Epigenetic Screening Libraries

Epigenetic regulation of gene expression is a dynamic and reversible process that implies acetylation, methylation, phosphorylation, ubiquitination, and other chromatin structure modifications that alter DNA transcription mechanisms. These modifications are known as epigenetic marks, and the most important of them are chromatin remodeling and DNA methylation. There are separate groups of proteins, known as “writers” (Lys, Arg, or DNA methyltransferases), “readers” (proteins/domains binding PTMs, ex. Bromodomains), and “erasers” (HDACs, SIRTs) of such marks.

Epigenetic “writers” catalyze the addition of chemical substituents onto either histone tails or DNA. These marks are not necessarily permanent modifications; they can be removed by “erasers.” In particular, the bromodomain-containing family of proteins recognizes or ”reads” modified lysine residues within histone proteins. These mechanisms collectively regulate gene expression to establish normal cellular phenotypes. On the other hand, they can contribute to or trigger the development of a number of syndromes and diseases (for example, various types of cancer).

Life Chemicals has developed two Epigenetic Screening Libraries designed with both ligand-based and structure-based approaches:

The Libraries contain drug-like screening compounds carefully selected by computational chemistry and virtual screening techniques to boost epigenetic-targeted drug discovery.

Epigenetic Focused Library by 2D Fingerprint Similarity Search

2D fingerprint similarity implies that a reference set of active compounds (with their activity data reported in the literature) and molecules of interest are represented as a set of small fragments encoded in bit strings (“fingerprints”). Both sets of “fingerprints” are compared to estimate their degree of similarity.

First, our reference set of known epigenetic modulator molecules was obtained from the ChEMBL database. The set included all available compounds with high experimental activity towards human epigenetic targets. Next, the 2D fingerprint similarity search against the Life Chemicals HTS Compound Collection was performed with Tanimoto index ≥ 0.85 (5 analogs max), resulting in 3,500 epigenetics-related screening compounds (Fig. 1). The library does not contain PAINS, toxic and reactive compounds.

Compound distribution targeting single protein within the Epigenetic Focused Library

Figure 1. Compound distribution targeting single protein within the Epigenetic Focused Library.

Epigenetic Targeted Library by Docking Screening

The Library contains 8,000 structurally-diverse screening molecules picked out by virtual molecular screening against the following epigenetic targets:

Jumonji domain-containing protein D3 (JMJD3, KDM6B)

The Jumonji domain-containing protein D3 (JMJD3), also known as lysine-specific demethylase 6B (KDM6B), is a member of the JmjC histone demethylases family, which together with ubiquitously transcribed X-chromosome tetratricopeptide repeat a protein (UTX), specifically demethylates histone 3 at lysine 27 (H3K27). The JMJD3 and UTX play essential roles in the epigenetic regulation of gene expression, altering cellular memory and reprogramming cells’ fate. Specifically, the JMJD3 is involved in several cellular processes, such as proliferation, differentiation, and apoptosis. The regulation of the JMJD3 is highly gene- and context-specific, and it is engaged in several tissue responses, such as vertebrate development, cancer, inflammatory and neurodegenerative diseases [1].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 4ASK
  • Constraints: metal coordination
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,273

Examples of virtual hit compounds (binding site: 4ASK) from the JMJD3 (KDM6B) compound set.

Figure 2. Examples of virtual hit compounds (binding site: 4ASK) from the JMJD3 (KDM6B) compound set.

Histone Methyltransferase EZH2

The enhancer of zeste homolog 2 (EZH2) is an enzymatic catalytic subunit of the polycomb repressive complex 2 (PRC2) that can modulate downstream target gene expression by trimethylation of Lys-27 in histone 3 (H3K27me3). Besides H3K27me3, PRC2 also methylates non-histone protein substrates, such as transcription factor GATA4. Additionally, EZH2 directly interacts with other proteins to activate downstream genes in a manner that is independent of PRC2. Functions of EZH2 in cell proliferation, apoptosis, and senescence have been identified. Important roles of EZH2 in the pathophysiology of cancer are widely concerned, and targeting EZH2 for cancer therapy is presently a hot research topic [2].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 5LS6
  • Constraints: no
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,465

Examples of virtual hit compounds (binding site: 5LS6) from the JMJD3 (KDM6B) compound set.

