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Accepted Manuscript

Combined multi-pharmacophore, molecular docking and molecular dynamic study for


discovery of promising MTH1 inhibitors

Duoqian Dai, Lu Zhou, Xiaohong Zhu, Rong You, Liangliang Zhong

PII: S0022-2860(17)30178-3
DOI: 10.1016/j.molstruc.2017.02.034
Reference: MOLSTR 23428

To appear in: Journal of Molecular Structure

Received Date: 20 December 2016


Revised Date: 9 February 2017
Accepted Date: 9 February 2017

Please cite this article as: D. Dai, L. Zhou, X. Zhu, R. You, L. Zhong, Combined multi-pharmacophore,
molecular docking and molecular dynamic study for discovery of promising MTH1 inhibitors, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.02.034.

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ACCEPTED MANUSCRIPT

Combined multi-pharmacophore, molecular docking and molecular

dynamic study for discovery of promising MTH1 inhibitors

Duoqian Dai, Lu Zhou*, Xiaohong Zhu, Rong You, Liangliang Zhong

College of Chemical Engineering, Sichuan University, Chengdu, 610065, China

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*Corresponding author: zhoulu@suc.edu.cn Tel +86 028 85405220 Fax Tel +86 028 85403397

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Abstract

MutT homolog 1 (MTH1), a nudix phosphohydrolase enzyme participates in the process of repairing of

DNA damage by hydrolyzing oxidized deoxy-ribonucleoside triphosphate in cancer cells, is regarded

as a potential target for anticancer therapy. In order to seek for promising inhibitor of MTH1,

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structured-based pharmacophore and 3D-QSAR pharmacophore hypotheses combine with the ADMET

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analysis and Lipinskis rule of five were used for screening the public molecules libraries (Asinex,

Ibscreen and Natural). Then molecular docking studies were performed on screened hits via various

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docking programs (Glide SP, GOLD and Glide XP), five molecules with three scaffolds were picked

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out as potential inhibitors against MTH1. Eventually, 20 ns molecular dynamics simulation was
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implemented on the potential inhibitors. The RMSD (Root Mean Square Deviation) values were used

to illustrate bind stability between potential molecules and MTH1. Therefore, the five hits may be
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considered as promising MTH1 inhibitors by all above studies.


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Keywords: MTH1, Pharmacophore, Virtual screening, Molecular docking, Molecular dynamics s


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1. Introduction

MutT homolog 1 (MTH1), a nudix phosphohydrolase enzyme participates in the process of repairing of

DNA damage by hydrolyzing oxidized deoxy-ribonucleoside triphosphate in cancer cells [1-3].

Oxidative stress could overproduce highly reactive molecules such as ROS (reactive oxygen species) in

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tumor cell. The result of the generated ROS was DNA damage which cause cell senescence or cell

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death [4-6]. MTH1 expressed increasing after the cell offered oxidative stress, and participated in DNA

repair by hydrolyzing oxidized 8-oxo-2-deoxy-guanosine 5-triphosphate (8-oxo-dGTP) to

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8-oxo-2-deoxy-guanosine 5-monophosphate (8-oxo-dGMP), which turns out to cut the damage or

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death of cancer cell down [7-10]. For cancer cell survival, MTH1 is required, while not requirement for
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normal cell survival [11]. So, a method which breaking down 8-oxo-dGTP hydrolysis pathway via

restraining the activities of MTH1 could make tumor cells death. Therefore, MTH1 is considered as an
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ideal therapeutic targets for cancer cells.


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As for the MTH1 inhibitor, TH287, TH588 (shown in Figure 1a and 1b) were reported to restrain
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activity of MTH1 and had conspicuous influence of tumor mice [11,12], (S)-crizotinib (shown in

Figure1c) is an approved drug for NSCLC treat, and also reported that it play role in MTH1 and
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considered as MTH1 promising inhibitor [13, 14]. While there are no report about the approved drugs
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for MTH1, so it is significance to discover more promising MTH1 inhibitors.


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a) TH287 b) TH588 c) (S)-crizotinib

Figure 1. Chemical structures of MTH1 inhibitors.


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A technology combined the pharmacophore, molecular docking and molecular dynamics is widespread

use for novel inhibitors discovery. In order to discover the promising inhibitors of MTH1,

structured-based pharmacophore and 3D-QSAR pharmacophore hypotheses were developed for MTH1

in the study, a method combined those two pharmacophore hypotheses was used for database screening

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to discover novel MTH1 inhibitors. Moreover, molecular docking method with various programs was

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employed to the docking between molecules and protein considering the requirement of active cavity.

Eventually, 20 ns molecular dynamics simulation was implemented on the most potential molecules.

