Serum-Stable and Selective Backbone-N-Methylated Cyclic Peptides That Inhibit Prokaryotic Glycolytic Mutases

Abstract

N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cis N-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library.

Figure 1.. tRNA tuning approach of N-MeAA incorporation using “pool-and-split” RaPID selection paradigm.

(a) N-methyl amide amino acid codons. (b) RaPID selection where rounds 1–3 utilize the pooled procaryotic iPGMs, whereas rounds 4 onward continue the selection process using individual iPGMs and the enriched library. (i) Puromycin ligation, in vitro translation, and reverse transcription. (ii) Negative selection of bead binders. (iii) Affinity selection with pooled or individual iPGMs. (iv) Transcription of enriched DNA sequences.

Figure 2.. Comparative functional and human serum stability characterization of select Sa-D and Mo-L/D synthetic cyclic peptides and ∆N-Me analogs.

(a–c) MCP CRCs on representative iPGM orthologs from bacteria, S. aureus (Sa), E. coli (Ec), H. pylori (Hp), M. orale (Mo) and M. pneumonia (Mp) and nematode, C. elegans (Ce), B. malayi (Bm) and W. bancrofti (Wb). Top plots represent N-Me containing MCPs and bottom plots correspond to ∆N-Me analogs. Data are representative of N≥3 independent experiments. (d–f). MCP stability time course where solid symbols represent N-methyl amide-containing MCPs and open symbols the corresponding amide analogs, where solid dashed lines represent analog pairs. MCPs pairs containing an internal standard peptide were incubated with human serum at 37°C as described in materials and methods. At 0, 1, 3, 9, 24, and 72 h, the human serum was reduced with 10 mM TCEP and relative intensity of each peptide to the standard peptide was determined by LC/MS. Relative intensity at 0 h was defined as 100%. The experimental data were fitted by non-linear regression one phase decay curve using GraphPad Prism 9.2.0.

Figure 3.. Functional, binding kinetics and thermal stability characterization of Sa-D2 and –D3 cyclic peptides and analogs.

(a) CRCs for activity of Sa-D2 on representative iPGM orthologs from the protozoan L. mexicana (Lm); bacteria S. aureus (Sa), E. coli (Ec), H. pylori (Hp) and M. orale (Mo), and nematode C. elegans (Ce) and W. bancrofti (Wb). (b, c) Sensorgrams for Sa-D2 (0.5 – 31 nM) or Sa-D2 (8 – 500 nM) binding kinetics derived from a C. elegans or S. aureus iPGM, respectively, conjugated sensor chip surface. (d) First derivative plots of the fluorescence emission as a function of temperature for either Sa-D2 (dashed line, T_m=48.7°C) or Sa-D3 (blue line, T_m=62.4°C) binding to S. aureus iPGM vs DMSO control (black line, T_m=45.7°C). (e, f) Sensorgrams for Sa-D3 (156 nM - 10 μM; K_D=790 nM, t_½ =27s) and Sa-D3 ∆N-Me (39 nM - 10 μM; K_D=280 nM, t_½ =25s) binding kinetics and saturation binding curve (inset) for S. aureus iPGM. The experimental SPR data (black) were globally fit to a 1:1 binding model (blue lines) using BIAevaluation, as described in Experimental procedures, to determine kinetic rate constants. Corresponding insets show the signal observed at equilibrium, R_eq, plotted as a function of the [MCP], fit to a hyperbolic, single-site binding equation. Data are representative of N≥3 independent experiments.

Figure 4.. Ipglycermides Ce-2 Y7F and Sa-D2 have unique C. elegans iPGM Zn²⁺-coordinating binding modes.

(a) Key protein-ligand interactions and Cys13 – Zn²⁺ coordination observed between Sa-D2 and C. elegans iPGM. N-Me amides are shown with hydrogens (white) for clarity. (b) Two-dimensional protein-cyclic peptide pharmacophore interactions. Phosphatase (apricot) and phosphotransferase (pastel blue) domain are indicated having H-bonding (black dashes), ionic (red dots), or π-cation (purple dots) interactions. (c) Superposition of C. elegans iPGM•Sa-D2 (dark blue/dim grey; PDB: 7TL7) and C. elegans iPGM•Ce-2 Y7F (ice blue/violet; PDB: 7KNG) structures fixed over the amino acid range of 27–95 and 339–538 (phosphatase domain). Zn²⁺ ions are sea blue. (d) Conformation of Sa-D2 as bound to C. elegans iPGM, with the two cis N-methyl amides at Tyr6 and Tyr8 indicated by arrows.

Figure 5.. Sa-D3 ipglycermide partially mimic 3PG binding to S. aureus iPGM.

(a) Conformation of Sa-D3 as bound to S. aureus iPGM, showing the trans N-methyl amide at _MeSer9 (shown with N-Me hydrogens (white) for clarity) and Asp13 interaction with R185, R191, R257 and R260 of the 3-phosphoglycerate (3PG) binding site. (b) Two-dimensional protein-cyclic peptide pharmacophore interactions. Phosphotransferase (pastel blue) domain and Hinge regions (celadon) are indicated having H-bonding (black dashes) or ionic (red dots) interactions. An edge-face aromatic F377 interaction from the phosphatase domain (apricot) is shown by purple dots. (c) Electrostatic surface representation of S. aureus iPGM • Sa-D3 complex where the cyclic peptide (grey) surface is rendered in yellow and the trans N-methyl amide circled. (d) Same as panel c but rotated 90° about horizontal axis to show 3PG binding site. (e) Superposition of the S. aureus iPGM - Sa-D3 (white; PDB: 7TL8) with 3PG bound (PDB 4NWJ) S. aureus iPGM (lilac). The Sa-D3 cyclic peptide is rendered as grey, and 3PG as green/magenta cylinders (chemical structure shown in inset). Prepared with CCP4MG, version 2.10.10.