**4. Current small molecule inhibitors of histone methyltransferases**

Development of small molecule inhibitors for histone methyltransferases has garnered remarkable attention over the past years due to their combined efficacy and potency in various cancer treatments. In this section, we will discuss small molecule inhibitors of a few HMTs that have either shown promising results in preclinical development, clinical trial stage, or that have been FDA approved.

## **4.1 Small molecule inhibitors of EZH2**

As aforementioned, EZH2 is a lysine methyltransferase that is overexpressed and found to contribute to many cancer progressions including but not limited to breast, prostate, colon, ovarian, liver, bladder, lymphoma, skin, and lung cancer. The overabundance of EZH2 causes hypersilencing of genes that restrain proliferation and promotes differentiation [67]. As a result, several studies have been conducted to understand the mechanism of action and structure of the enzyme so that appropriate drugs can be developed to inhibit its aberrant activity. For example, FDA has approved the use of tazemetostat (EPZ6438) (**Table 1**), an EZH2 small molecule inhibitor, for the treatment of epithelioid sarcoma (not qualified for resection) in 16 years and above patients. Tazemetostat has an inhibition constant (Ki) of 2.5nM and works by competitively inhibiting SAM binding site on EZH2 [32]. Another small molecule inhibitor, CPI-1205 (**Table 1**), completed phase 1 clinical trial for B-cell lymphoma and solid advanced tumor and is in phase 1b/2 clinical trial for metastatic castration-resistant prostate cancer (mCRPC) [130, 131]. Furthermore, another potent small molecule inhibitor, GSK2816126 (**Table 1**), which showed remarkable preclinical potential entered phase 1 clinical trial for the treatment of lymphoma and solid cancers but proved to be unsuitable target for inhibiting EZH2 due to its unfavorable pharmacokinetic profile [132]. More than 50 small molecule inhibitors for EZH2 are in preclinical development [133]. A few are highlighted in **Table 1**.

*Translational Research in Cancer*

H3 [124].

**3.4 In silico screen**

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

*In silico* screening or virtual screening is a common method used in drug discovery as a pre-filtering method for identifying promising compounds that can be used for experimental studies. Drug development using this method of screening is estimated to save approximately \$130 million and 0.8 years per drug [125]. The approach to virtual screening can be broadly divided into structure-based methods

*Schematic demonstrating the use of FRET for epigenetic screen. In the presence of histone methyltransferase and its methyl donor, SAM, a GFP-tagged Histone 3 becomes methylated on lysine-9 (K9) and undergoes FRET as terbium (Tb) conjugated antibody binds to the monomethylated K9 (K9me). This leads to increased GFP emission at 540nm wavelength (*Right arm of the diagram*), demonstrating the occurrence of K9 methylation. In contrast, when in the absence of K9 methylation mediated by HMTs, due to the addition of potent HMT inhibitors (*Lower arm of the diagram*), FRET does not occur as K9 cannot be methylated and thereby the antibody cannot bind to its epitope. As a result, the wavelength of GFP will be at lower end of the emission* 

**10**

**Figure 1.**

*spectra, 520 nm.*


#### **Table 1.**

*List of representative small molecule inhibitors for EZH2, hDOT1L, and PRMT5.*

**13**

*Discovery of Small Molecule Inhibitors for Histone Methyltransferases in Cancer*

There are over 20 hDOT1L small molecule inhibitors and can be categorized into four groups based on their mode of action: (i) SAH(S-adenosyl-L-homocysteine) mimicking compounds; (ii) benzimidazole or (iii) urea group-containing compounds; and (iv) carbamate-containing compounds [143]. The activity of human homolog of yeast DOT1L or hDOT1L is mostly dysregulated in a subset of acute myeloid leukemia that has MLL gene translocation. This results in an onco-MLL protein which aberrantly recruits hDOT1L to the promoter of MLL target genes. Together with other transcription factors, hDOT1L drives the overexpression of HoxA9 and HoxA7 which leads to leukemia [141]. Hence, the need for inhibitors of hDOT1L. The first potent inhibitor of this HMT was EPZ004777 (**Table 1**), but it failed to progress through clinical development due to poor pharmacokinetics [139]. Another small molecule inhibitor, EPZ5676 (pinometostat) (**Table 1**) with Ki of less than 0.08nM, was shown to have improved pharmacokinetics and has now

PRMT5 is overexpressed in a several types of cancers. There are currently over 50 PRMT5 small molecule inhibitors, and PRMT5 is emerging as a hotspot for cancer targeted therapy [144]. Some small molecule inhibitors of PRMT5 are currently undergoing assessment in phase 1 clinical trial for non-Hodgkin lymphomas and solid cancers include GSK3326595 and JNJ-64619178 (**Table 1**). PRMT5 has also been implicated in the progression and metastasis of pancreatic and colon cancer. As a result, a lead compound, PR5-LL-CM01 (**Table 1**), has been discovered by our group to have more potent inhibitory properties compared to EPZ015666 in pancreatic and colon cancer cells [114]. However, EPZ015666 showed high potency *in vitro* and in mantle lymphoma cells with an IC50 of 22nM (**Table 1**) [145]. Another potent inhibitor of PRMT5, LLY-283 (IC50 = 20 nM) (**Table 1**) showed an outstanding inhibition of breast, lung, skin, ovarian, and hematological cancer cells' proliferation [142].

Taken together, in this chapter, we discussed the important roles that epigenetic enzymes play in a variety of cancers. We also summarized several popular methods currently used for screening small molecule inhibitors of epigenetic enzymes. As shown in **Table 1**, we provided a list of representative small molecule inhibitors of HMT that are either FDA approved, or at preclinical or different stages of clinical trials. Notably, compared to the well-developed HDAC small molecule inhibitors, the development of small molecule inhibitors for HMTs is a rising and cutting-edge drug development area. We can envision that in the next 5~10 years, intense attention will continuously be drawn to the discovery of HMTs small molecule inhibitors. We have no doubt that many HMTs small molecule inhibitors will be shifted into clinical trials, more will be approved by FDA, and most likely, more members of HMTs will be targeted for cancer treatment. Additionally, it is very possible that novel HTS methods will emerge, which will further accelerate the discovery of anti-HMTs drugs. Moreover, it is viable that the clinical indications of HMTs small molecule inhibitors could be further expanded to other diseases beyond cancer. In summary, the development of new classes of anti-HMTs drugs will offer brand new

*DOI: http://dx.doi.org/10.5772/intechopen.92830*

completed phase 1 clinical trial [138].

**4.3 Small molecule inhibitors of PRMT5**

**5. Conclusion, perspective, and future directions**

and exciting opportunities for diseases treatment.

**4.2 Small molecule inhibitors of hDOT1L**
