**3.4 In silico screen**

*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

#### **Figure 1.**

*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 spectra, 520 nm.*

**11**

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

and ligand-based methods. Structure-based approach encompasses docking candidate molecules against available 3D structure of the target protein. When there is no crystal structure of the target protein, ligand-based approach is more useful because it relies on the screening of bioactive ligands of a similar chemical structure [126]. This approach is similar to pharmacophore-directed homology modeling: a process that involves superposing known active ligands for structurally similar targets and then extracting matching chemical properties of the ligand that are required for their bioactivity. Pharmacophore from different bioactive molecules can be generated using commercially available software such as HipHop, PHASE,

In computer-aided design of epigenetic drugs, there are a plethora of databases like ZINC containing over 35 million compounds available for screening. Other databases like *SPECS, Chembridge,* and *Enamine* have been used to identify inhibitors for most subsets of histone methyltransferases [128]. Molecular dynamics simulation also aids in drug discovery as they are employed to understand the conformational changes in the different domains of a target protein [129]. For instance, a study developed analogs of eosin, a template molecule known for having anti-methyltransferase activity, using pharmacophore methods. These molecules were docked on to PRMT1, SET7, and CARM1, and the AutoDock analysis revealed that compounds that target SAM substrate-binding site were more active in PRMT1 and CARM1 while those that target lysine and co-factor binding site were more

**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.

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**.

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

DISCO, HypoGen, among others [127].

**4.1 Small molecule inhibitors of EZH2**

promising in SET7 [126].

#### *Discovery of Small Molecule Inhibitors for Histone Methyltransferases in Cancer DOI: http://dx.doi.org/10.5772/intechopen.92830*

and ligand-based methods. Structure-based approach encompasses docking candidate molecules against available 3D structure of the target protein. When there is no crystal structure of the target protein, ligand-based approach is more useful because it relies on the screening of bioactive ligands of a similar chemical structure [126]. This approach is similar to pharmacophore-directed homology modeling: a process that involves superposing known active ligands for structurally similar targets and then extracting matching chemical properties of the ligand that are required for their bioactivity. Pharmacophore from different bioactive molecules can be generated using commercially available software such as HipHop, PHASE, DISCO, HypoGen, among others [127].

In computer-aided design of epigenetic drugs, there are a plethora of databases like ZINC containing over 35 million compounds available for screening. Other databases like *SPECS, Chembridge,* and *Enamine* have been used to identify inhibitors for most subsets of histone methyltransferases [128]. Molecular dynamics simulation also aids in drug discovery as they are employed to understand the conformational changes in the different domains of a target protein [129]. For instance, a study developed analogs of eosin, a template molecule known for having anti-methyltransferase activity, using pharmacophore methods. These molecules were docked on to PRMT1, SET7, and CARM1, and the AutoDock analysis revealed that compounds that target SAM substrate-binding site were more active in PRMT1 and CARM1 while those that target lysine and co-factor binding site were more promising in SET7 [126].
