A Screening Assay Cascade to Identify and Characterize Novel Selective Estrogen Receptor Downregulators (SERDs)

Abstract

Here, we describe an approach to identify novel selective estrogen receptor downregulator (SERD) compounds with improved properties such as oral bioavailability and the potential of increased efficacy compared to currently marketed drug treatments. Previously, methodologies such as Western blotting and transient cell reporter assays have been used to identify and characterize SERD compounds, but such approaches can be limited due to low throughput and sensitivity, respectively. We have used an endogenous cell-imaging strategy that has both the throughput and sensitivity to support a large-scale hit-to-lead program to identify novel compounds. A screening cascade with a suite of assays has been developed to characterize compounds that modulate estrogen receptor α (ERα)-mediated signaling or downregulate ERα levels in cells. Initially, from a focused high-throughput screening, novel ERα binders were identified that could be modified chemically into ERα downregulators. Following this, cellular assays helped determine the mechanism of action of compounds to distinguish between on-target and off-target compounds and differentiate SERDs, selective estrogen receptor modulator (SERM) compounds, and agonist ERα ligands. Data are shown to exemplify the characterization of ERα-mediated signaling inhibitors using a selection of literature compounds and illustrate how this cascade has been used to drive the chemical design of novel SERD compounds.

Antiestrogens: structure-activity relationships and use in breast cancer treatment

“…Although fulvestrant has failed to demonstrate improved responses in first-line treatment compared with SERMs or aromatase inhibitors (Howell et al . 2004 a ), a better understanding of the mechanisms of action of pure AEs is important in view of the development and current clinical testing of several new SERDs with improved oral bioavailability compared with fulvestrant (Callis et al . 2015, McDonnell et al .…”

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive andESR1-Mutant Breast Tumors in Preclinical Models

“…Binding, ER agonism, antagonism, downregulation, and cell proliferation assays were carried out as described previously (23). BIAcore affinity measurements and immunoblotting from compound-treated cells are described in Supplementary Methods.…”

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays

“…This enabled complex time course experiments, repeat dosing on long-term assays, and simple combination dosing, as well as allowed the Echo to be used to add ligands and reagents pre-or postcompound dosing for stimulated cell assays. For example, direct stimulatory ligand dosing was enabled for functional nuclear hormone receptor antagonism assays, 17 and this was utilized extensively on the Integrated Echo II System, enabling the timing and order of addition of compound and ligand to be explored during assay development. The system was also amenable to codosing of compound plus other modulators that could be used to discern compound mechanism of action in a routine manner.…”

“…The system was also amenable to codosing of compound plus other modulators that could be used to discern compound mechanism of action in a routine manner. 17 • • During the assay, the user was provided with continuously updated details of the running stage, incubation end time, and current location of each assay plate. At completion, all assay details and final timings were saved in an accessible database.…”

“…Although intermediate plates can be used (i.e., ARPs with added buffer as described above), this requires an additional, unnecessary compound handling step to transfer the diluted compounds into the final cell plate. Direct Echo compound dosing into cell-based assays has been reported by AstraZeneca 11,[15][16][17] as well as a number of other groups [18][19][20][21] and offers the advantage of a simplified workflow without any additional steps or tip/plate consumables, reducing costs and overall assay time. Direct dosing also removes any concerns related to the long-term storage of ARPs (e.g., evaporation issues that can lead to variations in assay data quality).…”