# Novel Design Strategy for Checkpoint Kinase 2 Inhibitors Using Pharmacophore Modeling, Combinatorial Fusion, and Virtual Screening

Authors: Chun-Yuan Lin; Yen-Ling Wang
Journal: BioMed Research International
 (2014)

---

## Abstract

Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r
test) with the value 0.816 by combining the Bes
t
train
Bes
t
test and Fas
t
train
Fas
t
test prediction results. The potential inhibitors were selected from NCI database by screening according to Bes
t
train
Bes
t
test + Fas
t
train
Fas
t
test prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study.


---

## Body

## 1. Introduction

DNA-damage is induced by ionizing radiation, genotoxic chemicals, or collapsed replication forks, and when DNA was damaged or the responses of cells were failure, the mutation associated with the breast or ovarian cancer of genes may occur. To prevent and repair the DNA-damage, mammalian cells will control and stabilize the genome by cell cycle checkpoint. The checkpoint pathway consists of several kinases, such as ataxia telangiectasia mutated protein (ATM [1, 2]), ataxia telangiectasia and Rad3-related protein (ATR [1, 2]), checkpoint kinase 1 (Chk1 [3, 4]), and checkpoint kinase 2 (Chk2 [5–8]). ATM and ATR are upstream kinases passing messages to downstream kinases and phosphorylating several proteins that initiate the activation of the DNA-damage checkpoint. Moreover, ATM is a primarily pathway to activate p53 (protein 53 [9]) by Chk2, and ATR may influence the phosphorylation of Chk1. Both Chk1 and Chk2 are key components in DNA-damage; however, their cellular activities are different. Chk1 is involved in S and G2 phases of the cell cycle with ATR pathway. By contrast, Chk2 is activated in all phases through ATM-dependent pathway and plays an important role in response to DNA double-strand breaks and related lesions. Furthermore, Chk1 is an unstable protein and lacks the forkhead-associated domain (FHA) which was involved in several processes that protect against cancer and can be found in Chk2. Therefore, we concentrate on Chk2 in this study.Chk2 is a protein containing 543 amino acid residues and the structure of Chk2 consists of some functional elements, including the N-terminal SQ/TQ cluster domain (SCD), FHA, and the N-terminal serine/threonine kinase domain (KD) [5–8]. The SCD is known to be the preferred site with the residue Thr68 for phosphorylation to respond to DNA-damage by ATM/ATP kinases. The FHA domain is a phosphopeptide recognition domain found in many regulatory proteins and thought to bind to the phosphoThr68 segment of SCD [5–8, 10–14]. Hence it is a good candidate for interactions of Chk2 with its upstream regulators or downstream targets in the cell-cycle-checkpoint signaling. The KD occupies almost the entire carboxy-terminal half of Chk2 and has been identified based on their homology with serine/threonine kinases. Some studies reported that when DNA was damaged, Chk2 is activated by ATM/ATR through the phosphorylation of residue Thr68. Moreover, Chk2 induces transautophosphorylation of residues Thr383 and Thr387 and then cis-phosphorylation of residue Ser516 [5–8, 10–14]. After that, Chk2 will phosphorylate several downstream substrates, such as BRCA1 (breast cancer 1, early onset [15, 16]), Cdc25A (cell division cycle 25 homolog A), Cdc25C, and p53 [7, 8, 10]. Several researches indicated that Chk2 phosphorylates Cdc25A which is considered an oncogene on the residue Ser123 in S phase of cell cycle, and it also phosphorylates Cdc25C on the residue Ser216 in G2 phase helping prevent mitotic entry in cells with damaged DNA [5]. Furthermore, BRCA1 and p53 are involved in DNA repair process in the breast or ovarian cancer. BRCA1 is a human caretaker gene and helps repair damaged DNA or destroys cells which cannot be repaired. The p53 is a tumor suppressor protein involved in preventing cancer in human and plays an important role in the G1 checkpoint in response to DNA damaging agents. We consider that the sites of the phosphorylations are important in the drug design for cell survival when DNA is damaged.Recently, several studies identified the inhibitors of Chk2 [6–8, 10–14], and they also showed the crystal structures of Chk2 complex, such as PDB: 1GXC, 2W7X, and, and so forth. They are selective, reversible, and ATP-competitive Chk2 inhibitors demonstrating that they effectively restrain the radiation-induced phosphorylation of Chk2. In addition, several selective Chk2 inhibitors have been also identified (two examples were shown in Figure 1) and the researches indicated that they are potential and selective inhibitors of Chk2 with chemotherapeutic and radiosensitization potential. On structure-based drug design, several developments of Chk2 were published [17, 18]. Quantitative structure-activity relationship model (QSAR model) is a regression or classification model and is an important technique in the rational drug design. It is used to correlate the structure properties of compounds with their biological activities. The method to predict the quality by QSAR was improved by considering the three-dimensional structure QSAR (3D-QSAR) [19–24] of targeted inhibitor. Therefore, the compound structure can be directly optimized in the 3D space. The comparative molecular field analyses (CoMFA) [18, 25–30] and the comparative molecular similarity indices analyses (CoMSIA) [18, 27–32] for Chk2 inhibitors were performed by ligand-based and receptor-guided alignment. They used the cocrystal structure from protein data bank (PDB code: 2CN8) [7], and then they identified new plausible binding modes used as template for 3D-QSAR [18]. There is another research of Chk2 studied in QSAR/QSPR [17] providing structures that will improve reducing the side effects of Chk2 inhibitors.Figure 1
Two-dimensional chemical structures of known Chk2 inhibitors. The experimental IC50 of PV1019 and NSC 109555 were 138 nM and 240 nM, respectively.Pharmacophore [20–24, 33–35] is a set of structural features responsible for the biological activity of a molecule. It allowed compounds with diverse structures to find the common chemical features by ligand pharmacophore mapping, and that is different from CoMFA and CoMSIA with the common structure constraint. Thus, pharmacophore can explain how diverse ligands bind to a receptor site by these features and visualize the feature of potential chemical interactions between ligands and receptors. Moreover, pharmacophore can easily and quickly identify candidate inhibitors for a target protein based on 3D query. Therefore, in this work, we first used 3D-QSAR study to build pharmacophore hypotheses (denoted as PhModels) for Chk2 inhibitors by HypoGen Best, Fast, and Caesar algorithms, respectively. Then we used the combinatorial fusion to select and combine prediction results for improving the predictive accuracy in biological activities of inhibitors. Virtual screening is a computational technique used in drug discovery research. There are two categories of screening techniques: ligand-based and structure-based. In this work, for ligand-based virtual screening, we used the selected PhModels as 3D structure query by pharmacophore hypothesis screening that each compound in National Cancer Institute (NCI) database will be mapped onto the pharmacophoric features of selected PhModels. When the chemical features of a compound fit the generated PhModels, it will be selected. All of feasible compounds in NCI database were selected in this work. Finally, the potential inhibitors were retrieved from selected compounds by using molecular docking program to predict the conformation and interaction energy between Chk2 and ligand. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design and can help medicinal chemists to identify or design new Chk2 inhibitors. Besides, the potential inhibitors of Chk2 retrieved in this work can be estimated by biologists for further study.


## 2. Materials and Methods

### 2.1. Biological Data Collection

In order to construct the PhModels, at first, we collected the Chk2 inhibitors with two-dimensional structures and the biological activity values from the ChEMBL database [36]. Then, according to the structure variations and chemical differences in the kinase inhibitor activity, 158 known Chk2 inhibitors were selected and retrieved. The biological activity of 158 known Chk2 inhibitors was represented as IC50 (nanomolar, nM). There are 260,071 compounds from the NCI database (release version 3, http://cactus.nci.nih.gov/download/nci/) which were used in the database screening and molecular docking approach in this work.