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The molecules with high Glide score and rational binding modes would considered as promising

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inhibitors for MTH1.
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2. Materials and methods

2.1 Crystal Structure Preparation for Structure-Based Pharmacophore Generation


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13 human X-ray crystal structures of MTH1 bound with ligands were selected form Protein Data Bank
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(http://www.rcsb.org). And the PDB ID code were 3ZR0, 4C9W, 4C9X, 4N1T, 4N1U, 5ANS, 5ANT,
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5ANU,5ANV, 5ANW, 3WHW, 3ZR1 and 3Q93.We look for available MTH1 crystal structure with

resolution less than 2.0. Ten structures can meet the condition. For a better crystal structure, a method
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combined cross-docking with self-docking of the MTH1 crystal structures was conducted with
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Glide-XP (extra precision) [15]. according to the data of self-docking (Table 1) and cross-docking,
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there are 9 MTH1 crystal structures with RSMD 2 . And 8 out of 9 MTH1 crystal structures were

selected for further study because all of the 9 crystal ligands could dock with.

2.2 Structure-Based Pharmacophore Hypotheses Generation

Interaction Generation protocol of Discovery Studio Client v2.50 was applied to generate

structure-based pharmacophores hypotheses [16-17]. To build a structure-based phramacophore

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hypothesis, analyzing interaction information between receptor and ligand is the most critical step. In

our study, 8 MTH1 crystal structures complex with known inhibitors were applied to generate

structure-based phramacophore hypotheses. Crystal ligand of each crystal structure was used to define

active site, an appropriate site sphere radius was defined by a criterion to cover all residues of active

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site. Interaction Generation protocol of Discovery Studio was employed to create pharmacophore

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features hypotheses, then Edit and Cluster Pharmacophore Features Tool combined with Heat-map

generate by screening all inhibitors from crystal structures was used to reduce the pharmacophore

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features with no or less catalytic importance.

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2.3 Dataset Preparation for 3D-QSAR Pharmacophore Generation
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All 3D-QSAR pharmacophore hypotheses were generated by the Discovery Studio Client v2.50.

Before generating the hypotheses [16-17], a dataset consist of 41 MTH1 inhibitors which collected
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from available literatures [18, 19] were used for training sets and test sets. All 41 inhibitors with their
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activity span over 7 orders (0.00008M-12.5M) and they belong to four different scaffolds. 19 of
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them were selected as training sets (shown in Figure 2) to develop the 3D-QSAR model, and others

were considered as test sets (shown in Supplementary Figure 1). According to the activity values, all of
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the compounds were categorized into three organizations show in Table 3 (poor active (IC50>0.1M,
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+), moderate active (0.001M<IC50<0.1M, ++) and high active (IC50<0.001M, +++)).
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1 (0.00008M) 2 (0.0008M) 3 (0.0003M)

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4 (0.0005M) 5 (0.0005M) 6 (0.0005M)

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7 (0.0014M) 8 (0.0016M) 9 (0.002M)
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10 (0.0045M) 11 (0.009M) 12 (0.009M)


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13 (0.084M) 14 (0.12M) 15 (0.140M)

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16 (0.332M) 17 (0.646M) 18 (3.32M)

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19 (12.5M)
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Figure 2. Chemical structures of training set molecules and their IC50 value.

2.4 3D-QSAR Pharmacophore Hypotheses Generation


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Before 3D-QSAR pharmacophore hypotheses generation, an Uncertain Value 2 and Active Value had
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been added for training sets molecules. For searching some appropriate pharmacophore features,
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Feature Mapping protocol of Discovery Studio was adopted to analyze the possible pharmacophore

features. According to the results of feature mapping, hydrogen bond donor (HBD), hydrogen bond
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acceptor (HBA), hydrophobic (HY) and ring aromatic (RA) were considered as appropriate
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pharmacophore features for hypotheses development. Then the BEST method was selected in
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Conformation Generation of the Generate Conformations protocol by considering the impact of

molecular flexibility, meanwhile, several parameters had been selected as follows: Maximum

conformations255, Energy Threshold10 kcal/mol. The training sets molecules were utilized to

develop pharmacphore hypotheses using the HypoGen algorithm. As a result, ten predictive hypotheses

were generated, the best predictive hypotheses was named Pharm-A.

