A Small-Molecule Pan-Id Antagonist Inhibits Pathologic Ocular Neovascularization

Abstract

SUMMARY Id helix-loop-helix (HLH) proteins (Id1–4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.

Anti-tumor effects of an Id antagonist with no acquired resistance

“…We tested whether AGX51 would have similar effects on pancreatic cancer cells. In line with the results obtained in other cancer models that we tested, AGX51 caused a depletion of the ID1 and ID3 proteins in the pancreatic cancer cell line 806, starting at a dose between 4 and 20 µM ( Figure 3A) with ID3 showing slightly less sensitivity as observed in other cellular and biophysical assays (Wojnarowicz et al, 2019). Furthermore, AGX51 decreased cell viability in a variety of pancreatic cancer cell and organoid lines, including mouse organoids ( Figure 3B), mouse cell lines 806, NB44, and 4279 ( Figure 3C), and human cell lines Panc1 and A21 ( Figure 3D) with an IC50 of 5.5-19.5 µM.…”

“…Id3 and Id4 are also lost albeit with delayed kinetics. Id2 is not expressed in 4T1 cells (data not shown) but we previously showed that ID2 levels decrease in response to AGX51 treatment in HCT116 cells (Wojnarowicz et al, 2019). Similar to 4T1 cells, HMLE RAS Twist cells (immortalized human mammary epithelial cells overexpressing Twist) also showed reduced levels of ID1, ID3 and ID4 when treated with 20 μM AGX51 over similar time courses ( Figure S1B).…”

“…An increase in E protein binding occurred at a time when no Id1 protein loss was observed ( Figure 1G), suggesting that the increase in E protein binding is due to disruption of the Id1-E PPI as opposed to decreased overall Id levels. This conclusion is supported by direct evidence of the disruption of the ID1-E47 interaction observed in HCT116 cells (Wojnarowicz et al, 2019).…”

“…We recently reported that AGX51 treatment of HUVEC and HCT116 cells leads to disruption of the Id-E protein-protein interaction (PPI), resulting in ubiquitination of Id1 and its subsequent degradation (Wojnarowicz et al, 2019). To further characterize the effects of AGX51 in cancer cells, we treated the 4T1 murine mammary cancer cell line with increasing concentrations of AGX51 (0-80 μM) for 24 hours and observed a significant decrease in Id1 protein levels starting at 40 μM ( Figure 1A).…”

“…We recently identified a small-molecule antagonist of Id proteins based on X-ray crystal structure coordinates and an in silico screen (Wojnarowicz et al, 2019). The top candidate from this screen, AGX51, caused a change in the secondary structure of purified Id1 and inhibited the ability of Id1 to associate with its target E proteins in cells.…”