### 2.2. Training and Testing Sets Selection

Before generating PhModels, we should divide the 158 Chk2 inhibitors into the training set and testing set, respectively. The rules used to select training set inhibitors are according to the following requirements as suggested by the Accelrys Discovery Studio. (1) All selected inhibitors should have clear and concise information including structure features and activity range. (2) At a minimum, 16 diverse inhibitors for training set were selected to ensure the statistical significance. (3) The training set should contain the most and the least active inhibitors. (4) The biological activities of the inhibitors spanned at least 4 orders of magnitude. Based on the above four rules, the 158 Chk2 inhibitors were divided, and the scatter diagram of training set and testing set inhibitors was shown in Figure2. Figure 2 demonstrates the distribution of the inhibitors in the training set and testing set, and the representative points of the testing set are close to those of the training set. The training set with 25 inhibitors is used to construct PhModels, and the IC50 values of these 25 inhibitors are ranged from 2.3 to 100,000 nM (Table 1). The testing set with remaining 133 inhibitors is used to test the predictive ability of generated PhModels, and the IC50 values of the 133 testing set inhibitors are ranged from 3.4 to 74,000 nM (Table 2). After selecting the training set and testing set inhibitors, we established PhModels at first, and then we used the correlation analysis to estimate the prediction abilities of PhModels.Table 1
Experimental and estimated IC50 values of training set inhibitors.
CHEMBL ID
ExperimentalIC50 (nM)
Estimated IC50(nM)
Besttrain
Fasttrain
Caesartrain
CHEMBL195041
2.3
15
9.9
1129
CHEMBL193990
6.6
6.8
6.2
942
CHEMBL248935
14
20
20
833
CHEMBL195320
18
8.5
6.2
942
CHEMBL176164
23
19
23
1151
CHEMBL250765
37
30
22
950
CHEMBL362677
47
23
23
1153
CHEMBL249959
70
110
20
1000
CHEMBL250992
72
47
6.9
72
CHEMBL251155
110
220
23
756
CHEMBL588536
270
670
790
78578
CHEMBL400772
470
2200
268
231
CHEMBL367390
640
2000
2237
1028
CHEMBL608262
830
1200
1456
94262
CHEMBL401105
900
1000
235
20
CHEMBL176115
1100
970
1044
1449
CHEMBL253542
1200
1100
189
3.8
CHEMBL592490
1800
860
1275
93360
CHEMBL589090
6700
1700
1419
3561
CHEMBL199299
15000
22000
233
1745
CHEMBL251629
19000
3600
615
411
CHEMBL259084
28000
6800
31827
5300
CHEMBL251628
37000
63000
1360
24786
CHEMBL438485
50000
16000
320
243
CHEMBL589501
100000
160000
48276
96926
Correlationcoefficient
(
r
train
)
0.955
0.840
0.238Table 2
Experimental and estimated IC50 values of testing set inhibitors.
CHEMBL ID
ExperimentalIC50 (nM)
Estimated IC50(nM)
Besttrain
Fasttrain
BesttrainBesttest + FasttrainFasttest
Besttest
Fasttest
Caesartest
Besttest
Fasttest
Caesartest
CHEMBL195177
3.4
3.9
5.2
5.1
14.8
6.2
12.3
6.2
CHEMBL359881
4.4
12.0
46.1
42.3
17.3
22.5
29.4
22.5
CHEMBL179717
4.5
9.0
42.1
43.2
14.3
21.9
29.3
21.9
CHEMBL175553
5.5
11.2
57.8
43.1
17.6
20.5
29.1
20.5
CHEMBL192161
7
183.4
74.6
36.7
258.7
253.9
291.4
253.9
CHEMBL191969
8.2
6.4
15.3
14.3
10.5
9.4
13.5
9.4
CHEMBL175472
9.8
7.9
48.4
9.9
12.4
22.5
29.1
22.5
CHEMBL361378
12
9.9
48.1
43.7
14.7
21.7
29.3
21.7
CHEMBL362255
12
11.9
55.4
42.3
15.5
22.9
29.4
22.9
CHEMBL369254
12
70.2
51.3
45.6
38.0
23.7
31.5
23.7
CHEMBL364978
13
4.8
4.9
5.1
13.5
6.5
12.3
6.5
CHEMBL195846
14
3.9
5.0
5.1
12.8
6.2
12.3
6.2
CHEMBL179583
16
12.2
48.5
43.1
13.4
23.2
28.9
23.2
CHEMBL178972
17
15.9
52.5
43.1
18.0
23.3
29.1
23.3
CHEMBL250360
23
19.8
46.8
43.7
17.6
21.7
29.4
21.7
CHEMBL175879
24
21.6
57.0
42.3
24.0
23.2
29.2
23.2
CHEMBL179267
31
7.4
63.5
42.8
20.5
21.2
29.4
21.2
CHEMBL192022
32
20.3
47.5
42.6
23.9
22.4
29.4
22.4
CHEMBL250158
39
10.1
20.4
43.7
19.1
21.5
29.3
21.5
CHEMBL363339
41
10.1
59.8
42.2
18.1
24.0
29.5
24.0
CHEMBL250555
45
26.3
48.2
42.9
24.5
22.7
29.4
22.7
CHEMBL250359
52
3.7
5.2
3.5
20.6
7.0
28.8
7.0
CHEMBL251585
52
4.5
3.0
3.1
11.9
4.9
10.7
4.9
CHEMBL398529
53
23.0
48.4
43.5
21.4
22.4
29.4
22.4
CHEMBL178971
55
62.4
44.9
43.1
41.7
22.1
29.3
22.1
CHEMBL427879
55
13.7
45.4
42.4
16.9
19.9
29.5
19.9
CHEMBL250963
57
8.6
44.1
42.9
17.8
21.5
27.2
21.5
CHEMBL251170
60
51.3
45.3
43.1
26.3
21.5
29.4
21.5
CHEMBL250759
61
45.6
47.4
43.3
36.6
23.2
29.5
23.2
CHEMBL367263
61
19.7
50.3
9.6
17.3
23.9
29.1
23.9
CHEMBL250159
67
55.7
45.3
43.1
25.7
17.2
29.4
17.2
CHEMBL398467
70
11.4
46.5
42.7
21.0
20.4
29.4
20.4
CHEMBL250796
73
3.3
3.4
4.2
14.7
6.0
14.4
6.0
CHEMBL250957
74
36.1
44.9
43.7
30.5
20.4
29.5
20.4
CHEMBL206609
77
25.9
51.0
44.0
9.6
16.3
17.8
16.3
CHEMBL400755
78
8.8
26.6
43.0
12.9
22.5
29.4
22.5
CHEMBL249569
80
11.1
48.3
43.3
17.0
22.9
28.0
22.9
CHEMBL193397
81
9.9
43.8
42.8
14.5
21.2
28.4
21.2
CHEMBL438868
82
18.9
42.0
43.1
13.1
19.0
29.3
19.0
CHEMBL249566
86
17.6
48.8
43.1
23.5
22.8
29.6
22.8
CHEMBL249345
90
23.2
47.6
43.0
20.9
20.0
25.0
20.0
CHEMBL399146
90
29.8
47.9
42.7
28.4
22.1
29.3
22.1
CHEMBL602931
92
483.6
560.1
506.8
645.9
594.0
588.2
594.0
CHEMBL249347
95
10.1
46.3
43.1
27.2
20.1
29.2
20.1
CHEMBL193476
100
951.1
914.5
925.8
2435.2
1027.4
1981.9
1027.4
CHEMBL250361
100
14.2
6.6
14.5
14.9
20.9
29.6
20.9
CHEMBL248934
109
20.0
52.5
43.3
14.0
22.4
29.4
22.4
CHEMBL249750
110
12.7
49.3
43.5
20.7
21.6
29.3
21.6
CHEMBL208463
133
10.4
46.0
41.0
968.6
2768.3
2670.7
2768.3
CHEMBL250566
140
5.2
17.3
26.3
18.4
20.9
29.6
20.9
CHEMBL251256
140
936.4
