Research on Active Constituents from Chinese Medicine against

Leukemia Targeting ENL based on Molecular Docking

Hao Sun

and Jian Tang

School of Chinese Medicine, Bozhou University, Bozhou, Anhui, China

Keywords: Leukaemia ENL Protein, Traditional Chinese Medicine Active Ingredients, Molecular Docking.

Abstract: Leukemia is a class of malignant clonal disease of hematopoietic stem cells. It poses a huge threat to human

health. Over the years, progress has been made in using traditional Chinese medicine (TCM) in China to

treat and relieve various leukemia symptoms. Erb and he Wan et al noted that ENL protein is a key factor

affecting the viability of MLL-r cells in leukemia. Molecular adding or removing molecular group

modification on the histone can regulate gene expression A prominent structural feature of the ENL protein

is a YEATS domain that identifies a specific acetyl (Ac) on the histone H3. Computer-aided Drug Design

(CADD) method has gradually mature and plays an increasingly important role in drug development.

Molecular docking is the most widely used and successful method in structure-based drug design. Molecular

docking generally refers to the process in which two or more molecules identify each other through

geometric and energy matching. Two major topics of molecular docking methods are spatial identification

and energy identification between molecules.

1 INTRODUCTION

Leukemia is a class of malignant clonal disease of

hematopoietic stem cells. It poses a huge threat to

human health. Scientists have found that the

regulatory protein ENL promotes leukemogenesis.

Over the years, progress has been made in using

traditional Chinese medicine (TCM) in China to

treat and relieve various leukemia symptoms.

Mixed lineage leukemia (MLL) is named for the

chromosome translocation of the MLL fusion

protein at 11q23.This type of leukemia has attracted

wide attention for its unique clinical and biological

characteristics.

The cause of leukemia is usually chromosomal

translocation, producing fusion proteins formed by

fragment junctions of two proteins that ultimately

cause disease. Fusion proteins in MLL are typical so

commonly present in aggressive childhood leukemia

and are associated with poor prognosis. Therefore, it

is very necessary to develop leukemia treatment

strategies based on MLL rearrangement (MLL-

rearranged, MLL-r) fusion proteins. Erb and he Wan

https://orcid.org/0000-0001-5748-7516

https://orcid.org/0000-0003-3289-3352

et al noted that ENL protein is a key factor affecting

the viability of MLL-r cells in leukemia (Wan 2017).

The histones containing DNA in the cells are

structural and signaling factors. Molecular adding or

removing molecular group modification on the

histone can regulate gene expression A prominent

structural feature of the ENL protein is a YEATS

domain that identifies a specific acetyl (Ac) on the

histone H3. This suggests that this "read" ability of

ENL to acetylated histone is essential for the

induction of MLL-r leukemia.

Another complementary mechanism for SEC and

DotCom stability was identified by Erb and he Wan

et al. They found that inactivation of ENL impaired

the SEC and DotCom function in MLL-r cells. The

ability of ENL to bind to SEC, DotCom drops a hint

that a model-ENL, through the YEATS domain,

recognizes that acetylated H3, enhances the stability

of binding of SEC and DotCom complexes to DNA

and regulates the activity of aberrant regions of the

genome.

Protein ENL is essential for MLL-r leukemia.

Some leukemias exist resulting from a mixture of

some MLL protein and part of another. The second

protein is typically part of the SEC (super elongation

complex) protein complex or the DotCom (DOT1L-

containing complex). Both protein complexes

Sun, H. and Tang, J.

Research on Active Constituents from Chinese Medicine against Leukemia Targeting ENL based on Molecular Docking.

DOI: 10.5220/0011211800003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 391-396

ISBN: 978-989-758-595-1

391

regulate gene transcriptional programs in MLL-r

leukemia (the intact and partially fused SEC/

DotCom complex is highlighted in red in Fig). The

ENL protein binds to both complexes while the

complex is fused to MLL in cells, and ENL interacts

both with unfused SEC/ DotCom and with fused

SEC/ DotCom. ENL contains a YEATS domain that

can recognize a specific acetyl group (Ac) on the

histone H3. Erb and Wan et al confirmed that the

YEATS domain of ENL protein helps stabilize the

binding of SEC and DotCom to DNA, promoting

gene expression in the driving leukemic manner.

