Rapid discovery of cyclic peptide protein aggregation inhibitors by continuous selection

Abstract

Protein aggregates are associated with numerous diseases. Here we report a platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli based on phage-assisted continuous evolution (PACE). We developed a new PACE-compatible selection for protein aggregation inhibition and used it to identify cyclic peptides that suppress amyloid-β42 and human islet amyloid polypeptide aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, greatly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically resynthesized in in vitro assays. Our platform provides a powerful approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities.

Figure 1.. PACE-based strategy for cyclic peptide discovery.

Selection phage (SP) carrying a library of genetically encoded cyclic peptides are used to infect host E. coli cells transformed with an “accessory plasmid.” The accessory plasmid encodes a selection that links a user-defined inhibitory activity to expression of phage protein pIII, which is required for phage reproduction. Upon infection, the cyclic peptide library member is expressed from the transduced SP genome and, if active, triggers production of pIII from the accessory plasmid. As a result, only SP that carry active library members propagate, allowing rapid enrichment of desired cyclic peptide sequences.

Figure 2.. Design of a selection for cyclic peptide protein aggregation inhibitors.

(a) Cartoon overview of a split T7 RNAP folding reporter strategy for identifying protein aggregation inhibitors. APP = aggregation-prone protein, T7n and T7c = N- and C-terminal halves of split-T7 RNAP, respectively. (b) Diagram of the genetic circuits that constitute the protein aggregation inhibitor selection. (c) Propagation activity of kan^R-encoding phage on host cells encoding the APP-T7n selection, where the APP is either Aβ42 or its non-aggregating F19S/L34P mutant. (d) Comparison of phage propagation activities of SP carrying Aβ42 aggregation inhibitor cyclo-CKVWQLL or scrambled negative control cyclo-CVQWLKL on host cells encoding the Aβ42-T7n selection. (c-d) Data reflect two biological replicates plotted as individual values. (e) Effect of the mutation of key residues in the cyclo-CKVWQLL sequence on SP propagation on host cells encoding the Aβ42-T7n selection. Data reflects mean and standard error (s.e.) of three (e) biological replicates. Fold phage propagation is calculated as the number of phage generated from an infected culture divided by the number of phage (10⁵) used to infect the culture.

Figure 3.. Selection of cyclic peptide Aβ42 aggregation inhibitors.

(a) Graphical overview of selection procedure. (b) Phage titers (solid lines, plotted on left y-axis) and propagation activity (dashed lines, plotted on right y-axis) of successive rounds of selection of an SP-encoded cyclo-CX₆ library on selection cells encoding Aβ42 as the target APP. “In” refers to the input SP pool used to infect selection cells and “out” refers to the output SP pool obtained after selection, for a given round of selection. pfu = plaque-forming units. (c) Top 10 enriched cyclic peptide sequences after 7 rounds of selection on Aβ42 as determined by high-throughput sequencing analysis. (d) Propagation activity of clonal phage encoding enriched sequences CRVWCAR and CRVYQVL on host cells encoding the Aβ42-T7n selection. Phage encoding active sequence CKVWQLL is shown for comparison. Data reflect two biological replicates plotted as individual values. (e) Propagation activity of clonal phage encoding single alanine substitutions of the CRVWCAR sequence on host cells encoding the Aβ42-T7n selection. (f) Evaluation of cyclic peptide sequences enriched by selection on Aβ42-T7n expressed from plasmid using the Aβ42-GFP folding reporter. CKVWQLL is shown for comparison. Cyclic peptides are induced at 0.04 mM IPTG. (e-f) Data reflects mean and s.e. of three biological replicates. (b, d-e) Fold phage propagation is calculated as the number of phage generated from an infected culture divided by the number of phage used to infect the culture. For (d-e), 10⁵ input phage were used to infect.

Figure 4:. Selection of cyclic peptide inhibitors of hIAPP aggregation.

