De novo design of D-peptide ligands: Application to influenza virus hemagglutinin

Abstract

D-peptides hold great promise as therapeutics by alleviating the challenges of metabolic stability and immunogenicity in L-peptides. However, current D-peptide discovery methods are severely limited by specific size, structure, and the chemical synthesizability of their protein targets. Here, we describe a computational method for de novo design of D-peptides that bind to an epitope of interest on the target protein using Rosetta's hotspot-centric approach. The approach comprises identifying hotspot sidechains in a functional protein-protein interaction and grafting these side chains onto much smaller structured peptide scaffolds of opposite chirality. The approach enables more facile design of D-peptides and its applicability is demonstrated by design of D-peptidic binders of influenza A virus hemagglutinin, resulting in identification of multiple D-peptide lead series. The X-ray structure of one of the leads at 2.38 Å resolution verifies the validity of the approach. This method should be generally applicable to targets with detailed structural information, independent of molecular size, and accelerate development of stable, peptide-based therapeutics.

Keywords: D-peptide; X-ray crystallography; computational design; hemagglutinin; influenza.

Fig. 1.

De novo structure-based design of D-peptides. (A) Schematic depiction of the mirror-image phage display methodology (21). [a → b] A target protein is synthesized with D-amino acids and folded. [b → c] Bacterial phages (gray) presenting a library of L-peptides or proteins (orange) are used to identify binders. [c → d] When synthesized with D-amino acids, the binders bind the original target L-protein. (B) Computational de novo D-peptide design methodology. [a] Structure of the target L-protein (blue) in complex with a ligand protein (yellow). [b] The target protein structure is mirror-inverted in silico. A library of L-polypeptide ligands (orange) is designed using different scaffolds to present the original ligand interaction hotspots (yellow). [c] The best ligands are selected based on a scoring function. [d] Corresponding D-peptides are synthesized and tested for binding to the L-target. (C) Blueprint of D-peptide ligand design against influenza A hemagglutinin. bnAb FI6v3 Fab (yellow) is shown in complex with HA (blue) from H1N1 A/California/04/2009 (H1/Cal) (PDB ID 3ZTN) (34). L- and D-enantiomeric disembodied hotspot residues Phe100D (L-Fh and D-Fh) and Trp100F (L-Wh and D-Wh) from FI6v3 HCDR3 are shown in space-filling view (C/O/N in yellow, red, and blue colors) and the peptide scaffold (brown) is in cartoon representation.

Fig. 2.

Enzymatic stability and immunogenicity of D-peptides. (A) Panel of peptides used for the enzymatic stability study. L- and D-enantiomers of the peptides were subjected to MDCK cell lysate and half-life calculated from degradation profiles (B). The values, reported in hours, are an average of two independent experiments. (B) Degradation profiles of L- and D-peptides in MDCK cell lysate. Colored lines correspond to the D-peptides while gray lines with the same symbols correspond to L-peptides. (C) Schema of the in vivo immunogenicity experiment. (D) IgG1 responses in mice immunized with L- or D-peptides in two different adjuvants (Alum, CFA/IFA). Phosphate-buffered saline (PBS) was used as solvent control. ELISA of the sera of individual mice (n = 5/group) was tested 41 d after the first injection in 1/50 dilution in duplicate. The peptide used for immunization was coated onto 96-well microtiter plates and the bound antibody was detected with peroxidase-conjugated goat anti-mouse IgG1. Mean values per mouse are shown. Animals immunized with L-peptides are grouped on the Left side and animals treated with corresponding D-peptides on the Right side. Sera that scored positive, i.e. the measured OD₄₅₀ value was above a predetermined cut-point (ranging from 0.163 to 0.265), determined with preimmune sera for each peptide separately (SI Appendix, Table S7), are shown in blue. Samples below the cut-point (negative) are shown in gray. (E) Dose–response curves for peptides (FSD1 and HP35) that showed the strongest responses in (panel D). Day 41 sera of mice immunized with FSD1 or HP35 peptides were serially twofold diluted, starting from a 1/50 dilution, and tested in single experiments with the same ELISA as described for panel D. Each colored titration curve corresponds to an individual mouse immunized with the indicated peptide.

Fig. 3.

