Supplementary Figure S1 from Palazestrant (OP-1250), A Complete Estrogen Receptor Antagonist, Inhibits Wild-type and Mutant ER-positive Breast Cancer Models as Monotherapy and in Combination
Authors: Parisian Alison D. , Barratt Susanna A. , Hodges-Gallagher Leslie , Ortega Fabian E. , Peña Guadalupe , Sapugay Judevin , Robello Brandon , Sun Richard , Kulp David , Palanisamy Gopinath S. , Myles David C. , Kushner Peter J. , Harmon Cyrus L. 
Published: March 2024

<p>OP-1250 specifically inhibits the ER without cross-reactivity with other hormone receptors. A, LanthaScreen co-activator recruitment assay using wild-type ERα LBD and fluorescein tagged PGC1a comparing OP-1250 and other approved and in-trial comparator molecules. Data are normalized to DMSO vehicle and 10 µM E2-treated wells (100% recruitment) and presented as mean and SD of at least two separate experiments. B, Reporter gene activity assay in CAMA-1 and MCF7 cells transiently transfected with a luciferase reporter gene construct containing an estrogen response element and minimal tk promoter. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to E2 vehicle. The dotted line represents activity in the absence of estrogen, the hashed line represents activity with E2 supplementation. Data shown represent mean and SD from a representative of at least three independent experiments. C, Reporter gene assay of ER-negative SK-BR-3 cells transiently transfected with wild-type or mutant ESR1 vectors along with a luciferase reporter gene cloned to 3× estrogen response element and TATA minimal promoter. Transcriptional activity was measured as luciferase activity. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to wild-type E2 vehicle and empty vector + E2. The dotted line represents activity with empty vector control, the hashed line represents activity following supplementation with 31.6 nM E2. Data shown represent mean and SD from a representative of at least three independent experiments. D, Reporter gene activity assay of HeLa cells transiently transfected with glucocorticoid, androgen, or progesterone receptor and a luciferase reporter cloned to 3× PRE and TATA minimal promoter, measuring the impact of OP-1250 on the glucocorticoid receptor in the presence and absence of 31.6 nM dexamethasone, progesterone receptor in the presence and absence of 500 nM MPA (medroxyprogesterone acetate), and androgen receptor in the presence and absence of 50 nM R1881. Data are normalized to the indicated amount of agonist (either MPA, dexamethasone, or R1881). Figures show data as the mean and SD from a representative of at least three independent experiments. E, Reporter gene assay of ER-negative SK-BR-3 cells transiently transfected with ESR1 or ESR2 along with a luciferase reporter gene cloned to 3× estrogen response element and TATA minimal promoter. Transcriptional activity was measured as luciferase activity. Cells were cultured in estrogen-depleted media and reporter gene activity was normalized to E2 vehicle. Data shown represent mean and SD from a representative of at least three independent experiments. F, Uterine wet weight assay following treatment with a dose response of OP-1250 or fulvestrant in the presence of E2 supplementation. Mean and SEM from six mice per treatment group are plotted with asterisks indicating significance relative to vehicle by one-way analysis of variance.
* represents adjusted p-value <0.05, *** represents adjusted p-value <0.001, **** represents adjusted p-value <0.0001. G, Simple western analysis of ERα protein levels in Ishikawa cells after 72-h treatment with listed compounds. Samples are normalized to untreated control. Data shown represent mean and SD from a representative of at least three independent experiments. H, Representative blot image from simple western assay shown in panel G after 72-h treatment with 300 nM compound or 1 nM E2 with indicated compound treatments and cell line. AR=androgen receptor; GR=glucocorticoid receptor; PR= progesterone receptor; WT=wild-type.</p>