(a) Phage titer (solid line, plotted on left y-axis) and propagation activity (dashed line, plotted on right y-axis) of successive rounds of selection of an SP-encoded SICLOPPS library on selection cells targeting hIAPP aggregation. “In” refers to the input SP pool used to infect selection cells and “out” refers to the output SP pool obtained after selection, for a given round of selection. pfu = plaque-forming units. Fold phage propagation is calculated as the number of phage generated from an infected culture divided by the number of phage used to infect the culture. (b) Propagation activity of SP pools from each round of the selection shown in (a) where input phage titers are normalized to 10⁵ pfu/mL. Data reflect two biological replicates plotted as individual values. (c) Top 10 enriched cyclic peptide sequences after 5 rounds of selection on hIAPP as determined by high-throughput sequencing analysis. (d) Structure of cyclo-CHVVGVI. (e) Effect of varying concentrations of chemically synthesized cyclo-CHVVGVI (left) or linear CHVVGVI (right) on aggregation of 10 μM hIAPP measured by ThT fluorescence. Assay was run in phosphate-buffered saline (PBS) pH 6.9 at 32 °C under quiescent conditions. Data reflect plots of individual values of three technical replicates. The hIAPP alone (0 μM peptide) data is the same in both plots. (f) Effect of cyclo-CHVVGVI on lag time (t_lag) of hIAPP aggregation. Data reflects t_lag fits from four biological replicates (4 separate ThT assays; see Supplementary Figure 7) plotted as individual values. P-values (two-tailed Student’s t-test between 0 μM peptide and each peptide concentration): *, P ≤ 0.05; **, P ≤ 0.01. (g-h) TEM analysis of 10 μM hIAPP incubated in the presence (g) of 40 μM cyclo-CHVVGVI or alone (h) for for 2 h. (i-j) TEM analysis of 10 μM hIAPP in the presence (i) of 40 μM cyclo-CHVVGVI or alone (j) for 14 h. (g-j) For compatibility with TEM, hIAPP incubations were run in 25 mM sodium phosphate buffer, pH 7.4; see Supplementary Figure 13 for hIAPP aggregation kinetics under TEM assay conditions. Scale bars = 200 nm. This experiment was not repeated.

Figure 5.. Negative selection to remove promiscuous cyclic peptide sequences.

(a) Propagation activity of the SP pool from round 5 of positive selection for hIAPP inhibition on selection cells encoding hIAPP, Aβ42, or an insoluble antibody fragment (scFv) fused to T7n. (b) Phage titer (solid line, plotted on the left y-axis) and propagation activity (dashed line, plotted on the right y-axis) of successive rounds of selection of the hIAPP round 5 SP pool on Aβ42-T7n negative selection cells. “In” refers to the input SP pool used to infect selection cells and “out” refers to the output SP pool obtained after selection, for a given round of selection. pfu = plaque-forming units. Fold phage propagation is calculated as the number of phage generated from an infected culture divided by the number of phage used to infect the culture. (c) Propagation activity of pre- and post-negative selection SP pools on selection cells encoding hIAPP or Aβ42 as the target APP. (d) Top 10 enriched cyclic peptide sequences after 4 rounds of negative selection as determined by high-throughput sequencing analysis. (e) Propagation of clonal phage encoding cyclic peptide sequences enriched by either positive selection on hIAPP only (CHVVGVI) or by hIAPP positive selection followed by negative selection against Aβ42 inhibitors (CDLGVFR and CRCVFSG) on selection cells encoding hIAPP or Aβ42 as the target APP. (a, c, e) Data reflect two biological replicates plotted as individual values. Propagations used an input of 10⁵ phage.

Figure 6.. Cyclic peptides identified by negative selection inhibit hIAPP aggregation.

(a-b) Structures of cyclo-CDLGVFR and cyclo-CRCVFSG. (c) Effect of varying concentrations of chemically synthesized cyclo-CDLGVFR (left) or linear CDLGVFR (right) on aggregation of 10 μM hIAPP measured by ThT fluorescence. Assay was run in phosphate-buffered saline (PBS) pH 6.9 at 32 °C under quiescent conditions. Data reflect plots of individual values of three technical replicates. The hIAPP alone (0 μM peptide) data is the same in both plots. (d) Effect of cyclo-CDLGVFR on lag time (tlag) of hIAPP aggregation. Data reflects tlag fits from four biological replicates (4 separate ThT assays; see Supplementary Figure 18) plotted as individual values. P-values (two-tailed Student’s t-test between 0 μM peptide and each peptide concentration): **, P ≤ 0.01. (e) Effect of varying concentrations of chemically synthesized cyclo- CRCVFSG (left) or linear CRCVFSG (right) on aggregation of 10 μM hIAPP measured by ThT fluorescence. Assay was run in phosphate-buffered saline (PBS) pH 6.9 at 32 °C under quiescent conditions. Data reflect plots of individual values of three technical replicates. The hIAPP alone (0 μM peptide) data is the same as in panel (c). One replicate of 40 μM linear CRCVFSG excluded due to high ThT signal at time = 0. (f) Effect of cyclo-CRCVFSG on lag time (t_lag) of hIAPP aggregation. Data reflects t_lag fits from four biological replicates (4 separate ThT assays; see Supplementary Figure 19) plotted as individual values. P-values (two-tailed Student’s t-test between 0 μM peptide and each peptide concentration): **, P ≤ 0.01. (g-h) TEM analysis of 10 μM hIAPP incubated in the presence of 40 μM cyclo-CDLGVFR (g) or cyclo-CRCVFSG (h) for 2 h. (i-j) TEM analysis of 10 μM hIAPP incubated in the presence of 40 μM cyclo-CDLGVFR (g) or cyclo-CRCVFSG (h) for 14 h. (g-j) For compatibility with TEM, hIAPP incubations were run in 25 mM sodium phosphate buffer, pH 7.4; see Supplementary Figure 13 for hIAPP aggregation kinetics under TEM assay conditions. Scale bars = 200 nm. This experiment was not repeated.