Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment

Abstract

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)-fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis

“…1A, top panels), an essential component of PNCs (15). Compounds able to reduce PNC prevalence by 50% (13) were filtered in secondary assays to eliminate those that induced apoptosis, DNA damage, generic cytotoxicity, or cell-cycle blockage (13, 16). Remaining hits were evaluated for invasion inhibition through Matrigel and anchorage-independent growth inhibition using soft agar assays.…”

“…Remaining hits were evaluated for invasion inhibition through Matrigel and anchorage-independent growth inhibition using soft agar assays. Compound MLS000556915 was selected for optimization based on its potency as a PNC inhibitor, soft agar growth inhibitor, ability to block invasion, and lack of cytotoxicity (13, 16). …”

“…Thus, being a complex, multi-component structure, PNC prevalence may better reflect the complex nature of malignant transformation than a single gene or gene product. Using PNC reduction as a surrogate metastatic phenotypic marker, we developed a lead compound upon optimization of hits from a high-content screen (13). Here we report the evidence for metarrestin as a promising anti-cancer compound.…”

Identification of Disease-Relevant Genes for Molecularly-Targeted Drug Discovery

“…Recent guidelines advocate for the assessment of key properties including stability, solubility, permeability, potency and selectivity to evaluate the fit-forpurpose suitability of a given small molecule tool [33]. The combination of chemical genetics and HCS has turned out to be a powerful tool for uncovering protein functions and dissecting signaling pathways [21,[34][35][36][37]. Recently, these approaches have been used extensively to interrogate stem cell biology.…”

Identifying New Targets for Personalised Cancer Therapy

“…The use of small molecule probes to gain insight into the biological processes has been extremely effective and is having an ever growing impact on basic research and drug discovery. The combination of chemical genetics and HCS has turned out to be a powerful tool for uncovering protein functions and dissecting signalling pathways (Baldwin et al, 2008;Kalen et al, 2009;Mani et al, 2008;Norton et al, 2009;Raccor et al, 2008).…”