Figure 3. Examples of virtual hit compounds (binding site: 5LS6) from the JMJD3 (KDM6B) compound set.

Polycomb protein EED

The polycomb repressive complex 2 (PRC2) is a crucial component in regulating gene expression, involved in cell development and differentiation. Abnormal activity of the PRC2 is observed in the progression of many human cancers. Its higher activity results in increased levels of the H3K27me3, which are linked to different types of cancer, including prostate, breast, lymphoma, and myeloma cancers. The WD40 repeat-containing protein EED is a core component of the PRC2, enhancing its activity through interaction with the H3K27me3. Pharmaceutical intervention against this component of the PRC2 has gained attention, as demonstrated by the development of several novel EED inhibitors [3].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 5U6D
  • Constraints: hydrogen bond
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,226

Examples of virtual hit compounds (binding site: 5U6D) from the EED compound set.

Figure 4. Examples of virtual hit compounds (binding site: 5U6D) from the EED compound set.

Protein arginine N-methyltransferase 6 (PRMT6)

The PRMT6 functions in various cellular pathways that include regulation of transcription and cell cycle, alternative splicing, and DNA repair regulation, all of which have been linked to breast cancer etiology. Published data reveal that the PRMT6 plays a significant role in breast cancer development. However, whether it functions as a promoter or inhibitor of breast cancer is yet to be determined. Discrepancies in the PRMT6 expression may arise due to n altered regulation of this target in different breast cancer subtypes. Therefore, the PRMT6 could potentially serve as a therapeutic marker in a subset of breast cancers, although a further examination is required to determine which breast cancers display an increased PRMT6 expression and how this increased expression affects specific cellular pathways [4].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 6W6D
  • Constraints: no
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,374

Examples of virtual hit compounds (binding site: 6W6D) from the PRMT6 compound set.

Figure 5. Examples of virtual hit compounds (binding site: 6W6D) from the PRMT6 compound set.

Histone-lysine N-methyltransferase EHMT2 (G9a)

The G9a is the primary enzyme for mono- and dimethylation at Lys 9 of histone H3. It predominantly forms the heteromeric complex as a G9a-GLP (G9a-like protein), afunctional histone lysine methyltransferase in vivo. A growing body of evidence suggests that the G9a catalyzes stone and nonhistone proteins’ methylation, which plays a crucial role in diverse biological processes and human diseases. It has been observed that it is overexpressed in a number of cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, aggressive lung cancer, brain cancer, multiple myeloma, and aggressive ovarian carcinoma. Its elevated levels were commonly correlated with higher methylation levels, leading to the suppression of important tumor suppressor genes. Thus, it is believed that targeting the G9a in cancer will lead to the re-expression of these genes [5].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 7BTV
  • Constraints: volume
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,356

Examples of virtual hit compounds (binding site: 7BTV) from the EHMT2 (G9a) compound set

Figure 6. Examples of virtual hit compounds (binding site: 7BTV) from the EHMT2 (G9a) compound set.

Poly [ADP-ribose] polymerase 1 (PARP-1)

The poly (ADP-ribose) polymerase 1 (PARP1) enzyme is one of the promising molecular targets for the discovery of antitumor drugs. The PARP1 is a common nuclear protein (1–2 million molecules per cell) serving as a “sensor” for DNA strand breaks. Increased PARP1 expression is sometimes observed in melanomas, breast cancer, lung cancer, and other neoplastic diseases. It has been proved that there is a correlation between high PARP1 expression and treatment-resistance of tumors. Since PARP1 inhibitors act as chemo- and radiosensitizers in the conventional therapy of malignant tumors, they are considered promising antitumor agents. Moreover, PARP1 inhibitors can be used as standalone, potent drugs against tumors with broken DNA repair mechanisms [6].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 7KK4
  • Constraints: no
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,397

Examples of virtual hit compounds (binding site: 7KK4) from the PARP1 compound set.

