Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1

Abstract

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 IC₅₀ 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.

Keywords: antibiotic resistance; computational design; drug design; peptide macrocycles; protein folding.

Fig. 1.

Computational design approach for generating peptide macrocycle inhibitors of NDM-1. (A) Structure of NDM-1 (PDB ID 4EXS), chain B. The active site binds catalytic zinc atoms and is flanked by an ordered FL and a flexible HL. Hydrophobic amino acid residues on the inner face of the HL, and metal-coordinating residues, are labeled. (B) Comparison of a subset of NDM-1 crystal structures. PDB IDs 3RKJ, 3S0Z, 3ZR9, and 4HL1 are shown in gray. In lavender and green are PDB ID 4EXS, chains A and B, respectively. Where most of the structure, including the FL, is rigid, the HL shows extensive conformational flexibility, putting inner-face hydrophobic side chains (labeled) in diverse positions. (C) Crystal structure of NDM-1 active site (green) with ʟ-captopril (purple) bound (PDB ID 4EXS, chain B). Active-site zinc atoms are shown beneath the surface as spheres. (D) In silico conversion of ʟ-captopril to a ᴅ-proline, ʟ-cysteine dipeptide (purple) flanked by polyglycine sequences (pink). (E) Rapid in silico sampling of closed conformations of a peptide macrocycle containing the ᴅ-cysteine, ʟ-proline stub (purple), and flanking sequences (pink) in the context of the NDM-1 active site, using the generalized kinematic closure approach. For each closed conformation, Rosetta design heuristics were used to find side-chain identities and conformations (represented here by spheres).

Fig. 2.

Designed eight-residue peptide macrocycle inhibitors of NDM-1, designated NDM1i-1A (A) through NDM1i-1G (G). (i) Amino acid sequences (AA) and backbone conformational bins (Bin) of designed peptides. In this and the following two columns, ʟ-amino acids are shown in cyan and ᴅ-amino acids in orange. Backbone conformational bins are described in SI Appendix, section 2.1.6. (ii) Peptide design computer models shown as stick representations. Intramolecular backbone hydrogen bonds are shown as green lines. Sequence numbering is as shown in i. (iii) Space-filling computer models of designed peptides in the NDM-1 active site, with NDM-1 shown in gray. The HL, FL, and interacting residues Phe70 and Val73 are indicated. (iv) Conformational landscape analysis performed with the Rosetta simple_cycpep_predict application, showing computed energy of the peptide modeled in isolation plotted against rmsd to its designed binding conformation. Each point represents a separate conformational sampling attempt. Colors indicate the number of intramolecular backbone hydrogen bonds observed in the sampled conformation. P_Near values are indicated, with the mean and SE of three independent large-scale conformational sampling simulation replicates reported. (v) Experimentally measured activity of NDM-1 (vertical axis) in the presence of varying concentrations of peptide (horizontal axis). Points are mean of three independent replicates, and error bars represent the SEM. Red curves show fits to the Hill equation, with IC₅₀ values and fit confidence indicated on each plot. (Insets) Fit residuals.

Fig. 3.

Comparison of computationally predicted metrics and experimentally measured IC₅₀ values for peptides NDM1i-1A through NDM1i-1G. The IC₅₀ value for ᴅ-captopril is shown as dashed gray lines. (A) Comparison of experimentally measured IC₅₀ values (vertical axis) with Rosetta-computed estimates of ΔG_binding (horizontal axis). Vertical error bars represent uncertainty in fitted parameters, and horizontal error bars represent SEM of 20 replicates of the computation, with optimization of side-chain conformations in bound and unbound states producing some variation from replicate to replicate. No correlation is observed. (B) Comparison between experimentally measured IC₅₀ values and estimates of ΔG_folding (–RT ln(P_Near/(1-P_Near))) as described in the SI Appendix) obtained from computed energy landscapes (for examples, see Fig. 2, column iv). Vertical error bars are as in A. Horizontal error bars represent the SEM of three independent landscape simulations. The blue line shows the empirical line of best fit with R² value indicated.

Fig. 4.