2453.5
2240.6
192.6
215.1
215.5
215.1
CHEMBL437331
142
471.8
521.6
450.6
64.2
222.1
60.7
222.1
CHEMBL249541
157
23.8
42.5
43.0
16.0
18.8
29.3
18.8
CHEMBL249776
158
13.9
47.2
43.7
16.7
20.9
29.4
20.9
CHEMBL249350
174
30.6
51.2
43.4
22.5
23.3
29.4
23.3
CHEMBL249546
176
10.6
47.3
42.8
17.9
21.0
29.4
21.0
CHEMBL251364
176
21.7
43.9
43.1
17.8
19.7
29.1
19.7
CHEMBL399933
180
14.8
45.0
43.5
20.8
18.9
29.4
18.9
CHEMBL400287
180
19.3
45.5
43.5
21.5
19.8
29.4
19.8
CHEMBL175780
200
61.7
47.5
42.3
36.0
21.9
29.5
21.9
CHEMBL176326
200
935.6
926.3
913.6
1896.0
986.2
1980.7
986.2
CHEMBL590335
210
791.5
721.8
807.4
616.3
639.0
644.7
639.0
CHEMBL398561
220
575.1
926.2
571.5
209.1
278.3
287.9
278.3
CHEMBL249777
231
15.0
45.7
42.5
25.5
21.7
29.4
21.7
CHEMBL442282
233
13.9
46.9
42.3
21.5
22.5
29.4
22.5
CHEMBL195599
250
981.5
908.0
925.8
2266.7
1057.6
1981.9
1057.6
CHEMBL176015
290
54.1
52.8
54.4
24.0
29.3
28.0
29.3
CHEMBL251284
310
484.8
429.7
533.7
189.6
196.8
217.4
196.8
CHEMBL600441
310
454.6
559.9
516.5
300.5
513.5
254.1
513.5
CHEMBL599581
410
594.7
496.6
509.1
506.1
252.9
299.2
252.9
CHEMBL592784
420
462.2
492.0
475.1
216.7
203.8
198.3
203.8
CHEMBL1197465
580
2492.8
8163.9
5925.9
995.1
896.8
488.9
896.8
CHEMBL590809
600
492.4
539.0
534.3
816.4
536.2
549.3
536.2
CHEMBL1197456
610
2871.8
7537.6
6733.2
615.7
4139.2
3514.6
4139.2
CHEMBL590637
610
1251.9
1786.7
1121.1
2379.0
1650.9
1262.3
1650.9
CHEMBL591518
680
1797.9
1804.0
1516.5
6075.0
4714.4
2726.5
4714.4
CHEMBL598973
700
12585.6
151896.0
84151.4
1047.3
396.5
1318.1
396.5
CHEMBL251368
710
27.7
45.9
43.8
13.4
21.2
29.1
21.2
CHEMBL1197303
800
5153.6
36481.1
6594.4
1277.8
681.9
4077.9
681.9
CHEMBL1197320
890
2537.8
7191.1
6002.7
1559.4
517.5
420.0
517.5
CHEMBL1197528
960
2752.7
7737.1
5925.9
765.7
654.9
559.0
654.9
CHEMBL215803
1000
3760.6
140257.0
74847.0
9672.7
50053.5
49263.9
50053.5
CHEMBL253324
1000
996.2
2416.5
603.1
264.7
553.1
272.4
553.1
CHEMBL589347
1100
458.3
560.2
482.3
209.7
299.1
196.7
299.1
CHEMBL604784
1100
1188.4
1365.6
1205.7
2305.3
1962.3
1307.9
1962.3
CHEMBL1197529
1120
2047.4
11678.5
8090.0
3368.8
3648.8
3659.9
3648.8
CHEMBL1197326
1130
12645.5
45428.8
7432.7
1138.2
465.0
416.0
465.0
CHEMBL176041
1200
925.0
906.8
913.6
1933.6
1040.0
1980.7
1040.0
CHEMBL590079
1350
548.9
548.5
550.6
1000.9
860.3
855.1
860.3
CHEMBL605083
1400
489.2
613.8
554.4
1224.9
1401.0
1345.6
1401.0
CHEMBL175481
1500
69.2
57.7
49.2
1853.9
1526.1
1852.1
1526.1
CHEMBL205906
1540
1696.1
1109.3
1153.0
980.6
17655.5
937.5
17655.5
CHEMBL590808
1600
1662.5
1799.1
1709.4
51882.2
48813.2
48302.3
48813.2
CHEMBL1170748
1700
1974.2
1940.4
1652.8
3251.4
2734.9
407.6
2734.9
CHEMBL253541
1800
43066.9
42831.1
79622.8
1255.9
1099.1
15566.3
1099.1
CHEMBL176554
1900
572.3
7226.3
9126.2
1096.1
3352.6
3926.3
3352.6
CHEMBL377597
2000
938.5
6719.7
5644.6
50.4
116.9
105.4
116.9
CHEMBL1170749
2200
10874.0
30873.8
3995.6
5835.1
25044.3
1090.5
25044.3
CHEMBL590336
2200
507.8
594.0
516.4
568.9
544.3
521.1
544.3
CHEMBL590807
2200
52944.6
70480.3
52639.4
44898.4
49196.7
36195.2
49196.7
CHEMBL600868
2200
1342.8
1603.8
1312.9
7373.6
5797.7
4864.3
5797.7
CHEMBL398759
2300
666.0
2847.2
1010.2
447.2
924.3
309.0
924.3
CHEMBL604459
2300
1176.9
2299.2
1369.3
614.2
877.8
682.5
877.8
CHEMBL179383
2400
15724.9
14621.7
14257.4
4508.5
4132.8
4023.9
4132.8
CHEMBL592489
2400
469.2
494.6
486.9
204.7
240.5
208.4
240.5
CHEMBL425904
2800
605.0
651.0
534.9
478.1
451.9
488.6
451.9
CHEMBL150894
3000
537.8
844.9
797.6
186.8
600.2
289.0
600.2
CHEMBL590793
3000
475.4
2480.4
1251.5
192.0
251.7
205.4
251.7
CHEMBL600865
4400
796.9
2072.3
1223.3
198.1
261.3
228.1
261.3
CHEMBL249253
5000
659.8
503.6
499.5
406.6
227.9
309.5
227.9
CHEMBL587506
5200
1050.8
1293.3
1083.2
2201.3
2162.7
1656.0
2162.7
CHEMBL204930
5800
755.5
1260.7
1105.5
47881.6
47912.3
47868.0
47912.3
CHEMBL554900
5900
6722.8
580526.0
807309.0
745.4
428.2
47897.1
428.2
CHEMBL176276
6000
3360.2
8955.9
8476.6
2075.6
2054.0
1970.7
2054.0
CHEMBL589091
6100
1319.5
1308.4
1172.0
579.2
603.1
600.9
603.1
CHEMBL559781
7400
91783.7
822917.0
1230010.0
414.5
6865.5
47911.9
6865.5
CHEMBL249252
8000
100671.0
38586.8
31129.8
590.5
491.6
467.9
491.6
CHEMBL589089
9800
1070.7
1321.5
1002.7
38589.3
48136.2
41143.2
48136.2
CHEMBL217090
10000
628.8
1010.9
1548.5
390.6
451.8
910.1
451.8
CHEMBL217092
10000
1030.2
1199.3
1908.8
379.9
625.6
707.2
625.6
CHEMBL382588
10000
1365.2
5880.3
5488.7
2560.7
3993.4
3649.4
3993.4
CHEMBL590581
10000
145206.0
149922.0
108067.0
50609.5
49348.5
48302.3
49348.5
CHEMBL242753
10300
1742.5
3393.1
1941.1
1202.5
2613.0
1297.3
2613.0
CHEMBL398758
11000
1582.8
187965.0
1573.6
237.0
4333.9
369.0
4333.9
CHEMBL399151
11000
812.1
1263.5
2069.9
334.5
1193.9
1271.6
1193.9
CHEMBL395080
13450
513.5
484.0
458.6
198.5
188.9
183.3
188.9
CHEMBL1171533
15000
349604.0
296515.0
159368.0
26784.5
26699.3
45506.4
26699.3
CHEMBL602729
17000
148756.0
149052.0
139465.0
224.1
324.7
229.4
324.7
CHEMBL249255
19000
182486.0
42615.8
41578.3
1698.2
616.1
683.9
616.1
CHEMBL202930
21730
12828.4
11945.6
12145.8
213.3
219.7
217.6
219.7
CHEMBL589986
22000
1167.1
1337.4
1143.5
53747.5
49155.6
49316.8
49155.6
CHEMBL251471
40000
3358.9
1946.2
2075.7
512.2
423.7
3427.4
423.7
CHEMBL560056
74000
152006.0
156723.0
208466.0
223.2
208.1
190.4
208.1
Correlationcoefficient  (
r
test
)
0.810
0.771
0.783
0.710
0.728
0.714
0.816Figure 2
The scatter diagram of training set and testing set inhibitors.


