**3.2 AlphaLISA screen (amplified luminescent proximity homogeneous assay-linked assay)**

AlphaLISA is a high throughput screening approach used to analyze and measure post-translational modifications, protein-protein interactions, and concentrations of analytes. The robust, highly sensitive, reproducible, miniaturized, scalable, and automated nature of AlphaLISA assays earned them their widespread application in research and drug discovery. The principle behind AlphaLISA technology is based on the mechanism of another methodology, Luminescent oxygen channeling immunoassay (LOCI). LOCI involves chemiluminescent reaction of a singlet oxygen transfer and was developed in 1994 by Ullman et al*.* to quantify latex particle binding [113]. Similarly, AlphaLISA assays employ biotinylated antibody bound to streptavidin donor beads and an acceptor bead bound to a second antibody. These antibodies are specific to different epitope on a protein (could also be product of a bimolecular interaction or a modified protein). The binding of these antibodies to the proteins brings the donor and the acceptor bead into proximity of at most 200nm. Upon excitation of donor beads at 680nm, a singlet oxygen is excited from the donor bead and this triggers singlet oxygen reaction with the chemical dyes (thioxene and europium) on the acceptor bead, which results in chemiluminescent emission at 615nm [114, 115]. Multiplate readers equipped with AlphaLISA screen detections can be used to record signals. A systematic method used to validate the quality of HTS assay output like AlphaLISA is called the Z-factor. The Z-factor is calculated by accounting for the positive and negative controls' mean signal-tomean background ratio. Hence, in the design of AlphaLISA assay, to ensure quality control, wells containing maximum signal and no signal solution mix must be included [116]. This assay is widely optimized to screen for small molecule modulators for different epigenetic enzymes. For example, in our lab, Prabhu et al. used an optimized AlphaLISA screen protocol to identify a lead compound capable of targeting PRMT5, and subsequently, the compound was shown to be more potent in reducing pancreatic and colon cancer cells' proliferation compared to another commercially available compound EPZ015666 [26]. Not only is this assay used to screen for compounds for epigenetic targeted therapy, but also they uncover new roles of different epigenetic enzymes in cancer. Consequently, together with other assays, it was uncovered using AlphaLISA HTS assay that an inhibitor of G9a lysine methyltransferase, A-366, limits cell growth and differentiation in leukemic cells [117]. Potent small molecule inhibitors for EZH2, a methyltransferase known to silence tumor suppressor genes, are also being identified using this particular HTS assay [118]. AlphaLISA kits specific for histone methyltransferase modifications, among many other epigenetic enzymes, are commercially available for research and drug development purposes [119].

## **3.3 FRET (Förster/fluorescent resonance energy transfer) assay**

Discovered in 1946 by Theodor Förster, FRET is cell-based assay that enables real-time observance of molecular interactions within cells [120]. This phenomenon

*Translational Research in Cancer*

to multiple oncogenic signaling pathways.

feedback loop involving NF-κB signaling [107].

tumorigenesis in cancer.

**in cancer treatment**

cancer and exert a crucial role in Wnt signaling through mediating the action of β-catenin on Wnt target genes as a transcriptional coactivator [100]. Moreover, c-fos is a proto-oncogene and overexpressed in a set of cancers. Some groups reveal that PRMT4 regulates transcriptional activation of c-fos, and that matrix metalloproteinases (MMPs), c-fos target genes, are significantly downregulated in CARM1-deficient cells [101]. Therefore, arginine methylation by PRMT4 is related

An important PRMT member that plays a critical role in cancer is PRMT5. As a primary type II PRMT, PRMT5 functions in the presence of other binding partners, such as MEP50. Hou et al. discovered that E-cadherin expression was remarkably repressed by SNAIL and PRMT5 recruited by bridge molecule AJUBA, which was favorable for tumor metastasis [102]. It was also reported that p53 can be methylated on multiple arginine sites by PRMT5 in response to DNA damage [103]. Scoumanne et al. proposed that PRMT5 inhibition may promote cancer cells to progress toward apoptosis under chemotherapy/radiotherapy [104]. Moreover, Cho et al*.* found methylation of E2F1 by PRMT5 weakened its ability to promote apoptosis and repress proliferation, indicating PRMT5 overexpression may enhance cancer cells' growth and survival [105]. It is well known that continuous NF-κB activation exists in most cancers. Our group uncovered that PRMT5 dimethylated the p65 subunit of NF-κB at arginine 30 (R30) to activate NF-κB pathway [106], an important transcription factor that is involved in the progression of many cancers. Also, PRMT5 is reported to promote its own overexpression in several cancers through a

Besides the PRMTs we discussed above, PRMT6 is widely taken as a transcriptional repressor. Neault et al*.* reported that embryonic fibroblast cells from the PRMT6 knockout mouse were subjected to a premature senescence, while the cellular senescence can be rescued in PRMT6 and p53 double knockout mouse embryonic fibroblast (MEF) cells [108], affirming growth suppression effect of excess p53 due to PRMT6 deficiency. Thus, PRMT6 actively suppresses p53 cascade to promote

Taken together, many members in the PRMT family have shown essential role in cancer development and progression. Thus, it is unsurprising that these PRMTs

**3. Discovery of small molecule inhibitors for histone methyltransferases** 

The timeline of drug development from conception of the idea to a feasible drug available in the market for treatment of diseases takes between 12 and 15 years and can cost up to \$1billion [109]. Drug discovery process begins with identifying a druggable biological target that contributes to a disease progression. These targets can be identified through text mining from online databases, microarray data mining using bioinformatic tools, proteomic data mining from proteomic databases, and chemogenomic data mining, which involves simultaneous exploration of multiple cell phenotypes by screening small molecules from chemical libraries to a number of biological targets [110]. Then, promising or known targets can be validated *in vitro* and *in vivo* to confirm that their activity influences phenotype associated with a disease. This step is followed by screening or high throughput screening (HTS), which describes the process of sifting through compound libraries for molecules with high affinity for a target of interest [111]. The two approaches

have become the rising targets in cancer therapeutics in recent years.

**3.1 Screening assays for epigenetic drug discovery**

**8**

depends on the proximal interaction (1-10nm) of a donor fluorophore and an acceptor fluorophore, whereby upon excitation, the donor fluorophore transfers energy to the acceptor, increasing its emission wavelength [121] (**Figure 1**). The measure of FRET is taken as the ratio of the intensities of the donor/acceptor fluorophore. Although highly sensitive in distance, FRET assay does not permit the level of sensitivity and high throughput as AlphaLISA [122]. A type of FRET, FLIM-FRET (fluorescence lifetime imaging FRET) was used to conduct epigenetic biomarker screening in ER-positive breast cancer cell line and patients. The utilization of FRET in this study was based on the presence of certain histone modifications around ERα in the nucleus. Consequently, the assay revealed H3K27ac and H4K12ac interaction with ERα, making HATs a potential therapeutic target in compound screening [123]. Another study optimized TR-FRET (time-resolved FRET) for high throughput screening, following the treatment with HDAC inhibitors, to detect the methylation levels of histone 3(H3) in U-2 OS cells using terbium-tagged antibodies specific to a particular H3 modification and green fluorescent protein (GFP)-tagged H3 [124].