This pathway of action presents a way: using a

small molecule inhibitor targeting the ENL protein

YEATS domain as a drug molecule can selectively

kill leukemia MLL-r cells and then treat leukemia.

Other cell types seem to largely tolerate ENL loss,

but both SEC, DotCom and ENL are expressed in

multiple cells, so it is important that when

developing such drugs is to understand this

difference in tolerance.

In MLL-r leukemia, the importance of ENL is

consistent with multiple studies. These studies show

that poor regulation of DOT1L viability is required

for the survival and proliferation of MLL-r cells.

H3K79 methylation has long been associated with

gene transcriptional viability and is a regulatory

mechanism controlling DOT1L activity. A key

unanswered question is how these histone

modifications can directly affect transcription.

Methyl-lysine signaling is often coupled to

downstream processes by a similar mechanism to the

methylation modification of the YEATS domain

read. All the reader domains of the major histone

methylation sites have been identified except for

H3K79. Furthermore, methylated lysine can be

dynamically regulated by demethylases, but

demethylases that remove methyl groups at H3K79

remain to be characterized. The study by Erb and he

Wan et al further encouraged researchers to identify

and identify "readers" and demethylases for H3K79

methylation because these enzymes cross with ENL-

mediated signaling pathways and may also become

therapeutic targets for MLL-r leukemia.

There is currently a new theory that epigenetic

regulators can play an important role in disease.

Driven by this awareness, a growing number of

academics and businesses are working to develop

inhibitors of these mechanisms to treat cancer.

Clinical trials of MLL-r leukemia have evaluated

DOT1L inhibitors, and Erb et al. found that in

cellular models, using DOT1L inhibitors, plus

knockin of ENL mutant genes that do not recognize

acetylated lysine, was more effective than inhibiting

gene expression programs that drive leukemenesis in

both methods alone. This suggests that there is a

synergy between the two therapies.

Wan et al. also investigated the potential of

combinatorial therapies for MLL-r leukemia by

targeting the ENL YEATS domain with the bromine

domain of another reader of lysine acetylation, BET

family proteins. BET proteins typically interact with

SEC. And also promotes transcriptional elongation.

BET inhibitors disrupt the binding of the BET

protein to the acetyl-lysine fraction, and there are

about 20 clinical trials testing the efficacy of these

drugs in cancer treatment. Moreover, the

combination of the YEATS domain inhibitor of ENL

with the BET inhibitor JQ1 is highly toxic to MLL-r

leukemia cells.

The effects of these combinatorial therapies

suggest that multiple histone modification signals

act together to form a characteristic epigenetic state

of MLL-r leukemia. Therefore, multiple targeted

therapies are more effective and can reduce the

emergence of-with resistance, which is one of the

risks of monotherapy.

Drug developers have long focused mainly on

targeting enzymatic activity, rather than protein –

protein interactions. However, the development of

"reader" therapies targeting multiple apparent

modification groups has become increasingly

popular, thanks in part to the success of BET

inhibitors. The binding site of the YEATS domain is

also a very attractive target for drug development.

TCM treatment has made progress in the

treatment of leukemia for many years, and has

developed continuously, while the treatment of

leukemia is not limited to western medicine

technology. At present, the application of TCM has

become an important treatment means of leukemia.

According to researchers, there are six TCM that

have a good anti-leukemia effect, and specifically

list the relevant active ingredients.

Computer-aided Drug Design (CADD), is a

computer chemistry based approach to predicting

testing and computing the relationship between

ligand and receptor biological macromolecules

through computer simulations, enabling optimization

and design of lead compounds (Xie 2019). At

present, CADD method has gradually mature and

plays an increasingly important role in drug

development, which can greatly shorten the

development cycle of new drugs and reduce

development costs.

Molecular docking is the most widely used and

successful method in structure-based drug design.