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2.5 Pharmacophore comparison

The structure-based pharmacophore hypotheses were generated on the basis of the interactive

information between receptor and ligand, while 3D-QSAR phramacophore hypotheses were generated

according to the inhibitory information [20-23]. Selecting a suitable hypothesis from 8 structure-based

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pharmacophore hypotheses for screening, and considering the similarity between 3D-QSAR

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pharmacophore and structure-based pharmacophore, the generated 3D-QSAR pharmacophore

hypotheses would compare with 8 structure-based pharmacophore hypotheses by utilizing

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pharmacophore comparison protocol of Discovery Studio. The results of RMSD value could be

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adopted to compare the similarity among the pharmacophore hypotheses, the lower RMSD value
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means the higher similarity of two pharmacophores, and the suitable structure-based pharmacophore

hypothesis with lowest RMSD value was named Pharm-B.


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2.6 Pharmacophore Validation


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Different methods were applied to validate the quality between Pharm-A and Pharm-B. Which
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including (a) test set prediction, (b) decoy set verification and (c) Fisher validation for Pharm-A, while

(d) crystal ligand prediction and (e) decoy set verification for Pharm-B.
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2.6.1. For Pharm-A


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Test set prediction


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Firstly, all test set molecules were added the activity values which collected form literatures as the

training set molecules. Ligand Pharmacophore Mapping protocol has been used to verify the Pharm-A

with related parameters were set as follows: Fitting Method Flexible, Conformation

GenerationBEST. Different statistical parameters including predicted IC50, error, fit value and

correlation coefficient (r2) could be a criteria to evaluate the Pharm-A.

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Decoy set validation

Decoy set validation was adopted to check the screening capability of Pharm-A. A decoy set which

comprises 1000 unknown activity molecules and 40 active MTH1 inhibitors was employed as a

screening database which used for Pharm-A. After screening, Goodness of fit (GF) value and

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Enrichment factor (EF) value would evaluate the capacity that the Pharm-A differentiate active

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compounds from decoy set.

Fischers randomization method

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Fishers validation method was employed to validate the statistics rationality of Pharm-A by shuffling

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the activity values of the all training set molecules. 19 pharmacophore hypotheses were generated
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randomly to attain 95% confidence level. It would be considered as a rational pharmacophore

hypothesis that the total costs of Pharm-A are lower than the total costs of 19 pharmacophore
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hypotheses.
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2.6.2. For Pharm-B


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Crystal ligand prediction

Firstly, a database consisted of 8 crystal ligands (shown in Supplementary Figure 2) extracted from the
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MTH1 crystal structures would be built. Screen library protocol has been employed to verify the
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Pharm-B and related parameters were set as follows: Minimum FeaturesFlexible, Conformation
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GenerationBEST, Energy Threshold20.0 kcal/mol. The number of retained crystal ligand and the

best fit feature would be used to evaluate the rationality of Pharm-B.

Decoy set validation

This method has been mentioned above, the same decoy set was employed as a screening database

which was also used for Pharm-B.

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2.7 Virtual screening and Molecular docking study

Considering the different influences of Pharm-A and Pharm-B, and the rationality of retained hits. A

method combined Pharm-A and Pharm-B was adopted to discover promising molecules. Firstly, the

validated Pharm-B was employed as 3D query to search potentially active compounds in total 971,228

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molecules downloaded from Zinc database by Ligand Phramacophore Mapping protocol. Considering

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the drug-likeness, the Lipinskis rule of five and ADMET properties were used for all hits which

screened from Pharm-B. Then, those hits met all above requirements would be used for further virtual

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screening based on Pharm-A. With related parameters of Ligand Phramacophore Mapping protocol

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were set as follows: Fitting MethodFlexible, Conformation GenerationFAST, and Maximum
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Omitted Features0. After screening by Pharm-A, and those hits which the fit value is greater than

12.5 would be employed to analyze binding interactions with protein through docking study.
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In molecular docking study, diverse docking programs (Glide SP (standard precision), GOLD and
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Glide XP (extra precision)) with their various scoring functions were used. The use of various docking
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programs could eliminate the impact of false positive in some degree and make results more rational.

Firstly, a crystal structure of MTH1 should be prepared used for molecular docking study. As the result
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of cross-docking and self-docking experiment of the MTH1 crystal structure shows, the crystal
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structure of MTH1 PDB ID: 5ANU with high docking score, low RMSD and all crystal ligand could
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match with, was selected in 8 crystal structures of MTH1. Then molecular docking was performed

using the GOLD v5.0 software [24] and Glide SP protocol. The docking score from self-docking of

5ANU in two software would be a criterion to retain hits. The hits with docking score higher or close to

self-docking score in two different procedures would be picked out for further docking study by Glide

XP protocol. Finally, the top docking score hits in Glide XP would be retained and considered as

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promising MTH1 inhibitors. Moreover, the binding interactions and docking score of those promising

MTH1 inhibitors would be analyzed and make a comparison with the crystal ligand.