### 2.3. Pharmacophore Generation

The workflow of PhModel generation for Chk2 inhibitors was shown in Figure3. In this study, we used the HypoGen program [37] in Accelrys Discovery Studio 2.1 to generate PhModels. At the initial step, 3D conformations of the training set inhibitors were generated by using “3D-QSAR Pharmacophore Generation protocol” with the Best, Fast, and Caesar generating algorithms, respectively, based on the CHARMm-like force field. The conformational-space energy was constrained ≤20 kcal/mol which represented the maximum allowed energy above the global minimum energy. For each training set inhibitor, the number of the diverse 3D conformations was set to ≤255. All other parameters were set as default values. Following the above rules, the 3D conformations were generated, and then we can construct the PhModel by using “Ligand Pharmacophore Mapping protocol.” Each of the ten PhModels using HypoGen Best, Fast, and Caesar algorithms were generated in this study.Figure 3
The workflow of PhModel generation for Chk2 inhibitors.


### 2.4. Combinatorial Fusion

In this study, we use a combinatorial fusion technique to facilitate prediction results selection and combination for improving predictive accuracy in biological activities of inhibitors. The combinatorial fusion we take is analogous to that used in information retrieval [38, 39], pattern recognition [40], molecular similarity searching and structure-based screening [41], and microarray gene expression analysis [42]. These works have demonstrated the following remark [43].Remark 1.
For a set of multiple scoring systems, each with a score function and a rank function, we have that (a) the combination of multiple scoring systems would improve the prediction accuracy only if (1) each of the systems has a relatively high performance, and (2) the individual systems are distinctive (or diversified), and (b) rank combination performs better than score combination under certain conditions.Given an inhibitor and for each prediction resultA, let s
A be a function as the predicted biological activity and it is represented as a real number. We view the function s
A as the score function. Since s
A only assigns a number not a set of numbers, in this work, no rank function would be used for an inhibitor. Therefore, the rank combination and the rule (b) in Remark 1 are not considered in the study. Suppose we have m prediction results (m scoring functions). There are combinatorially 2
m
-
1 combinations for all m individual prediction results (
∑
k
=
1
m
(
m
k
)
=
2
m
-
1
) with score functions. The total number of combinations to be considered for predicting biological activity of an inhibitor is 2
m
-
1. This number of combinations can become huge when the number of prediction results m is large. Moreover, we have to evaluate the predictive power of each combination across all inhibitors. This study would start with combining only two prediction results which still retain fairly good prediction power.Supposem prediction results A
i, i
=
1,2
,
…
,
m, are given with score function s
A
i; there are several different ways of combination. Among others, there are score combination, voting, linear average combination, and weighted combination [38–42]. Voting is computationally simple and better than simple linear combinations when applied to the situation with large number of prediction results. However, a better alternative is to reduce the number of prediction results to a smaller number and then these prediction results are combined. In this paper, we reduce the set of prediction results to those which perform relatively well and then use the rank/score function to decide whether to combine by score. In this paper, we use the rules (a) (1) and (a) (2) stated in Remark 1 as our guiding principle to select prediction results and to decide on the method of combination. After generating each of the ten PhModels by using HypoGen Best, Fast, and Caesar algorithms for training set inhibitors, each of the best PhModel (denoted as Besttrain, Fasttrain, and Caseartrain) was evaluated by its correlation coefficient of the training set (
r
train
). Then these best PhModels were used to predict the biological activities of testing set inhibitors by using HypoGen Best, Fast, and Caesar algorithms. Therefore, there are nine prediction results (denoted as Z
train
×
Z
test, Z
=
{
Best
,
Fast
,
Caesar
}, that is, BesttrainBesttest) generated for testing set inhibitors. Using data fusion, results from various prediction results are combined to obtain predictions with larger accuracy rate. The diversity rank/score function is used to select the most suitable prediction results for combination. If these three best PhModels were selected, there are nine prediction results and then there are 2
9
-
1
=
511 combinations. According to the rule (a) (1) in Remark 1, the r
train of Caseartrain is far less than those of Besttrain and Fasttrain (Table 1); then, the Caseartrain was not considered in the combinations. Therefore, there are six prediction results (Z
1
train
×
Z
2
test, Z
1
=
{
Best
,
Fast
} and Z
2
=
{
Best
,
Fast
,
Caesar
}) and 2
6
-
1
=
63 combinations. A special diversity rank/score graph was used to choose the best discriminating prediction results for further combination.For an inhibitorp
i in the testing set P
=
{
p
1
,
p
2
,
…
,
p
t
} and the pair of prediction results A and B, the diversity score function d
i
(
A
,
B
) is defined as d
i
(
A
,
B
)
=
∑
|
s
A
-
s
B
|. When there are q prediction results selected (in this study, q
=
6), there are (
q
2
)
=
q
(
q
-
1
)
/
2 (in this study, the number is 15) diversity score functions. If we let i vary and fix the prediction result pair (
A
,
B
), then d
i
(
A
,
B
) is the diversity score function s
(
A
,
B
) from P
=
{
p
1
,
p
2
,
…
,
p
t
}. Sorting s
(
A
,
B
) into descending order would lead to the diversity rank function r
(
A
,
B
). Consequently, the diversity rank/score function f
(
A
,
B
) is defined as f
(
A
,
B
)
=
(
s
(
A
,
B
)
∘
r
(
A
,
B
)
-
1
)
(
j
)
=
s
(
A
,
B
)
(
r
(
A
,
B
)
-
1
(
j
)
), where j is in T
=
{
1
,
2
,
3
,
…
,
t
}. We note that the set T is different from the set P which is the testing set considered. The set T is used as the index set for the diversity rank function value and |
T
|
=
t is indeed the cardinality of P. The diversity rank/score function f
(
A
,
B
) so defined exhibits the diversity trend of the prediction result pair (
A
,
B
) across the whole spectrum of input set of t inhibitors and is independent of the specific inhibitor under study. For two prediction results A and B, the graph of the diversity rank/score functionf
(
A
,
B
)
(
j
) is called the diversity rank/score graph. This study aims to examine all the q
(
q
-
1
)
/
2 diversity rank/score graphs to see which pair of prediction results would give the larger diversity measurement according to the rule (a) (2) in Remark 1.


### 2.5. Database Screen

After examining 15 diversity rank/score graphs, the PhModelsA and B determined from the best prediction result pair were used to screen the NCI database for new Chk2 inhibitor candidates. Under the PhModel, pharmacophore hypothesis screening can be used to screen small molecule database to retrieve the compounds as potential inhibitors that fit the pharmacophoric features. In this study, the “Search 3D Database protocol” with the Best/Fast/Casear Search option in Accelrys Discovery Studio 2.1 was employed to search the NCI database with 260,071 compounds. We could filter out and select the compounds in the NCI database based on the estimated activity and chemical features of PhModel.


### 2.6. Molecular Docking

After the database screening approach, the selected compounds can be further estimated according to the interaction energy between a receptor and a ligand through the molecular docking approach. In this study, selected compounds in the NCI database were docked into Chk2 active sites by CDOCKER docking program, and then their CDOCKER interaction energies were estimated. Finally, new potential candidates were retrieved from the NCI database with high interaction energy. The workflow of database screening and molecular docking approach was shown in Figure4.Figure 4
The workflow of database screening and molecular docking approach for new Chk2 inhibitor candidates.


## 2.1. Biological Data Collection

In order to construct the PhModels, at first, we collected the Chk2 inhibitors with two-dimensional structures and the biological activity values from the ChEMBL database [36]. Then, according to the structure variations and chemical differences in the kinase inhibitor activity, 158 known Chk2 inhibitors were selected and retrieved. The biological activity of 158 known Chk2 inhibitors was represented as IC50 (nanomolar, nM). There are 260,071 compounds from the NCI database (release version 3, http://cactus.nci.nih.gov/download/nci/) which were used in the database screening and molecular docking approach in this work.