Molecular docking generally refers to the process in

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

392

which two or more molecules identify each other

through geometric and energy matching. Two major

topics of molecular docking methods are spatial

identification and energy identification between

molecules. Spatial matching is the basis of

intermolecular interactions, and energy matching is

the basis for maintaining a stable binding between

molecules. For geometric matching calculation,

lattice calculation, fragment growth are usually used,

while energy calculation uses simulated annealing

and genetic algorithm.

When the binding site of the ligand to the

receptor protein is unknown, its site can be predicted

by CADD methods and can guide mutation

experiments and drug design. It is therefore

important to identify the sites where the receptor

protein surface interacts with the ligands for drug

design.

The molecular docking technology has been used

to virtually screen the bioactive constituents from

TCM and determine the targets in recent years. Xu

Cao and Singh have made outstanding contributions

in the drug field using molecular docking (Cao 2021,

Singh 2012). Which is efficient in ‘narrowing’ the

chemical database before pharmacological assays in

vivo or in vitro.

2 METHODS AND MATERIALS

2.1 Software

ChemOffice Professional (PerkinElmer Inc., USA),

AutoDock Tools1.5.6 and AutoDock Vina (The

Scripps Research Institute, USA), PyMOL (TM)

1.7.4.5 Edu (Schrodinger, LLC Inc., USA) and

Discovery Studio 2016 client (BIOVIA Co., USA).

2.2 Establish A Ligand Database

The Chinese medicinal materials with the effect of

treating leukemia contain multiple active

ingredients, and the ligand database required for this

test can be summarized.

Seven Chinese medicine materials, including

Paridis Rhizoma (Guan 2007), Sophorae

Flavescentis Radix (Yan 2014), Scutellariae

Barbatae Herba (Niu 2021), Herba Hedyotidis (Qin

2008), Paeoniae Radix Rubra (Lu 2015), Salviae

Miltiorrhiza Radix et Rhizoma (Xu 2021),

Glycyrrhizae Radix et Rhizoma (Huang 2017), were

chosen for the remarkable effects in the treatment of

leukemia. The total 50 main constituents from these

herbs were collected via reviewing the related

articles. The structural files of these materials were

all downloaded from the PUBCHEM (PubChem

(nih.gov) and processed with MM2 in Chem3D,

stand as mol2 documents. Followed by Autodock,

all the mol2 documents saved as pdbqt documents.

2.3 Locking the ENL Protein Active

Site

We used Autodock tools to process ENL with

dehydration molecules, deligand, etc, and exported

them as pdbqt files. The ENL protein has a YEATS

domain (gully) that recognizes the acetylation

modification. It can serve as an active site for

molecular docking. By operating on the software,

the active site of the ENL was determined as

center_x =0.954 nm, center_y = -0.12 nm, center_z

= 10.368 nm.

2.4 Molecular Docking of ENL with

Small Moleculares

The PDBQT format file of the ENL protein as the

ligand of the ligand and the ligand library was

imported into the Autodock. vina software, and the

computer then scored each group. The acceptable

ligand GKN displayed by the ENL protein in the

RCSB PDB database was exported from GKT to the

PDBQT format and used as a ligand as a positive

control. The two groups of scores were finally

compared, and ligand scores near or beyond the

GKT, score were selected as the final screening

results, and could be used as a valuable reference for

the development of ENL inhibitor drugs for

leukemia.

3 RESULTS

The original ligand-GKT in the ENL complex (PDB

ID: 6HPW) showed an affinity of -7.2 kcal/mol.

Among the 50 constituents, 26 compounds had

higher binding affinity than GKT, respectively listed

in the table 1 below.

Research on Active Constituents from Chinese Medicine against Leukemia Targeting ENL based on Molecular Docking

393

Table 1: Affinity scores of molecular docking of ENL

with 26 compounds.

NO.

