Use of PROTACS as molecular probes of angiogenesis

Abstract

Small molecules designed to specifically activate or inactivate protein functions have been useful to study biological processes. PROTACS are small molecule chimera which comprise a ligand and a peptide recognition motif for an E3 ligase. These novel reagents exploit the ubiquitin-mediated proteasome degradation pathway to target the ligand-bound protein for intracellular degradation. Here, we report that an estrogen receptor (ER)-targeting PROTACS that causes degradation of ER is able to potently inhibit endothelial cell differentiation in a three-dimensional angiogenic sprouting assay. These findings support the use of ER-targeting PROTACS as probes of angiogenesis.

Figure 1

Endothelial cell spheroids were seeded in collagen I gels in a 96 well plate and stimulated with vascular endothelial growth factor (VEGF; 20 ng/ml) to induce angiogenic sprouting. In replicate wells, VEGF-treated spheroids were co-incubated with 12 µM of HIF-1α octapeptide, E2-octa-[Ala] or E2-octa. Representative photographic images of spheroids taken after 20 h show invasive growth of vessel structures with octapeptide (A) or E2-octa-[Ala] (B) co-treatment, but potent inhibitory effect with E2-octa (C) co-treatment.

Figure 2

E2-penta exerts the most potent inhibition of sprouting. VEGF-stimulated spheroids were co-treated with 3 µM of E2-PROTAC, which contain the HIF-1α peptide of differing length. The percentage of angiogenic sprouting was quantified by measuring total number and length of sprouts as done previously.

Figure 3

HUVECs were treated with vehicle (Con) or E2-penta at 0.5 and 2 µM for 24 h. Total cell protein was fractionated on polyacrylamide gels and Western blotted using a polyclonal ERα-specific antibody. The blots were also probed with anti-β-actin antibody to control for protein loading.

Scheme 1

Estradiol (E2)-based PROTAC containing a pentapeptide derived from HIF-1α recruits estrogen receptor (ER) to the pVHL complex for ubiquitination and degradation.