Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist

Abstract

We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.

Figure 1

Chemical structures of Grb2-SH2 domain binding antagonists and analogues.

Figure 2

Inhibition of cell migration by 1 and 3. (A) Inhibition of HGF-stimulated (50 ng/mL; white bar), or unstimulated (black bar) PC3M cell migration by 1 and 3. Values represent mean number of migrated cells per 10× microscopic field, expressed as percent maximum, +/- standard deviation. (B) Dose-response analysis of inhibition of PC3M cell migration by 3.

Figure 3

Protein capture by 3 and 4. (A) SDS PAGE and anti-Grb2 immunoblot analysis of lysates prepared from the human leiomyosarcoma cell line SK-LMS-1. Following target protein capture using 3 (left) or 4 (right), SA-coated beads were washed with buffer containing NaCl at the concentrations (M) indicated above each lane. (B) SDS-PAGE and immunoblot analysis of SK LMS-1 lysates (left lane) for known Grb2-associated proteins (top to bottom) Nck1, Nck2, Gab1 and Sos1 captured using 4 (middle lane) or 3 (right lane).

Figure 4

Representative MS/MS spectra of two [M+2H]²⁺ tryptic peptides from Grb2, which represent 10.1% sequence coverage, captured by 3-conjugated beads but not 4-conjugated beads. The b ion series is shown in red and the y ion series in blue. Sequest statistics: (A) Xcorr score 3.707, ΔCn=0.44 and p=1.9e-9; (B) Xcorr score = 2.98, ΔCn=0.28 and p=1.4e-5, where Xcorr is the peptide correlation score and ΔCn is the difference in correlation between the top two peptide matches.

Figure 5

Compound 1 inhibits PAK1 activation and nuclear translocation. (A) PC3M cells were serum-deprived and treated with 1 (+;1 uM), or left untreated (-), as indicated for 16 h, then left without further treatment (left lanes) or stimulated briefly with HGF (50 ng/mL; right lanes) for the times indicated. Cell lysates were analyzed by SDS-PAGE and immunoblot analysis with anti-phospho-PAK1 antibody (upper panel), or anti-PAK1 antibody to control for protein loading (lower panel). (B) PC3M cells were treated with compound 1 for 16 h and nuclear extracts were prepared using high-salt buffer. Samples were analyzed by SDS-PAGE and immunoblot analysis with anti-PAK1 antibody (upper panel), or anti-CREB antibody (lower panel).

Figure 6

Compound 1 inhibits lamellipodia formation. Alexa 488-phalloidin (green) and DAPI staining (blue) of PC3M cells (left panels) and MDCK cells (center and right panels) treated with HGF alone (top panels, control) or HGF + 1 (1 uM; lower panels). For PC3M cells, arrowheads indicate regions of cellular extension into the artificial wound. For MDCK cells, advancing lamellipodia observed in center panels are outlined in white in the right panels for clarity.

Scheme 1

Synthesis of the upper segment 10^{a a}Reagents, conditions and yields: (a) Tf₂NPh, 2,4,6-collidine, DMAP, DMF-CH₂Cl₂, reflux, 12 h, 47%; (b) Pd(OAc)₂, DPPP, Et₃N, reflux, 14 h, 81%; (c) LiAlH₄, THF, rt., overnight, 54%; (d) (i) Boc-Asn-OH, DIPCDI, HOBt, DMF, rt, 12 h 87%; (ii) TFA, CH₂Cl₂, rt, 1 h, 79%; (iii) Fmoc-1-amino-cyclohexenecarboxylic acid, EDC, HOBt, DMF, rt, 12 h, 79%; (iv) piperidine, CH₃CN, rt, 2 h, 86%.

Scheme 2

Synthesis of inhibitor 2 and biotinylated ligands 3 and 4^{a a}Reagents, conditions and yields: (a) EDC, HOBt, DMF, rt, 12 h (79% for 12a; 74% for 12b); (b) (i) piperidine, CH₃CN, rt, 2 h; (ii) ^tbutyloxalyl chloride, ⁱPr₂NEt, DMF, rt, 12 h, (two steps, 43% for 13a; 80% for 13b). (c) TFA-TES-H₂O, rt, 1 h 42%; (d) EDC, DMAP, DMF, rt, 12 h (78% for 14a; 42% for 14b); (e) TFA-HS(CH₂)₂SH-H₂O, rt, 1 h (41% for 3; 56% for 4).