2.8 Molecular Dynamics studies

In order to verify the stability between the hits and protein complex, molecular dynamics (MD)

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simulation was implemented in the top Glide score hits and MTH1 crystal structure 5ANU. MD

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simulation was carried out by the GROMACS 4.5.5 with GROMOS96 43A1 force field. Prior to

molecular dynamics simulation, generating the topology for complex is crucial. The topology of

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protein was generated by GROMACS software, while the hits topology was gained via PRODRG [25].

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After topology of protein-hits system gained, the system would be enveloped by a dodecahedral water
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box, and Na+ or Cl- would be added to for charge balance. Energy minimization was adopted to the

system to ensure the normal structure of system and reasonable distance between the atoms. 100ps NPT
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and 100ps NVT equilibration were conducted with pressure of 1 bar and temperature of 300K. 20ns
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production MD were performed to collect data every 2ps.


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3. Results and Discussion

3.1 Pharmacophore modeling


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3.1.1 Structure-based Pharmacophore Hypotheses


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Structure-based pharmacophore hypotheses were generated based on the potential binding site with
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protein and also considered the spatial arrangement between potential binding sites with amino acid

residues. 8 MTH1 crystal structures were selected from cross-docking and self-docking. The Docking

score value was obtained from self-docking. The lower docking scores indicated the higher

reproducibility of crystal structure, and RMSD value were shown in Table 1, all the crystal structures

with RMSD value were lower than 2 which indicated a high reproducibility of crystal structures. On

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the basis of Interaction Generation protocol of DS, 8 structure-based pharmacophore hypotheses were

developed, and their pharmacophoric features had been also showed in Table 1. All structure-based

pharmacophore hypotheses contain hdrogen bond donor (HBD), hydrogen bond acceptor (HBA) and

hydrophobic (HY). Through investigating the crystal ligands and the interaction with proteins (shown

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in Supplementary Figure 3-10), all crystal ligands have nitrogen atoms in the rings or amidogen in

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chemical structure, and nitrogen atoms or amidogen are easy to form hydrogen bonds with the oxygen

atoms of carboxyl groups of Asp120A and the hydrogen atom of carboxyl groups of Asp119A, which

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could well interpret the reason why all structure-based pharmacophore models own HBD and HBA.

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The hydrogen bonds not only meet the special requirement of crystal ligand, but also have significant
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role in the stability of crystal ligand. For Hydrophobic (HY), we could find that the crystal ligand from

hydrophobic interaction with Trp117A and Met 81A as a reason that the 8 crystal ligands own benzene
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ring or other hydrophobic groups.


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Table 1.Crystal structure of MTH1 and feature structure-based pharmacophore


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PDB ID Resolution( ) Ligand name Docking scorea RMSD( ) Featuresb

3ZR0 1.80 80G -13.445 0.319 AADHH


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4C9X 1.20 VHS -10.535 1.973 ADHHH


4N1T 1.60 2GD -7.973 0.096 ADDHH
4N1U 1.60 2GE -6.148 0.124 ADHH
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5ANS 1.60 RX8 -9.302 0.237 ADDHH


5ANU 1.80 58T -11.508 0.335 ADHH
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5ANV 1.16 RGJ -8.355 0.417 ADHH


5ANW 1.37 9CQ -9.538 0.184 ADDHH
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Docking score: The score of self-docking;

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Features: A, Hydrogen bond acceptor; D, Hydrogen bond donor; H, Hydrophobic.

3.1.2 3D-QSAR Pharmacophore Hypotheses

All ten pharmacophore hypotheses were developed by 19 training set molecules. Several statistical

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parameters such as correl value, RMSD, Total cost value, cost and pharmacophoric features for ten

hypotheses are presented in Table 2. Each pharmacophore hypothesis included HBA, HBD and HY

pharmacophoric feature. Which indicated that HBA, HBD and HY pharmacophoric features were vital

for pharmacophore hypotheses, the same results gained as analysis in structure-based pharmacophore

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hypotheses. A best pharmacophore hypothesis, which owns the highest Correl value, the lowest RMSD

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and the high cost value, would be selected and named Pharm-A. As was presented in Table 2. The

best pharmacophore hypothesis (Hypo1) owned the highest Correl value of 0.98, which indicated good

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predictive capacity compare with other 9 pharmacophore hypotheses. The cost value is an

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important indicator for pharmacophoric estimation. There were over 90% credibility for a
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pharmacophore hypothesis with the cost value greater than 60, while Hypo1 owned high cost

value of 194.39. As another parameter including a good configuration value of 14.24 and a lowest
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RMSD of 0.93 interpret that Hypo1 may be an excellent hypotheses. Figure3a and 3b show the
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Pharm-A which includes HBA (represented by green color), HBD (represented by magenta color)
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and two HY (represented by cyan color) pharmacophore features and the distances between the

pharmacophoric features.
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Table 2. Statistical parameters and pharmacophoric features of ten pharmacophore hypotheses.


