (consisting of a sequence and structure segment mirrored at each of three successive repeats) designed to bind zinc reveal a large‐scale zinc‐driven conformational change from an S4‐symmetric apo‐state to a nearly inverted S4‐symmetric holo‐state almost identical to the design model. These symmetric structures provide promising starting points for applications ranging from design of cyclic peptide based metal organic frameworks to creation of high affinity binders of symmetric protein homo‐oligomers. More generally, this work demonstrates the power of computational design for exploring symmetries and structures not found in nature, and for creating synthetic switchable systems.
PDB Code(s): 6UFU, 6UG2, 6UG3, 6UG6, 6UGB, 6UGC, 6UCX, 6UD9, 6UDR, 6UDW, 6UDZ, 6UF4, 6UF7, 6UF8, 6UFA and 6UF9;
The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-beta-lactamase 1 (NDM-1), a bacterial enzyme that degrades beta-lactam ...antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of L- and D-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a pep-tide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC.sub.50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets. antibiotic resistance | drug design | protein folding | peptide macrocycles | computational design
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.
PDF