Figure 7. Examples of virtual hit compounds (binding site: 7KK4) from the PARP1 compound set.

Lysine-specific demethylase 4A (KDM4A)

Histone demethylation, controlled by histone demethylase enzymes, is vital in the epigenetic regulation of gene expression. The lysine-specific demethylase 4A (KDM4A) plays important roles in both normal and cancer cells. The discovery of KDM4s inhibitors is a potential therapeutic strategy against various diseases, including cancer. High expressions of the KDM4s are considered to promote oncogenesis in some types of cancers, including prostate, breast, and colon. Downregulation of the KDM4s by means of molecular biology methods or inhibition of their catalytic activity by small molecule inhibitors is a confirmed strategy for oncotherapy. In recent years, increasing attention has been paid to the development of KDM4s inhibitors as antitumor agents [7].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 5VGI
  • Constraints: metal coordination
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,229

Examples of virtual hit compounds (binding site: 5VGI) from the KDM4 compound set.

Figure 8. Examples of virtual hit compounds (binding site: 5VGI) from the KDM4 compound set.

Menin/MLL

The Menin is an essential co-factor of oncogenic MLL fusion proteins, and the menin-MLL interaction is critical for the development of acute leukemia in vivo. Targeting the menin-MLL interaction with small molecules represents an attractive strategy to develop new anticancer agents. The latest achievements, including the publication of the menin crystal structure and development of potent small molecule and peptidomimetic inhibitors, have demonstrated feasibility of targeting the menin-MLL interaction. However, biochemical and structural studies revealed that the MLL binds to the menin in a complex bivalent mode engaging two MLL motifs. Thus, inhibition of this protein-protein interaction has turned out to be a difficult task. Recent efforts on targeting the menin-MLL interaction have revealed potential benefits of blocking menin in cancer [8].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 5DB3
  • Constraints: no
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 943

Examples of virtual hit compounds (binding site: 5DB3) from the Menin/MLL compound set.

Figure 9. Examples of virtual hit compounds (binding site: 5DB3) from the Menin/MLL compound set.

References:

  1. Burchfield JS, Li Q, Wang HY, Wang RF. JMJD3 as an epigenetic regulator in development and disease. Int J Biochem Cell Biol. 2015, 67:148-157.
  2. Duan, R., Du, W. & Guo, W. EZH2: a novel target for cancer treatment. J Hematol Oncol 13, 104 (2020).
  3. Dong H, Liu S, Zhang X, et al. An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor Progression by Modulating the Immune Response. Cancer Research. 2019, 79(21):5587-5596.
  4. Alan Morettin, R. Mitchell Baldwin, Jocelyn Côté, Arginine methyltransferases as novel therapeutic targets for breast cancer. Mutagenesis. 2015, 30(2), 177–189.
  5. Casciello F, Windloch K, Gannon F, Lee JS. Functional Role of G9a Histone Methyltransferase in Cancer. Front Immunol. 2015, 6:487.
  6. Malyuchenko NV, Kotova EY, Kulaeva OI, Kirpichnikov MP, Studitskiy VM. PARP1 Inhibitors: antitumor drug design. Acta Naturae. 2015, 7(3):27-37.
  7. Lin H, Li Q, Li Q, et al. Small molecule KDM4s inhibitors as anti-cancer agents. J Enzyme Inhib Med Chem. 2018, 33(1):777-793.
  8. Cierpicki T, Grembecka J. Challenges and opportunities in targeting the menin-MLL interaction. Future Med Chem. 2014, 6(4):447-462.
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