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Hypothesis Correl value RMSD Total cost cost Pharmacophoric features


1 0.98 0.93 86.74 194.39 ADHH
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2 0.94 1.63 102.47 178.66 ADHH


3 0.96 1.41 103.64 177.49 ADHH
4 0.93 1.80 105.05 176.08 ADHH
5 0.93 1.84 108.82 172.31 ADHH
6 0.92 1.90 109.92 171.21 ADHH
7 0.91 2.06 111.65 169.48 ADHHH
8 0.92 1.95 119.08 162.05 DHH
9 0.90 2.16 119.80 161.33 ADHH
10 0.89 2.27 119.86 161.27 ADHH
Configuration value=14.24, Fixed cost=70.5228, Null cost=281.13, cost =Null cost - total cost.
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In addition, several statistical data of training set compounds were adopted to evaluate the best

pharmacophore hypotheses Pharm-A. Table 3 lists the experimental activities collected from

literatures and the estimated activities obtained from phramacophore generation. Error value

represented the ratio between the experimental activities value with estimated activities value, all

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Error value were less than 3 except compound 18 (error value = -6.49) and explain the good

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capability of Pharm-A to predict the activity values of training set compounds. And Fit value was

a parameter indicated the match quality between the training set compounds with Pharm-A, the

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higher match quality owns higher score. As Table 3 shown that the high active molecules own

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high score, and the low active molecules own low score. The most active compound 1 and the
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least active compound 19 mapped with the Pharm-A were depicted in Figure 3c and Figure 3d.

Apparently, the compound 1 could map all pharmacophore features of Pharm-A, and compound
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19, the least active compound, mapped three pharmacophore features without HBD features.
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Moreover, the correlation coefficient (r2) was calculated for the training set compounds in
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estimated and experimental activity values and the value is equal to 0.965 (shown in Figure 4).

Pharm-A showed a good capability in predicting the activity of training set compounds in
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conclusion.
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Figure 3. (a) The best pharmacophore hypothesis, Pharm-A. (b) Distances between the
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pharmacophoric features of Pharm-A. (c) Mapping of the most active compound 1 in training set

on Pharm-A. (d) Mapping of the least active compound 19 in training set on Pharm-A.
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Table 3. Experimental, estimated activities and Fit values of training set compounds obtained from
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Pharm-A.
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Compound Exp.a Esti.b


Error Fit value Exp.a scale Esti.b scale
No. IC50(M) IC50(M)
1 0.00008 0.00008 1.00 10.11 +++ +++
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2 0.0008 0.00064 2.14 9.20 +++ +++


3 0.0003 0.00066 2.20 9.19 +++ +++
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4 0.0005 0.00033 -1.51 9.49 +++ +++


5 0.0005 0.00039 -1.29 9.42 +++ +++
6 0.0005 0.00077 1.55 9.12 +++ +++
7 0.0014 0.0019 1.35 8.74 ++ ++
8 0.0016 0.0037 2.34 8.49 ++ ++
9 0.002 0.0039 2.00 8.41 ++ ++
10 0.0045 0.0046 1.03 8.35 ++ ++
11 0.009 0.0039 -2.30 8.42 ++ ++
12 0.009 0.0079 -1.14 8.11 ++ ++
13 0.084 0.055 -1.53 7.27 ++ ++

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14 0.12 0.137 1.14 6.87 + +
15 0.14 0.121 -1.15 6.93 + +
16 0.332 0.301 -1.10 6.53 + +
17 0.646 0.531 -1.22 6.29 + +
18 3.32 0.512 -6.49 6.30 + +
19 12.5 20.58 1.65 4.70 + +
a b
Experimental activity. Estimated activity
+++ represents high active compound with IC50<0.001M, ++ represents moderate active

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compound with IC50 <0.1M and 0.001M<IC50, + represents poor active compound with
IC50>0.1M.

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Training set
Test set
5.0 Linear fit of training set ---- correlation coefficient (r2) = 0.965
4.5 Linear fit of test set ---- correlation coefficient (r2) = 0.755

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4.0
3.5
Estimated Activity (pIC50)

3.0

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2.5
2.0
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1.5
1.0
0.5
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0.0
-0.5
-1.0
D

-1.5
-2.0
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-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Experimental Activity (pIC50)
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Figure 4. Linear relation between estimated and experimental activity pIC50 values for training set

and test set compounds.