Comparison of computational design model and X-ray crystal structure (PDB ID 6XBF) of peptide NDM1i-1G bound to NDM-1. In all panels, peptide ʟ- and ᴅ-amino acid residues are shown as cyan and orange sticks, respectively. (A) Design model of NDM1i-1G (pink surface) in the active-site cleft of NDM-1 (green surface) with peptide residues ʟ-Leu3 and ʟ-Ile6 making contact with Met67 and Phe70 of the HL. The side chain of residue ᴅ-Arg1 was not resolved. (Top Inset) Peptide ᴅ-Arg2 projects toward the FL, making contact with Glu152 and Asp223. (Lower Inset) Stub residues ʟ-Pro5 and ᴅ-Cys6 occlude the active site as ʟ-captopril does, with ᴅ-Cys6 coordinating both active-site zinc atoms. (B) X-ray crystal structure of NDM1i-1G (pink surface) bound to NDM-1 (green surface). Crystallographic water molecules are shown as blue surfaces. Peptide residues ʟ-Leu3 and ʟ-Ile6 contact HL residues Met67 and Phe70, albeit in a slightly different configuration than designed. The ᴅ-Arg1 side chain was not resolved. (Top Inset) Glu-152 and Asp-223 coordinate a zinc cation, displacing the side chain of ᴅ-Arg2. (Bottom Inset) The ʟ-Pro5, ᴅ-Cys6 stub occludes the active site as designed. (C) Overlay of design (lighter colors) and crystal structure (darker colors). The flexible HL undergoes a 3.1-Å shift (green arrow), while the peptide rotates slightly about its base, resulting in a 1.8 Å rmsd (orange arrow). (D) Overlay of peptide portion of design (lighter colors) aligned to peptide portion of crystal structure (darker colors). The peptide’s internal conformation matches the design to a backbone heavyatom rmsd of 0.3 Å with side-chain rotamers of ʟ-Leu3 and ʟ-Ile6 closely aligning. All four designed internal hydrogen bonds (green lines) were present in the experimentally observed conformation.

Fig. 5.

Comparison of design model and crystal structure (PDB ID 6XCI) of peptide NDM1i-3D bound to NDM-1. (A) Design model of NDM1i-3D (pink surface, with ʟ- and ᴅ-amino acid residues shown in cyan and orange, respectively) in the NDM-1 active site (green). ʟ-2-aminomethyl phenylalanine (ʟ-A34) and ʟ-norleucine (ʟ-Nlu) residues make hydrophobic contacts with the inner face of the HL. (Inset) ᴅ-Cys at position 5 coordinates active-site zinc atoms. (B) X-ray crystal structure of NDM1i-3D bound to NDM-1. The peptide is rotated nearly 180° relative to the design model with ʟ-Nlu and ʟ-A34 residues in opposite positions. Water molecules are shown as sticks with blue surfaces. (Inset) ʟ-Glu at position 8 coordinates the active-site zinc. Cadmium is observed in place of zinc at the adjacent site. (C) Overlay of X-ray crystal structure (darker colors) and design model (lighter colors). NDM-1 is shown in green; ʟ- and ᴅ-amino acids in NDM1i-3D are shown in cyan and orange, respectively, and stub residues are shown in purple. The crystal structure’s positions are labeled in black, and the design model’s positions in white. As shown, the rotation of the design model puts ʟ-Glu-8 (red arrows) where ᴅ-Cys-4 (orange arrows) would be. The motion of the peptide displaces it by an rmsd of 9.4 Å, while the HL moves by 1.2 Å. (D) Overlay of aligned peptide portions of the crystal structure (darker colors) and design model (lighter colors). Cyan and orange represent ʟ- and ᴅ-amino acids, as before. Despite the change in binding orientation, the crystal structure peptide conformation matches the design to a backbone heavyatom rmsd of 0.4 Å. (E) Overlay of crystal structure with peptide design circularly permuted by four residues. The rough symmetry of the backbone conformation allows ᴅ-Pro 1 to occupy the space that would be occupied by ʟ-Pro 5 (green arrows), ᴅ-Cys 4 to occupy the space that would be occupied by ʟ-Glu 8 blue arrows), and ʟ-Nlu 3 to occupy the space that would be occupied by ʟ-A34 6 (red arrows), possibly explaining why the peptide was able to bind to the same site in a very different binding mode.