## 2.2. Training and Testing Sets Selection

Before generating PhModels, we should divide the 158 Chk2 inhibitors into the training set and testing set, respectively. The rules used to select training set inhibitors are according to the following requirements as suggested by the Accelrys Discovery Studio. (1) All selected inhibitors should have clear and concise information including structure features and activity range. (2) At a minimum, 16 diverse inhibitors for training set were selected to ensure the statistical significance. (3) The training set should contain the most and the least active inhibitors. (4) The biological activities of the inhibitors spanned at least 4 orders of magnitude. Based on the above four rules, the 158 Chk2 inhibitors were divided, and the scatter diagram of training set and testing set inhibitors was shown in Figure2. Figure 2 demonstrates the distribution of the inhibitors in the training set and testing set, and the representative points of the testing set are close to those of the training set. The training set with 25 inhibitors is used to construct PhModels, and the IC50 values of these 25 inhibitors are ranged from 2.3 to 100,000 nM (Table 1). The testing set with remaining 133 inhibitors is used to test the predictive ability of generated PhModels, and the IC50 values of the 133 testing set inhibitors are ranged from 3.4 to 74,000 nM (Table 2). After selecting the training set and testing set inhibitors, we established PhModels at first, and then we used the correlation analysis to estimate the prediction abilities of PhModels.Table 1
Experimental and estimated IC50 values of training set inhibitors.
CHEMBL ID
ExperimentalIC50 (nM)
Estimated IC50(nM)
Besttrain
Fasttrain
Caesartrain
CHEMBL195041
2.3
15
9.9
1129
CHEMBL193990
6.6
6.8
6.2
942
CHEMBL248935
14
20
20
833
CHEMBL195320
18
8.5
6.2
942
CHEMBL176164
23
19
23
1151
CHEMBL250765
37
30
22
950
CHEMBL362677
47
23
23
1153
CHEMBL249959
70
110
20
1000
CHEMBL250992
72
47
6.9
72
CHEMBL251155
110
220
23
756
CHEMBL588536
270
670
790
78578
CHEMBL400772
470
2200
268
231
CHEMBL367390
640
2000
2237
1028
CHEMBL608262
830
1200
1456
94262
CHEMBL401105
900
1000
235
20
CHEMBL176115
1100
970
1044
1449
CHEMBL253542
1200
1100
189
3.8
CHEMBL592490
1800
860
1275
93360
CHEMBL589090
6700
1700
1419
3561
CHEMBL199299
15000
22000
233
1745
CHEMBL251629
19000
3600
615
411
CHEMBL259084
28000
6800
31827
5300
CHEMBL251628
37000
63000
1360
24786
CHEMBL438485
50000
16000
320
243
CHEMBL589501
100000
160000
48276
96926
Correlationcoefficient
(
r
train
)
0.955
0.840
0.238Table 2
Experimental and estimated IC50 values of testing set inhibitors.
CHEMBL ID
ExperimentalIC50 (nM)
Estimated IC50(nM)
Besttrain
Fasttrain
BesttrainBesttest + FasttrainFasttest
Besttest
Fasttest
Caesartest
Besttest
Fasttest
Caesartest
CHEMBL195177
3.4
3.9
5.2
5.1
14.8
6.2
12.3
6.2
CHEMBL359881
4.4
12.0
46.1
42.3
17.3
22.5
29.4
22.5
CHEMBL179717
4.5
9.0
42.1
43.2
14.3
21.9
29.3
21.9
CHEMBL175553
5.5
11.2
57.8
43.1
17.6
20.5
29.1
20.5
CHEMBL192161
7
183.4
74.6
36.7
258.7
253.9
291.4
253.9
CHEMBL191969
8.2
6.4
15.3
14.3
10.5
9.4
13.5
9.4
CHEMBL175472
9.8
7.9
48.4
9.9
12.4
22.5
29.1
22.5
CHEMBL361378
12
9.9
48.1
43.7
14.7
21.7
29.3
21.7
CHEMBL362255
12
11.9
55.4
42.3
15.5
22.9
29.4
22.9
CHEMBL369254
12
70.2
51.3
45.6
38.0
23.7
31.5
23.7
CHEMBL364978
13
4.8
4.9
5.1
13.5
6.5
12.3
6.5
CHEMBL195846
14
3.9
5.0
5.1
12.8
6.2
12.3
6.2
CHEMBL179583
16
12.2
48.5
43.1
13.4
23.2
28.9
23.2
CHEMBL178972
17
15.9
52.5
43.1
18.0
23.3
29.1
23.3
CHEMBL250360
23
19.8
46.8
43.7
17.6
21.7
29.4
21.7
CHEMBL175879
24
21.6
57.0
42.3
24.0
23.2
29.2
23.2
CHEMBL179267
31
7.4
63.5
42.8
20.5
21.2
29.4
21.2
CHEMBL192022
32
20.3
47.5
42.6
23.9
22.4
29.4
22.4
CHEMBL250158
39
10.1
20.4
43.7
19.1
21.5
29.3
21.5
CHEMBL363339
41
10.1
59.8
42.2
18.1
24.0
29.5
24.0
CHEMBL250555
45
26.3
48.2
42.9
24.5
22.7
29.4
22.7
CHEMBL250359
52
3.7
5.2
3.5
20.6
7.0
28.8
7.0
CHEMBL251585
52
4.5
3.0
3.1
11.9
4.9
10.7
4.9
CHEMBL398529
53
23.0
48.4
43.5
21.4
22.4
29.4
22.4
CHEMBL178971
55
62.4
44.9
43.1
41.7
22.1
29.3
22.1
CHEMBL427879
55
13.7
45.4
42.4
16.9
19.9
29.5
19.9
CHEMBL250963
57
8.6
44.1
42.9
17.8
21.5
27.2
21.5
CHEMBL251170
60
51.3
45.3
43.1
26.3
21.5
29.4
21.5
CHEMBL250759
61
45.6
47.4
43.3
36.6
23.2
29.5
23.2
CHEMBL367263
61
19.7
50.3
9.6
17.3
23.9
29.1
23.9
CHEMBL250159
67
55.7
45.3
43.1
25.7
17.2
29.4
17.2
CHEMBL398467
70
11.4
46.5
42.7
21.0
20.4
29.4
20.4
CHEMBL250796
73
3.3
3.4
4.2
14.7
6.0
14.4
6.0
CHEMBL250957
74
36.1
44.9
43.7
30.5
20.4
29.5
20.4
CHEMBL206609
77
25.9
51.0
44.0
9.6
16.3
17.8
16.3
CHEMBL400755
78
8.8
26.6
43.0
12.9
22.5
29.4
22.5
CHEMBL249569
80
11.1
48.3
43.3
17.0
22.9
28.0
22.9
CHEMBL193397
81
9.9
43.8
42.8
14.5
21.2
28.4
21.2
CHEMBL438868
82
18.9
42.0
43.1
13.1
19.0
29.3
19.0
CHEMBL249566
86
17.6
48.8
43.1
23.5
22.8
29.6
22.8
CHEMBL249345
90
23.2
47.6
43.0
20.9
20.0
25.0
20.0
CHEMBL399146
90
29.8
47.9
42.7
28.4
22.1
29.3
22.1
CHEMBL602931
92
483.6
560.1
506.8
645.9
594.0
588.2
594.0
CHEMBL249347
95
10.1
46.3
43.1
27.2
20.1
29.2
20.1
CHEMBL193476
100
951.1
914.5
925.8
2435.2
1027.4
1981.9
1027.4
CHEMBL250361
100
14.2
6.6
14.5
14.9
20.9
29.6
20.9
CHEMBL248934
109
20.0
52.5
43.3
14.0
22.4
29.4
22.4
CHEMBL249750
110
12.7
49.3
43.5
20.7
21.6
29.3
21.6
CHEMBL208463
133
10.4
46.0
41.0
968.6
2768.3
2670.7
2768.3
CHEMBL250566
140
5.2
17.3
26.3
18.4
20.9
29.6
20.9
CHEMBL251256
140
936.4
2453.5