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3.2 Pharmacophore Comparison

An ideal pharmacophore hypothesis must consider the interactive information between receptor and

ligand, as well as the information of inhibitors. In order to seek an ideal structure-based pharmacophore

hypothesis, the generated pharmacophore hypotheses were compared with Pharm-A. The results

RMSD values (shown in Supplementary Table 1) explain the similarity between two pharmacophore

hypotheses, the lower value indicated the higher similarity between two pharmacophore hypotheses.
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Structure-based pharmacophore 5ANV was considered as an ideal pharmacophore hypothesis with the

lowest RMSD value of 2.26 showing the highest similarity with Pharm-A, and named Pharm-B. Figure

5 depict the stacked hypothesis of Pharm-A and Pharm-B (HBA represented by green color, HBD

represented by magenta color and HY represented by cyan color). Three pharmacophoric features

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were well stacked in space and distance, which showed the high similarity of Pharm-A and

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Pharm-B.

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Figure 5. Stacked hypothesis of Pharm-A and Pharm-B with RMSD value of 2.26.
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As for Pharm-B, Figure 6a shown the Pharm-B which included HBA, HBD and two HY
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pharmacophore features the same pharmacophore features as Pharm-A, and the distances between

the pharmacophoric features were shown in Figure 6b. The crystal ligand RGJ mapped with all
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pharmacophoric features of Pharm-B (shown in Figure 6c) which indicated that development of

Pharm-B made full use of the information of interaction between receptor and ligand.

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Figure 6. (a) The ideal structure-based pharmacophore hypothesis, Pharm-B. (b) Distances between

the pharmacophoric features of Pharm-B. (c) Mapping of the crystal ligand of 5ANV on Pharm-B.
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3.3 Phramacophore Validation


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3.3.1 For Pharm-A


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Test set method

Pharm-A was validated using test set compounds to confirm the capability of predicting the
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molecular activity. Total 22 known MTH1 inhibitor were retrieved from literature and used to examine
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the capability of Pharm-A retaining active compounds. The test set compounds were categorized based
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on the activity value as well as training set. All experimental and estimated activities value of test set

derived from Pharm-A were listed in Table 4. It was observed that 1 high active and 3 moderate active

molecules were underestimated in the activities, while 1 poor active molecule overestimated as

moderate active molecule. Total 17 out of 22 molecules were estimated correctly in their active range.

The correlation coefficients r2 value for the test set compounds is equal to 0.755 (shown in Figure

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4). In addition, the fit values of all compounds shown the same range as training set compounds.

All above statistical database indicated the Pharm-A owned a great capability to predict the

molecular activity.

Table 4. Experimental, estimated activities and Fit values of test set compounds obtained from

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Pharm-A.

Compound Exp.a Esti.b Exp.a


Esti.b scale

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Fit value Error
No. IC50(M) IC50(M) scale
1 0.0002 0.0027 8.58 13.38 +++ +++

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2 0.0002 0.0047 8.34 23.44 +++ +++
3 0.0005 0.0037 8.45 7.33 +++ +++
4 0.0006 0.0014 8.85 2.41 +++ ++
5 0.0009 0.00065 9.20 -1.38 +++ +++

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6 0.0018 0.0024 8.63 1.35 ++ ++
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7 0.0031 0.0026 8.60 -1.21 ++ ++
8 0.01 0.023 7.65 2.32 ++ ++
9 0.01 0.39 6.42 39.12 ++ +
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10 0.013 0.0083 8.09 -1.50 ++ ++


11 0.021 0.66 6.19 31.26 ++ +
12 0.022 0.032 7.51 1.46 ++ ++
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13 0.053 0.55 6.27 10.47 ++ +


14 0.15 0.090 7.06 -1.69 + ++
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15 0.30 0.39 6.42 1.30 + +


16 0.41 0.40 6.41 -1.03 + +
17 0.54 0.44 6.37 -1.23 + +
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18 0.89 0.59 6.24 -1.51 + +


19 1.22 0.42 6.38 -2.87 + +
20 2.06 0.82 6.10 -2.51 + +
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21 2.81 3.12 5.52 1.11 + +


22 2.98 0.82 6.10 -3.63 + +
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a
Experimental activity. b Estimated activity.
+++ represents high active compound with IC50<0.001M, ++ represents moderate active
compound with IC50 <0.1M and IC50>0.001M, + represents poor active compound with
IC50>0.1M.

Decoy set approach

Decoy set comprise total 1040 molecules was employed to confirm the screening capability of

Pharm-A, The false positive (FP) values, false negative (FN) values, goodness of fit (GF) value and

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enrichment factor (EF) value were calculated by statistical data obtained from Pharm-A screening with

following formulas: FP=Ht-Ha, FN=A-Ha, EF=(A/D)(Ha/Ht), GF=[1-FP/(D-A)] (3Ha/4Ht+Ha/4A).

