Peptide bicycles that inhibit the Grb2 SH2 domain

Abstract

Developing short peptides into useful probes and therapeutic leads remains a difficult challenge. Structural rigidification is a proven method for improving the properties of short peptides. In this work, we report a strategy for stabilizing peptide macrocycles by introducing side-chain-to-side-chain staples to produce peptide bicycles with higher affinity, selectivity, and resistance to degradation. We have applied this strategy to G1, an 11-residue peptide macrocycle that binds the Src homology 2 (SH2) domain of growth-factor-bound protein 2 (Grb2). Several homodetic peptide bicycles were synthesized entirely on-resin with high yields. Two rounds of iterative design produced peptide bicycle BC1, which is 60 times more potent than G1 and 200 times more selective. Moreover, BC1 is completely intact after 24 hours in buffered human serum, conditions under which G1 is completely degraded. Our peptide-bicycle approach holds promise for the development of selective inhibitors of SH2 domains and other phosophotyrosine (pTyr)-binding proteins, as well as inhibitors of many other protein-protein interactions.

Figure 1

A head-to-tail macrocycle based on G1. A) Structures of G1, G1-Pro, and HT1-6. Side-chains shown in red are implicated in direct SH2 domain binding in the known structure-activity relationships of previous G1 analogs.^{[15b, 15d]} Amino acids that differ from the parent peptide G1 are shown in blue. B) Competition assay data for G1, G1-Pro, HT1, HT2, and HT4. Data for HT3, HT5 and HT6 are similar to those for HT2 and HT4 and are shown in Figure S2. IC₅₀ values reflect the curve fits shown.

Figure 2

Models help identify side-chain pairs for intramolecular cross-linking. A) Energy-minimized model of HT1 (ball-and-stick, nitrogens shown in blue, oxygens in red, carbons in gray, and hydrogens omitted) bound to Grb2-SH2 (surface colored to indicate electrostatics). Side-chains of HT1 not involved in direct Grb2-SH2 binding are omitted for clarity. This energy-minimized model is based on the structure of a native peptide ligand and established SAR data on G1 and its analogs.^{[14c, 18]} Modeling and energy minimization was performed using Molecular Operating Environment (Chemical Computing Group) using AMBER99 and OLPS-AA force fields (negligible differences seen between the two). Selected local water molecules from the crystal structure were included explicitly (not shown). Soft constraints were used on the protein and the peptide was allowed to move freely within the pocket. B) The identical model as in A), but rotated to show relative positions of Leu3 and Met9 (purple), and Val7 and Tyr10 (green). C) Structure of HT1 with side-chains color-coded as in B).

Figure 3

Peptide bicycles based on HT1. A) Chemical structures of BC1-4. Side-chains shown in red are implicated in direct SH2 domain binding in the known structure-activity relationships of G1. Purple and green side-chains comprise staples in positions modelled to be in close proximity, as shown in Fig. 2. B) Competition assay data for G1, HT1, and BC1-4. IC₅₀ values reflect the curve fits shown.

Figure 4

Peptide bicycles are resistant to degradation in human serum. Degradation of peptide macrocycles and bicycles was monitored after zero, 1, 7, 24 and 48 hours in buffered human serum. Percent peptide remaining was calculated by integrating HPLC peaks and normalizing to the zero time point.

Figure 5

Inhibition of tensin-SH2 by peptide macrocycles and bicycles. This fluorescence polarization competition assay used the same dye-labeled ligand as the Grb2-SH2 competition assay. IC₅₀ values reflect the curve fits shown.

Figure 6

Anti-pTyr dot blot assay. Peptides were normalized to 750 μM, then serially diluted as shown and spotted onto nitrocellulose. The membranes were blocked with bovine serum albumin, probed with anti-pY antibodies 4G10 (above) or PY20 (see Fig. S4), probed with secondary antibody, and then images were developed using chemoluminescent detection. Controls included EGF-stimulated cell lysate (highest concentration is 1 mg/mL total protein), the dye-labeled, pTyr-containing probe peptide used in the fluorescence polarization assays (pTyr-containing ligand), and vehicle (50% DMSO).