2240.6
192.6
215.1
215.5
215.1
CHEMBL437331
142
471.8
521.6
450.6
64.2
222.1
60.7
222.1
CHEMBL249541
157
23.8
42.5
43.0
16.0
18.8
29.3
18.8
CHEMBL249776
158
13.9
47.2
43.7
16.7
20.9
29.4
20.9
CHEMBL249350
174
30.6
51.2
43.4
22.5
23.3
29.4
23.3
CHEMBL249546
176
10.6
47.3
42.8
17.9
21.0
29.4
21.0
CHEMBL251364
176
21.7
43.9
43.1
17.8
19.7
29.1
19.7
CHEMBL399933
180
14.8
45.0
43.5
20.8
18.9
29.4
18.9
CHEMBL400287
180
19.3
45.5
43.5
21.5
19.8
29.4
19.8
CHEMBL175780
200
61.7
47.5
42.3
36.0
21.9
29.5
21.9
CHEMBL176326
200
935.6
926.3
913.6
1896.0
986.2
1980.7
986.2
CHEMBL590335
210
791.5
721.8
807.4
616.3
639.0
644.7
639.0
CHEMBL398561
220
575.1
926.2
571.5
209.1
278.3
287.9
278.3
CHEMBL249777
231
15.0
45.7
42.5
25.5
21.7
29.4
21.7
CHEMBL442282
233
13.9
46.9
42.3
21.5
22.5
29.4
22.5
CHEMBL195599
250
981.5
908.0
925.8
2266.7
1057.6
1981.9
1057.6
CHEMBL176015
290
54.1
52.8
54.4
24.0
29.3
28.0
29.3
CHEMBL251284
310
484.8
429.7
533.7
189.6
196.8
217.4
196.8
CHEMBL600441
310
454.6
559.9
516.5
300.5
513.5
254.1
513.5
CHEMBL599581
410
594.7
496.6
509.1
506.1
252.9
299.2
252.9
CHEMBL592784
420
462.2
492.0
475.1
216.7
203.8
198.3
203.8
CHEMBL1197465
580
2492.8
8163.9
5925.9
995.1
896.8
488.9
896.8
CHEMBL590809
600
492.4
539.0
534.3
816.4
536.2
549.3
536.2
CHEMBL1197456
610
2871.8
7537.6
6733.2
615.7
4139.2
3514.6
4139.2
CHEMBL590637
610
1251.9
1786.7
1121.1
2379.0
1650.9
1262.3
1650.9
CHEMBL591518
680
1797.9
1804.0
1516.5
6075.0
4714.4
2726.5
4714.4
CHEMBL598973
700
12585.6
151896.0
84151.4
1047.3
396.5
1318.1
396.5
CHEMBL251368
710
27.7
45.9
43.8
13.4
21.2
29.1
21.2
CHEMBL1197303
800
5153.6
36481.1
6594.4
1277.8
681.9
4077.9
681.9
CHEMBL1197320
890
2537.8
7191.1
6002.7
1559.4
517.5
420.0
517.5
CHEMBL1197528
960
2752.7
7737.1
5925.9
765.7
654.9
559.0
654.9
CHEMBL215803
1000
3760.6
140257.0
74847.0
9672.7
50053.5
49263.9
50053.5
CHEMBL253324
1000
996.2
2416.5
603.1
264.7
553.1
272.4
553.1
CHEMBL589347
1100
458.3
560.2
482.3
209.7
299.1
196.7
299.1
CHEMBL604784
1100
1188.4
1365.6
1205.7
2305.3
1962.3
1307.9
1962.3
CHEMBL1197529
1120
2047.4
11678.5
8090.0
3368.8
3648.8
3659.9
3648.8
CHEMBL1197326
1130
12645.5
45428.8
7432.7
1138.2
465.0
416.0
465.0
CHEMBL176041
1200
925.0
906.8
913.6
1933.6
1040.0
1980.7
1040.0
CHEMBL590079
1350
548.9
548.5
550.6
1000.9
860.3
855.1
860.3
CHEMBL605083
1400
489.2
613.8
554.4
1224.9
1401.0
1345.6
1401.0
CHEMBL175481
1500
69.2
57.7
49.2
1853.9
1526.1
1852.1
1526.1
CHEMBL205906
1540
1696.1
1109.3
1153.0
980.6
17655.5
937.5
17655.5
CHEMBL590808
1600
1662.5
1799.1
1709.4
51882.2
48813.2
48302.3
48813.2
CHEMBL1170748
1700
1974.2
1940.4
1652.8
3251.4
2734.9
407.6
2734.9
CHEMBL253541
1800
43066.9
42831.1
79622.8
1255.9
1099.1
15566.3
1099.1
CHEMBL176554
1900
572.3
7226.3
9126.2
1096.1
3352.6
3926.3
3352.6
CHEMBL377597
2000
938.5
6719.7
5644.6
50.4
116.9
105.4
116.9
CHEMBL1170749
2200
10874.0
30873.8
3995.6
5835.1
25044.3
1090.5
25044.3
CHEMBL590336
2200
507.8
594.0
516.4
568.9
544.3
521.1
544.3
CHEMBL590807
2200
52944.6
70480.3
52639.4
44898.4
49196.7
36195.2
49196.7
CHEMBL600868
2200
1342.8
1603.8
1312.9
7373.6
5797.7
4864.3
5797.7
CHEMBL398759
2300
666.0
2847.2
1010.2
447.2
924.3
309.0
924.3
CHEMBL604459
2300
1176.9
2299.2
1369.3
614.2
877.8
682.5
877.8
CHEMBL179383
2400
15724.9
14621.7
14257.4
4508.5
4132.8
4023.9
4132.8
CHEMBL592489
2400
469.2
494.6
486.9
204.7
240.5
208.4
240.5
CHEMBL425904
2800
605.0
651.0
534.9
478.1
451.9
488.6
451.9
CHEMBL150894
3000
537.8
844.9
797.6
186.8
600.2
289.0
600.2
CHEMBL590793
3000
475.4
2480.4
1251.5
192.0
251.7
205.4
251.7
CHEMBL600865
4400
796.9
2072.3
1223.3
198.1
261.3
228.1
261.3
CHEMBL249253
5000
659.8
503.6
499.5
406.6
227.9
309.5
227.9
CHEMBL587506
5200
1050.8
1293.3
1083.2
2201.3
2162.7
1656.0
2162.7
CHEMBL204930
5800
755.5
1260.7
1105.5
47881.6
47912.3
47868.0
47912.3
CHEMBL554900
5900
6722.8
580526.0
807309.0
745.4
428.2
47897.1
428.2
CHEMBL176276
6000
3360.2
8955.9
8476.6
2075.6
2054.0
1970.7
2054.0
CHEMBL589091
6100
1319.5
1308.4
1172.0
579.2
603.1
600.9
603.1
CHEMBL559781
7400
91783.7
822917.0
1230010.0
414.5
6865.5
47911.9
6865.5
CHEMBL249252
8000
100671.0
38586.8
31129.8
590.5
491.6
467.9
491.6
CHEMBL589089
9800
1070.7
1321.5
1002.7
38589.3
48136.2
41143.2
48136.2
CHEMBL217090
10000
628.8
1010.9
1548.5
390.6
451.8
910.1
451.8
CHEMBL217092
10000
1030.2
1199.3
1908.8
379.9
625.6
707.2
625.6
CHEMBL382588
10000
1365.2
5880.3
5488.7
2560.7
3993.4
3649.4
3993.4
CHEMBL590581
10000
145206.0
149922.0
108067.0
50609.5
49348.5
48302.3
49348.5
CHEMBL242753
10300
1742.5
3393.1
1941.1
1202.5
2613.0
1297.3
2613.0
CHEMBL398758
11000
1582.8
187965.0
1573.6
237.0
4333.9
369.0
4333.9
CHEMBL399151
11000
812.1
1263.5
2069.9
334.5
1193.9
1271.6
1193.9
CHEMBL395080
13450
513.5
484.0
458.6
198.5
188.9
183.3
188.9
CHEMBL1171533
15000
349604.0
296515.0
159368.0
26784.5
26699.3
45506.4
26699.3
CHEMBL602729
17000
148756.0
149052.0
139465.0
224.1
324.7
229.4
324.7
CHEMBL249255
19000
182486.0
42615.8
41578.3
1698.2
616.1
683.9
616.1
CHEMBL202930
21730
12828.4
11945.6
12145.8
213.3
219.7
217.6
219.7
CHEMBL589986
22000
1167.1
1337.4
1143.5
53747.5
49155.6
49316.8
49155.6
CHEMBL251471
40000
3358.9
1946.2
2075.7
512.2
423.7
3427.4
423.7
CHEMBL560056
74000
152006.0
156723.0
208466.0
223.2
208.1
190.4
208.1
Correlationcoefficient  (
r
test
)
0.810
0.771
0.783
0.710
0.728
0.714
0.816Figure 2
The scatter diagram of training set and testing set inhibitors.