In general, its a good screening capability of pharmacophore with the GF value over 0.6, and the

Pharm-A owned GF value of 0.71 listed in Table 5.

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Fischers randomization method

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Fischers randomization method, in this step, was adopted to interpret that Hypo1 was not generated

stochastic. Total cost of all 19 random hypotheses and Hypo1 were given in Figure7. It could be noted

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that total cost of all 19 random hypotheses were higher than Hypo1 which indicated Hypo1 owned a 95%

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confidence level. The Fishers randomization method could explain Hypo1 was not generated
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stochastic.
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Hypo1
280 random1
random2
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random3
240 random4
random5
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random6
random7
Total Cost

200 random8
random9
random10
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160 random11
random12
random13
120 random14
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random15
random16
random17
80
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random18
random19
0 1 2 3 4 5 6 7 8 9 10 11
Pharmacophore Hypotheses

Figure 7. Total costs of Hypo1 and 19 random hypotheses obtained from Fischers randomization

method

3.3.2 For Pharm-B

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Crystal ligand prediction

A database consisted of 8 crystal ligands was employed to screen based on Pharm-B, and the results

explained the rationality of Pharm-B. All 8 crystal ligands were retained with Pharm-B screening, and 7

of 8 ligands mapped with at least three pharmacophoric features of Pharm-B. The best matched

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molecule RGJ with Pharm-B was shown in Figure 6c. The above information could prove that

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Pharm-B is rational.

Decoy set approach

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Decoy set approach in this step was employed to confirm the screening capability of Pharm-B with

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similar decoy set molecules for Pharm-A, and the results of decoy set approach for Pharm-B were
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listed in Table 5. The EF value (16.1), the GF value (0.68) is more over 0.6, which indicated that

Pharm-B could serve as an ideal structure-based pharmacophore hypothesis for the discovery of
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promising MTH1 inhibitors.


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Table 5. Several parameters of decoy set approach by screening decoy set molecules based on Pharm-A
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and Pharm-B

No. Parameter Pharm-A Pharm-B


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1 Number of decoy set molecules, D 1040 1040


2 Number of active molecules, A 40 40
3 Number of hits, Ht 61 58
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4 Number of active compounds in hits, Ha 39 36


5 Enrichment factor, EF 16.6 16.1
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6 False positives, FP 22 22
7 False negatives, FN 1 4
8 Goodness of fit, GF 0.71 0.68

3.4. Virtual screening and Molecular docking

To obtain promising leads, Pharm-A and Pharm-B were used to screen Zinc databases with total

971,228 molecules. Firstly, 67,056 hits were reserved after screening based on Pharm-B, and fulfilled

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the criteria of Lipinskis rule of five and ADMET properties. Then, 59,848 of 67,056 hits were reserved

via screening based on Pharm-A. 2,315 molecules were selected with fit value over 12.5 and

considered for further docking study.

In molecules docking study, the remaining 2,315 hits combined with the MTH1 crystal structure (PDB

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CODE: 5ANU) were docked with the help of Glide SP protocol and GOLD5.0 software. According to

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the self-docking scores of MTH1 in two software, those hits with Glide score lower than -8.0 or Dock

score over 89 were considered as a database for further docking study by Glide XP. 122 of 2,315 hits

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met the requirement and used for further study. To get the promising inhibitors and optimal interaction

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with active site, Glide XP protocol was applied to 122 hits. All 122 hits were docked by Glide XP.
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Considering the binding interactions and docking score, 5 hits (shown in Figure 8) were selected as

promising MTH1 inhibitors, ultimately.


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ZINC08994256 ZINC12603519
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ZINC00796598 ZINC19792111

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ZINC19792112

Figure 8. Chemical structures of 5 hits with their ZINC code.

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As the Figure 8 was shown, all hits shared nitrogen in chemical structure, which showed a great

similarity to inhibitors. ZINC08994256, and ZINC00796598 had same scaffold, as well as the crystal

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ligand 58T, it may draw a conclusion that molecules possess this scaffold would gain inhibiting effect
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on MTH1. ZINC19792111, and ZINC19792112 had different conformations of the same molecule, the
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former owned a better docking scores. Also, a good score was acquired for ZINC12603519. The

docking score obtained from ZINC08994256, ZINC12603519, ZINC00796598, ZINC19792111,


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ZINC19792112 and crystal ligand 58T by Glide XP protocol were listed in Table 2 of Supplementary.
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It was noted that 5 hits with higher Glide score than ligand 58T, which indicated the activities of 5 hits
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were more superior than 58T (IC50=0.0005M). ZINC08994256 owned a highest Glide score of 13.69

and was considered as the most promising inhibitor.