Compounds

Binding Affinity

(kcal/mol)

1 Cryptotanshinone -8.5

2 Glabrene -8.4

3 Tanshinone IIA -8.3

4 Trifolirhizin -8.2

5 Paeoniflorigenone -8.2

6 Polyphyllin I -8.1

7 Oleanolic acid -8.1

8 Norkurarinone -7.9

9 Baicalin -7.9

10 Ursolic acid -7.9

11 Benzoylpaeoniflorin -7.8

12 Pseudoproto-Pb -7.7

13 Enoxolone -7.7

14 Glabridin -7.7

15 Paeoniflorin -7.6

16 Polyphyllin II -7.4

17 Quercetin -7.4

18 Albiflorin -7.4

19 Benzoyloxypaeoniflorin -7.4

20 Stigmasterol -7.4

21 Baicalein -7.3

22 Naringenin -7.3

23 Oroxylin A -7.3

24 Wogonin -7.3

uraridin -7.2

26 Oxypaeoniflorin -7.2

Cryptotanshinone has a strong binding affinity -

8.5 (kcal/mol) with 6HPW, Above the threshold

value, The molecular docking plan (Figure 1b)

shows the Van Der Waals ineraction between it and

the amino acids such as CYS42 in the 6HPW

protein; The quinone ring has a Pi-Alkyl interaction

between cryptotanshinone and ARG37; The benzene

ring between cryptotanshinone has a Pi-Alkyl

interaction and ARG37; Alkyl functions between the

fat loop of cryptotanshinone and ARG37, PRO70,

LYS72, Pi-Alkyl interaction between it and PHE35;

Conventional Hydrogen Bond interaction between 2

of cryptotanshinone-O and TYR71, a week

hydrogen Bond interaction between it and PRO70;

Pi-Sigma interaction between 11-C of

cryptotanshinone and PHE35.

Figure 1a: 3D model.

Figure 1b: 2D modle

Figure 1: Molecular docking model for Cryptotanshinone

and ENL.

Figure 2a: 3D model

Figure 2b: 2D modle

Figure 2: Molecular docking model for Glabrene and

ENL.

Glabrene has a strong binding affinity energy

with 6HPW, Above the threshold value, Molecular

docking plan (Figure 2b) shows a van der Waals

interaction between it and TYR71 et al. in the

6HPW protein; The B loop of Glabrene and GLY38

have a Pi-Donor hydrogen Bond interaction, Pi-

Sigma interaction between both and ILE44, PRO69,

ARG37; The carbon-carbon double bond on the C

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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loop of Glabrene and PRO70, ARG37 both have

Alkyl interactions; Alkyl interaction between the D

loop of ligand and PRO70, Pi-Sigma interaction

between it and LYS72, Pi-Sulfur interaction with

MET33zhijian; The carbon on the A loop CH3 of

Glabrene and ARG37 have Alkyl interaction; The

Conventional Hydrogen Bond interaction occurs

between O on the D loop of Glabrene and LYS72.

The structures of compounds in the top 8 affinity

scores of molecular docking are listed in Figure 3.

Figure 3: Chemical structures of compounds with top 8

affinity scores.

4 CONCLUSIONS

The results showed that the active TCM components

represented by Cryptotanshinone, Glabrene were

well and stably bound with the ENL associated with

leukemia. The results show that the TCM active

Chinese ingredients represented by

cryptodenshinone and photoglycyrrhizin bind well

and stably to the ENL associated with blood

carcinogenesis.This series of molecules represented

by cryptodanshinone and photoglycyrrhizin can

efficiently bind to ENL proteins and potentially

affect the function of ENL proteins. In addition the

effect of Cryptotanshinone and Glabrene on cancer

have been confirmed to some extent by Yuqing Ge

and Yuting Huang (Ge 2015, Huang 2017).

ACKNOWLEDGEMENTS

We thank Wentao Zhang for software analysis, and

Yutong Han for the supply and literature database.

This work was supported by Key Laboratory of

Chinese Medicine Materials Research of Anhui

Higher Education Institutes (Bozhou University,

KLAHEI18032) and Bozhou University Research

Grant (BZQD201901). Hopefully, one day, my

Research on Active Constituents from Chinese Medicine against Leukemia Targeting ENL based on Molecular Docking

395

experimental data can be based on more help, which

is also a good thing for me.

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