Design, synthesis, and testing of D-peptide libraries against HA from group 1 influenza A/California/07/2009 (H1N1) virus. (A) Structures of scaffolds for the 10 identified peptide classes are shown in cartoon representation. 1ACWΔC is derived from 1ACW by introducing two mutations (C6A and C10A) and an 8 amino acid C-terminal truncation. (B) Binding competition IC₅₀ and calculated K_i values for selected peptides from the 10 scaffold classes. Different combinations of FI6v3 and CR9114 hotspots were used and between 3-14 peptide variants were synthesized for each scaffold class. (C) Affinity and specificity of all synthesized peptides with different symbols corresponding to different peptide scaffolds. Hit area with affinity (K_i) <30 µM and specificity >10 is colored in yellow. K_i values were estimated from the pIC₅₀’s and based on the concentration and affinity of the small protein HB80.4 that binds to the HA stem (44) using the Cheng–Prusoff equation. Specificity values were defined and calculated as the ratio K_i^2D1/K_i^HB80.4, where K_i^2D1 is the approximated K_i in competition with head binding antibody 2D1 and K_i^HB80.4 with stem binder HB80.4. (D) AlphaLISA curves for DP93 and DP99 in binding competition with HB80.4 and the negative control, HA head binding antibody 2D1. (E) Sequence of selected peptides from six different scaffold classes. Letters represented as uppercase are L-amino acids and lowercase are D-amino acids. Noncanonical amino acids are shown beside the table. H– and –OH in the peptide sequence indicate that N- and C-termini are free and uncapped, whereas H- and -NH₂ in the peptide sequences indicate that N and C termini are uncapped and amidated, respectively.

Fig. 4.

Crystal structure of D-peptide DP93 with influenza H1/PR8 HA. (A) The crystal structure of DP93 in complex with influenza group-1 H1 HA from H1N1 A/Puerto Rico/8/1934 (H1/PR8) strain. The HA trimer is represented as a molecular surface with one protomer colored (HA1; pink and HA2; blue) and the other two protomers in whitish gray. DP93 is shown in a tube backbone representation (red) and glycans on the HA surface are in cyan sticks. (B and C) A zoomed-in view of one of three DP93 binding sites in the HA trimer is shown, with the atoms of DP93 in red and S in yellow, respectively. (D) 2D-representation of DP93. The amino acid sequence is represented in lowercase single letters for D amino acids and uppercase for L amino acids. Disulfide bonds are represented in black solid lines and dPCA-π as an N-terminal tag. (E) Computationally predicted binding mode of DP93. All C, and side chain O and S atoms of DP93 are in purple, red, and yellow, respectively. The overall Calpha RMSD between the model and the crystal structure of DP93 is 4.9 Å, mainly as a result of higher RMSDs for peptide regions that do not contact the HA. (F) Superimposition of the interacting side chains of the DP93 computational model (purple) and X-ray crystal structure (red) with HA. The hydrophobic interacting sidechains of the model and crystal structure are generally in close agreement. Sidechains of HA interacting residues of DP93 are shown and highlighted with dotted ellipses. (G) Superimposition of the hotspot phenylalanine residue from FI6v3 Fab of the HA–Fab complex (PDB 3ZTN) and DP93 crystal structure. Hotspot residues dPhe² and ф¹⁹ from DP93 occupy the same conserved hydrophobic pocket on HA as Phe^100D and Trp^100F from HCDR3 of Fab FI6v3. (H) Molecular interactions of DP93 in complex with H1/PR8 HA. Polar interactions are depicted in black dotted lines and measured in Å. HA1 (pink ribbon), HA2 (blue cartoon), water (green sphere), and potassium (orange sphere). Polar interactions were also incorporated into our design workflow, although the designed hydrophobic interactions in (F) dominated as expected for binding to the HA hydrophobic stem region. Abbreviations for D-amino acid residues are as follows: dAla (a), D-alanine; dCys (c), D-cysteine; dAsp (d), D-aspartic acid; dGlu (e), D-glutamic acid; dPhe (f), D-phenylalanine; dHis (h), D-histidine; dIle (i), D-isoleucine; dLys (k), D-lysine; dLeu (l), D-leucine; dMet (m), D-methionine; dAsn (n), D-asparagine; dPro (p), D-proline; dGln (q), D-glutamine; dArg (r), D-arginine; dSer (s), D-serine; dThr (t), D-threonine; dVal (v), D-valine; dTrp (w), D-tryptophan, and dTyr (t), D-tyrosine. Noncanonical D-amino acids abbreviated as dPCA, D-pyroglutamic acid; π, D-propargylglycine; b, D-homoleucine; j, D-aminobutyric acid; ф, D-homo-phenylalanine.