A New Methodology for Incorporating Chiral Linkers into Stapled Peptides

Abstract Stapled peptides have arisen as a new class of chemical probe and potential therapeutic agents for modulating protein–protein interactions. Here, we report the first two‐component i,i+7 stapling methodology that makes use of two orthogonal, on‐resin stapling reactions to incorporate linkers bearing a chiral centre into a p53‐derived stapled peptide. Post‐stapling modifications to the chain were performed on‐resin and enabled rapid access to various peptide derivatives from a single staple. The stapled peptides have increased helicity, protease stability and in vitro binding affinities to MDM2 compared to the equivalent unstapled peptide. This approach can be used to generate a library of diverse stapled peptides with different properties starting from a single stapled peptide, with scope for much greater functional diversity than that provided by existing stapling methodologies.

The reaction mixture was stirred overnight, followed by quenching with water (6 mL

Fluorescence Polarisation Assays
Direct FP: For direct FP assays, FITC-labelled peptides were dissolved in DMSO as stock solutions and diluted with the assay buffer (phosphate buffered saline, pH 7.4, 2 Mm DTT) to a final concentration of 100 nM. MDM2 (residues 2-125) was prepared to a concentration of 10 μM and serially diluted 2-fold with the assay buffer for a 16-point titration in triplicate. FITC-labelled peptide (100 nM, 20 μL) was then mixed with each serially diluted MDM2 solution (top concentration of 10 μM, 20 μL) in the microplate and incubated at 25°C for 30 min before the measurement was taken. The following filters were used for measuring the FP signals from FITC: an excitation filter 482-16 nm, a dichroic mirror LP504 and an emission filter 530-40 nm.
Data were analysed on GraphPad Prism 5.0 and the dissociation constant, Kd, with standard deviation was determined using the following equation assuming the ratio between the concentration of the bound and that of the total FITC-labelled peptide is proportional to the fluorescence polarisation change: Where FP is the fluorescence polarisation, FPmin is the minimum FP, FPmax is the maximum FP, L0 is the total concentration of TAMRA-labelled peptide, P0 is the total concentration of protein and Kd is the dissociation constant. Table S1. Binding affinities for peptides SP1-βA-F (A/B) and SP2-βA-F (B) determined by direct FP.

SP1-βA-F (B)
10.5 ± 10 SP2-βA-F (B) 46.2 ± 5.3 [a] SP2-βA-F (A) could not be used in direct FP as not enough peptide was available for the assay. Data were fitted in GraphPad Prism 5.0 using the following equations described in the literature: [5] ܽ Where F is the fluorescence polarisation, x is the concentration of the non-labelled peptide or nutlin-3a, Ki is the dissociation constant of the non-labelled peptide or nutlin-3a, Kd is the known dissociation constant of the TAMRA tracer, L0 is the total concentration of the TAMRA tracer, P0 is the total concentration of MDM2, F0 is the fluorescence polarisation when no TAMRA tracer is bound to MDM2 and Fmax is the fluorescence polarisation when all TAMRA tracer is bound. [6] The unit for Ki is consistent with that of L0, P0 and Kd. Figure S2. Competitive FP titration curve for peptides and nutlin-3a. Kd values can be found in Table 1. * nutlin-3a IC50 value was generated using a non-linear fit one site analysis as this provided a superior fit to the data points.

SP2 (A)
69.04 ± 9.95    Figure S4. Crude HPLC chromatographs of A) SP0, reaction mixture after B) conjugating with Linker 1 using CuAAC conditions and C) stapling using RCM conditions. The blue arrows denote the product peptide peak/s. Figure S5. Crude HPLC chromatograph of A) SP1-K and B) SP1-βA-F. The blue arrows denote the product peptide peak.