## 2.3. Pharmacophore Generation

The workflow of PhModel generation for Chk2 inhibitors was shown in Figure3. In this study, we used the HypoGen program [37] in Accelrys Discovery Studio 2.1 to generate PhModels. At the initial step, 3D conformations of the training set inhibitors were generated by using “3D-QSAR Pharmacophore Generation protocol” with the Best, Fast, and Caesar generating algorithms, respectively, based on the CHARMm-like force field. The conformational-space energy was constrained ≤20 kcal/mol which represented the maximum allowed energy above the global minimum energy. For each training set inhibitor, the number of the diverse 3D conformations was set to ≤255. All other parameters were set as default values. Following the above rules, the 3D conformations were generated, and then we can construct the PhModel by using “Ligand Pharmacophore Mapping protocol.” Each of the ten PhModels using HypoGen Best, Fast, and Caesar algorithms were generated in this study.Figure 3
The workflow of PhModel generation for Chk2 inhibitors.


## 2.4. Combinatorial Fusion

In this study, we use a combinatorial fusion technique to facilitate prediction results selection and combination for improving predictive accuracy in biological activities of inhibitors. The combinatorial fusion we take is analogous to that used in information retrieval [38, 39], pattern recognition [40], molecular similarity searching and structure-based screening [41], and microarray gene expression analysis [42]. These works have demonstrated the following remark [43].Remark 1.
For a set of multiple scoring systems, each with a score function and a rank function, we have that (a) the combination of multiple scoring systems would improve the prediction accuracy only if (1) each of the systems has a relatively high performance, and (2) the individual systems are distinctive (or diversified), and (b) rank combination performs better than score combination under certain conditions.Given an inhibitor and for each prediction resultA, let s
A be a function as the predicted biological activity and it is represented as a real number. We view the function s
A as the score function. Since s
A only assigns a number not a set of numbers, in this work, no rank function would be used for an inhibitor. Therefore, the rank combination and the rule (b) in Remark 1 are not considered in the study. Suppose we have m prediction results (m scoring functions). There are combinatorially 2
m
-
1 combinations for all m individual prediction results (
∑
k
=
1
m
(
m
k
)
=
2
m
-
1
) with score functions. The total number of combinations to be considered for predicting biological activity of an inhibitor is 2
m
-
1. This number of combinations can become huge when the number of prediction results m is large. Moreover, we have to evaluate the predictive power of each combination across all inhibitors. This study would start with combining only two prediction results which still retain fairly good prediction power.Supposem prediction results A
i, i
=
1,2
,
…
,
m, are given with score function s
A
i; there are several different ways of combination. Among others, there are score combination, voting, linear average combination, and weighted combination [38–42]. Voting is computationally simple and better than simple linear combinations when applied to the situation with large number of prediction results. However, a better alternative is to reduce the number of prediction results to a smaller number and then these prediction results are combined. In this paper, we reduce the set of prediction results to those which perform relatively well and then use the rank/score function to decide whether to combine by score. In this paper, we use the rules (a) (1) and (a) (2) stated in Remark 1 as our guiding principle to select prediction results and to decide on the method of combination. After generating each of the ten PhModels by using HypoGen Best, Fast, and Caesar algorithms for training set inhibitors, each of the best PhModel (denoted as Besttrain, Fasttrain, and Caseartrain) was evaluated by its correlation coefficient of the training set (
r
train
). Then these best PhModels were used to predict the biological activities of testing set inhibitors by using HypoGen Best, Fast, and Caesar algorithms. Therefore, there are nine prediction results (denoted as Z
train
×
Z
test, Z
=
{
Best
,
Fast
,
Caesar
}, that is, BesttrainBesttest) generated for testing set inhibitors. Using data fusion, results from various prediction results are combined to obtain predictions with larger accuracy rate. The diversity rank/score function is used to select the most suitable prediction results for combination. If these three best PhModels were selected, there are nine prediction results and then there are 2
9
-
1
=
511 combinations. According to the rule (a) (1) in Remark 1, the r
train of Caseartrain is far less than those of Besttrain and Fasttrain (Table 1); then, the Caseartrain was not considered in the combinations. Therefore, there are six prediction results (Z
1
train
×
Z
2
test, Z
1
=
{
Best
,
Fast
} and Z
2
=
{
Best
,
Fast
,
Caesar
}) and 2
6
-
1
=
63 combinations. A special diversity rank/score graph was used to choose the best discriminating prediction results for further combination.For an inhibitorp
i in the testing set P
=
{
p
1
,
p
2
,
…
,
p
t
} and the pair of prediction results A and B, the diversity score function d
i
(
A
,
B
) is defined as d
i
(
A
,
B
)
=
∑
|
s
A
-
s
B
|. When there are q prediction results selected (in this study, q
=
6), there are (
q
2
)
=
q
(
q
-
1
)
/
2 (in this study, the number is 15) diversity score functions. If we let i vary and fix the prediction result pair (
A
,
B
), then d
i
(
A
,
B
) is the diversity score function s
(
A
,
B
) from P
=
{
p
1
,
p
2
,
…
,
p
t
}. Sorting s
(
A
,
B
) into descending order would lead to the diversity rank function r
(
A
,
B
). Consequently, the diversity rank/score function f
(
A
,
B
) is defined as f
(
A
,
B
)
=
(
s
(
A
,
B
)
∘
r
(
A
,
B
)
-
1
)
(
j
)
=
s
(
A
,
B
)
(
r
(
A
,
B
)
-
1
(
j
)
), where j is in T
=
{
1
,
2
,
3
,
…
,
t
}. We note that the set T is different from the set P which is the testing set considered. The set T is used as the index set for the diversity rank function value and |
T
|
=
t is indeed the cardinality of P. The diversity rank/score function f
(
A
,
B
) so defined exhibits the diversity trend of the prediction result pair (
A
,
B
) across the whole spectrum of input set of t inhibitors and is independent of the specific inhibitor under study. For two prediction results A and B, the graph of the diversity rank/score functionf
(
A
,
B
)
(
j
) is called the diversity rank/score graph. This study aims to examine all the q
(
q
-
1
)
/
2 diversity rank/score graphs to see which pair of prediction results would give the larger diversity measurement according to the rule (a) (2) in Remark 1.


## 2.5. Database Screen

After examining 15 diversity rank/score graphs, the PhModelsA and B determined from the best prediction result pair were used to screen the NCI database for new Chk2 inhibitor candidates. Under the PhModel, pharmacophore hypothesis screening can be used to screen small molecule database to retrieve the compounds as potential inhibitors that fit the pharmacophoric features. In this study, the “Search 3D Database protocol” with the Best/Fast/Casear Search option in Accelrys Discovery Studio 2.1 was employed to search the NCI database with 260,071 compounds. We could filter out and select the compounds in the NCI database based on the estimated activity and chemical features of PhModel.


## 2.6. Molecular Docking

After the database screening approach, the selected compounds can be further estimated according to the interaction energy between a receptor and a ligand through the molecular docking approach. In this study, selected compounds in the NCI database were docked into Chk2 active sites by CDOCKER docking program, and then their CDOCKER interaction energies were estimated. Finally, new potential candidates were retrieved from the NCI database with high interaction energy. The workflow of database screening and molecular docking approach was shown in Figure4.Figure 4
The workflow of database screening and molecular docking approach for new Chk2 inhibitor candidates.


## 3. Results

### 3.1. PhModel Generation Results

Each of the ten PhModels using 25 training set inhibitors and HypoGen Best, Fast, and Caesar algorithms was generated by selecting hydrogen bond acceptor (A), hydrogen bond donor (D), and hydrophobic (H) and hydrophobic aromatic (HYAR) features. Each of the best PhModels, Besttrain, Fasttrain, and Caseartrain, was evaluated with the best r
train, and the predicted biological activities of training set inhibitors and r
train were listed in Table 1, respectively. From Table 1, the Besttrain obtained better r
train of value 0.955 than those by Fasttrain and Caseartrain. Moreover, the r
train of Caseartrain is far less than those of Besttrain and Fasttrain. Hence, HypoGen Best algorithm was used individually to generate the PhModels for most of target genes in the past. According to rule (a) (1) in Remark 1, the Caseartrain was not considered to be used for the prediction of testing set inhibitors.


### 3.2. Correlation Analysis of Testing Set Inhibitors

The testing set inhibitors were predicted by Besttrain and Fasttrain with HypoGen Best, Fast, and Caesar algorithms. Therefore, there are six prediction results, BesttrainBesttest (denoted as BB), BesttrainFasttest (denoted as BF), BesttrainCaseartest (denoted as BC), FasrtrainBesttest (denoted as FB), FasttrainFasttest (denoted as FF), and FasttrainCaseartest (denoted as FC), for testing set inhibitors. The predicted biological activities of testing set inhibitors and r
test by these six prediction results were listed in Table 2, respectively. From Table 2, for the Besttrain, the best r
test of value 0.81 was achieved by the BesttrainBesttest; for the Fasttrain, the best r
test of value 0.728 was achieved by the FasttrainFasttest. However, the BesttrainBesttest obtained the best r
test in overall; moreover, the prediction results in the Besttrain all outperform those in the Fasttrain.


