The Androgen Receptor (AR) is a member of the family of nuclear receptors responsive to steroid hormones. AR activity is regulated through the binding of androgenic hormones (eg., testosterone and di-hydroxy testosterone). In turn, AR functions to regulate the expression of a myriad of genes involved in metabolic processes, cell proliferation/apoptosis, and male sexual differentiation and development. AR dysfunction often has severe health implications, such as androgen insensitivity syndrome and prostate cancer. Consequently, AR commands much attention as a target for the discovery and development of improved drugs. In particular, anti-androgens comprise an important class of drugs used in the treatment prostate cancer. The aim of the present work was to devise, validate and perform a preliminary (small-scale) automated HTS screening campaign to identify novel modulators of AR activity. The study incorporated a new whole-cell, luciferase-based assay to quantify human Androgen Receptor activity. The performance and specificity of this AR assay were first confirmed using a small commercial library of known nuclear receptor modulators. The validated AR assay was then scaled-up and deployed to screen a library comprising 506 diverse small-molecule compounds of natural origin. The natural compound library was functionally interrogated to identify both activators and inhibitors of AR. Chemicals identified in the primary screen as putative AR inhibitors were further analyzed using a live cell multiplex assay. The multiplexed assay incorporates a fluorescent Calcein-AM assay to monitor viable cell number, as well as a luminescent AR assay, which were performed sequentially in the same assay well. Using this format, cytotoxic compounds that elicited false-positive ‘hits’ were eliminated from further consideration. Test compounds displaying novel bio-activities to AR, either agonist or antagonist, were further analyzed to establish relative potency values.  Automation of the initial library screens was carried out using an easy-to-use, robust instrumentation. An 8-channel liquid handler was used to prepare serial dilutions of library and reference compounds, followed by their transfer into 384-well assay plates. Subsequent additions of AR reporter cells and detection reagent were performed using a high-speed non-contact reagent dispenser. The small footprint of the instrument, as well as capability to autoclave the dispensing pathway, allowed for sterile manipulations during the assay process. A combination washer/dispenser, capable of media and reagent removal, as well as dispensing cells and other components was then used to perform the live cell multiplex assay. Results demonstrate how the combination of the novel, cell-based chemistries and automation can be used to rapidly, and accurately identify modulators of this important drug target.

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Date of publication: 07 February 2012

Author information: Brad Larson (1); Bruce Sherf (2); & Peter Banks (1)

(1) BioTek Instruments, Inc., Winooski, Vermont, USA
(2) INDIGO Biosciences, Inc., State College, Pennsylvania, USA


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