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The 2D and 3D binding modes of ZINC08994256 and crystal ligand 58T in the active cavity have

shown in Figure 9 and Figure 11 (Supplementary). It is observed that total 5 H-bonds, 2 cation-

interactions, and a - interactions had formed between ZINC08994256 and MTH1 crystal structure,

while 4 H-bonds and 2 - interactions of 58T. More intermolecular interaction formed compared with

58T indicated more stable of ZINC08994256. In previous studies, Asp119 and Trp117 were crucial

residues in active cavity. As for the binding mode of ZINC08994256, one H-bond formed between the
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hydrogen atom in amidogen and Asp119, and cation- interactions appeared between amidogen and

Trp117. For other residues, four H-bonds formed between the purine derivatives group of the

ZINC08994256 and Gly37 as well as three waters; - interactions appeared in the purine derivatives

group of the ZINC08994256 and the benzenes of Phe27; a cation- interactions appeared in the

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benzenes of the ZINC08994256 and amidogen. Anther hydrophobic interactions, such as Phe74,

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Trp117, Ile70, Phe72 and Phe27 form a hydrophobic pocket which ZINC08994256, also considered.

The above intermolecular interaction played an important role in the stability of ZINC08994256 and

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receptor. Therefore, ZINC08994256, the promising inhibitor acquired high Glide scores and ideal

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binding mode would be employed to molecular dynamics simulation, as well as ZINC12603519.
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Figure 9. The 2D binding modes of ZINC08994256 and crystal ligand 58T in the active site of MTH1
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3.5 Molecular dynamics

ZINC08994256 and ZINC12603519, the two top Glide score hits were subjected to molecular

dynamics studies. The root mean square deviation (RMSD) of ZINC08994256 and ZINC12603519

were shown in Figure 10. In first 5ns, the RMSD of ZINC08994256 raised to 0.25nm, and fluctuated in

the 0.27 or so in last 15ns; and the RMSD of ZINC12603519 reached a stable level of 0.15nm in first

8ns, and fluctuated in the 0.18 or so in last 12ns. Those above parameters could indicated that stable
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binding modes of ZINC08994256 and ZINC12603519 were defined in MTH1 active cavity. In other

words, the stability of the protein-hit complex verified the reliability of molecular docking studies.

0.40 0.40
MTH1---ZINC08994256 MTH1---ZINC12603519
0.35 0.35

0.30 0.30

RMSD (nm)
RMSD (nm)

0.25 0.25

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0.20 0.20

0.15 0.15

0.10 0.10

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0.05 0.05

0.00 0.00
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20

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Time (ns) Time (ns)

Figure 10.RMSD of ZINC08994256 and ZINC12603519 complex with MTH1 derived by dynamics

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simulation.
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4. Conclusion

Multiple pharmacophore hypotheses were developed based on the MTH1 crystal structures and
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inhibitors via structure-based and 3D-QSAR approaches. Pharm-A, the ideal structure-based
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pharmacophore hypothesis was selected with lowest RMSD of 2.26 by pharmacophore comparison.
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Pharm-B, the best 3D-QSAR pharmacophore hypothesis with highest cost, lowest RMSD and

rational correl value was validated by various methods as well as Pharm-A. After that, an approaches
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combined Pharm-A and Pharm-B screened the public database with total 971,228 molecules, with the
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criteria of Lipinskis rule of five and ADMET properties. 2,315 molecules were selected with fit value
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over 12.5 for docking study. Different docking programs were adopted for 2,315 molecules docking.

Considering the binding modes and Glide scores, 5 promising MTH1 inhibitor (ZINC08994256,

ZINC12603519, ZINC00796598, ZINC19792111 and ZINC19792112) were discovered. Finally, 20 ns

MD studies were performed on the MTH1 crystal structure combined with the two top Glide scores hits

ZINC08994256 and ZINC12603519. The results of MD studies indicated that the binding modes of

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two hits was rational. In conclusion, the combined pharmacophore hypotheses, Pharm-A and Pharm-B,

have the capability to identify promising MTH1 inhibitors from molecules database, and the 5 hits have

activities to against MTH1.

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Acknowledgements

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I would like to acknowledge those who contributed to study of MTH1 to provide much date. And

express my sincere gratitude to the listed authors, as well as College of Chemical Engineering, Sichuan

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University.

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Highlights:
 A novel cancer therapeutic targets for drug disgen.
 A new method combined structured-based pharmacophore with 3D-QSAR
pharmacophore hypotheses was used.
 A variety of computer simulation software were applied for molecular docking.
 Several novel and promising MTH1 inhibitors were discovered for the
development of cancer drug.

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