### 3.3. Combinatorial Fusion Analysis

Under the six prediction results, the diversity score functiond
i
(
A
,
B
) was calculated for each testing set inhibitor by a pair of prediction results (
A
,
B
). There are 15 diversity score functions s
(
A
,
B
) that were performed at first and then these diversity score functions were sorted to become the diversity rank function r
(
A
,
B
), respectively. Finally, 15 diversity rank/score functions f
(
A
,
B
) were represented as diversity rank/score graphs shown in Figure 5. Among 15 diversity rank/score graphs, there are several combinations (gray color) that have less diversity scores than those by others, such as BB + BC, BB + BF, and FB + FB, shown in Figure 5. It means that these combinations may have less r
test than those by others according to rule (a) (2) in Remark 1. In other words, several combinations, such as BB + FC (purple color), BB + FF (blue color), and BF + FF (orange color), may have larger r
test than those by others due to larger diversity scores. For the six prediction results, all of the 63 combinations were preformed and evaluated by its r
test, respectively, as shown in Figure 6. In Figure 6, for 15 pairs of two prediction results, the combinations BB + FB, BB + FC, and BB + FF have larger r
test than those by others. Moreover, the combination BB + FF has best r
test of value 0.816 among 15 combinations, even for 63 combinations. Besides, the average r
test by the combinations is larger than the individual prediction results. It means that the predictive accuracy for Chk2 inhibitors may be improved by considering the Besttrain and Fasttrain concurrently.Figure 5
The diversity rank/score graphs for 15 combinations of prediction results.Figure 6
Ther
test for all of 63 combinations from six prediction results.


### 3.4. Database Screen Results

The best PhModels, Besttrain and Fasttrain, were used to screen the NCI database with 260,071 compounds for new Chk2 inhibitor candidates by using HypoGen Best and Fast algorithms, respectively. The BesttrainBesttest and FasttrainFasttest prediction results for NCI database were combined in order to filter out possible false positive candidates. Of the 260,071 compounds, 191,505 passed the screening and best fitted to the chemical features in 3D space. 23 drug-like compounds that had an estimated IC50 value of less than 2 nM were studied in a molecular docking study (Figure 4).


### 3.5. Molecular Docking Results

23 drug-like compounds along with the training set compounds were docked into the active sites that were defined based on the bound inhibitor, PV1019, in a crystal structure of Chk2 (PDB: 2W7X). We used CDOCKER program to confirm that inhibitor candidates bind to the receptor. CDOCKER uses molecular dynamics (MD) in conjunction with the CHARMm force field to individually dock the compounds into the binding sites. The coordinates of Chk2 from the Chk2/PV1019 crystal structure were used after removing PV1019 and solvent molecules and adding protein hydrogen atoms. After docking each screened compound, its interaction energy value was calculated. The PV1019 was redocked into the Chk2 binding site by the CDOCKER program. Its-CDOCKER interaction energy was calculated by CDOCKER and determined to be 37.786 (kal/mol). The 23 drug-like compounds were docked into the Chk2 binding sites. Finally, there are 21 drug-like compounds with CDOCKER interaction energies greater than 37.786 (kal/mol). In addition, 11 drug-like compounds had high interaction value greater than 50 (kal/mol) (Figure4) and the top 2 are NSC136954 with 61.239 (kal/mol) and NSC70804 with 58.967 (kal/mol), respectively, kept for future characterization as inhibitors. The 21 drug-like compounds with their estimated IC50 values and CDOCKER interaction energy greater than 37.786 (kal/mol) were shown in Table 3.Table 3
The 21 drug-like compounds with their estimated IC50values and CDOCKER interaction energy greater than 37.786 (kal/mol).
Name
Estimated IC50(nM)
Interaction energy (kal/mol)
NSC 136954
1.989
61.239
NSC 70804
1.682
58.967
NSC 158029
1.885
57.944
NSC 603427
1.87
56.963
NSC 57782
1.6855
56.54
NSC 16739
1.5385
56.342
NSC 720227
1.914
55.839
NSC 618702
1.862
55.196
NSC 195178
1.7015
51.351
NSC 653142
1.557
51.19
NSC 653143
1.577
50.055
NSC 32200
1.901
49.439
NSC 342015
1.6515
47.327
NSC 343685
1.7615
46.436
NSC 205750
1.875
45.542
NSC 96538
1.705
44.344
NSC 210455
1.7935
42.258
NSC 314654
1.947
42.082
NSC 179894
1.6135
41.707
NSC 91710
1.701
40.533
NSC 370907
1.8785
40.502The structures and characteristics of the top 2 compounds are given in Table4, and we can find that some active site residues were identified from the Chk2 complex. The interaction sites of NSC136954 were Leu226, Val234, Ala247, Lys249, Ile251, Glu273, Ile274, Leu277, Ile286, Ile288, Ile299, Leu301, Leu303, Met304, Glu305, Gly306, Gly307, Glu308, Leu354, Thr367, Asp368, Phe369, and Gly370. On the other hand, the interaction sites of NSC70804 were Leu226, Leu227, Val234, Ala247, Ile248, Lys249, Ile251, Glu273, Leu277, Ile299, Leu301, Leu303, Met304, Gly307, Glu308, Glu351, Asn352, Leu354, Thr367, and Asp368. Several studies indicated that they are involved in hydrophobic interactions with Val234, Ile251, Leu354, Ile299, and the aliphatic portions of the side chains of Lys249, Thr367, and Asp368, in addition to several interactions of van der Waals or hydrophobic with Leu226, Val234, Leu303, Gly307, Leu354, and the aliphatic portions the side chains of Met304 and Glu308 [10, 11]. Furthermore, the quinazoline was sandwiched between the lipophilic side chains of Val234 and Leu354, with the side chains of Ala247, Leu301, and Leu303 also contributing to a hydrophobic surface surrounding the core and an interaction between the pyrazole and Lys249 is likely to account for the increase in Chk2 potency [12]. And residue Thr367 of Chk2 is a serine in Chk1. Portions of the glycine-rich P-loop in Chk2, which is located directly above the inhibitor, are disordered (residues 229–231), whereas this loop is well defined in the structure of Chk1, and Leu301 in Chk2 corresponds to the “gatekeeper” residue in many kinases, which has been found to form contacts with bound inhibitors and is poorly conserved [44].Table 4
The structures and characteristics of the top 2 compounds.
NSC 136954
NSC 70804
Structure
Superposition
Binding sites
Docking results


## 3.1. PhModel Generation Results

Each of the ten PhModels using 25 training set inhibitors and HypoGen Best, Fast, and Caesar algorithms was generated by selecting hydrogen bond acceptor (A), hydrogen bond donor (D), and hydrophobic (H) and hydrophobic aromatic (HYAR) features. Each of the best PhModels, Besttrain, Fasttrain, and Caseartrain, was evaluated with the best r
train, and the predicted biological activities of training set inhibitors and r
train were listed in Table 1, respectively. From Table 1, the Besttrain obtained better r
train of value 0.955 than those by Fasttrain and Caseartrain. Moreover, the r
train of Caseartrain is far less than those of Besttrain and Fasttrain. Hence, HypoGen Best algorithm was used individually to generate the PhModels for most of target genes in the past. According to rule (a) (1) in Remark 1, the Caseartrain was not considered to be used for the prediction of testing set inhibitors.


## 3.2. Correlation Analysis of Testing Set Inhibitors

The testing set inhibitors were predicted by Besttrain and Fasttrain with HypoGen Best, Fast, and Caesar algorithms. Therefore, there are six prediction results, BesttrainBesttest (denoted as BB), BesttrainFasttest (denoted as BF), BesttrainCaseartest (denoted as BC), FasrtrainBesttest (denoted as FB), FasttrainFasttest (denoted as FF), and FasttrainCaseartest (denoted as FC), for testing set inhibitors. The predicted biological activities of testing set inhibitors and r
test by these six prediction results were listed in Table 2, respectively. From Table 2, for the Besttrain, the best r
test of value 0.81 was achieved by the BesttrainBesttest; for the Fasttrain, the best r
test of value 0.728 was achieved by the FasttrainFasttest. However, the BesttrainBesttest obtained the best r
test in overall; moreover, the prediction results in the Besttrain all outperform those in the Fasttrain.


## 3.3. Combinatorial Fusion Analysis

Under the six prediction results, the diversity score functiond
i
(
A
,
B
) was calculated for each testing set inhibitor by a pair of prediction results (
A
,
B
). There are 15 diversity score functions s
(
A
,
B
) that were performed at first and then these diversity score functions were sorted to become the diversity rank function r
(
A
,
B
), respectively. Finally, 15 diversity rank/score functions f
(
A
,
B
) were represented as diversity rank/score graphs shown in Figure 5. Among 15 diversity rank/score graphs, there are several combinations (gray color) that have less diversity scores than those by others, such as BB + BC, BB + BF, and FB + FB, shown in Figure 5. It means that these combinations may have less r
test than those by others according to rule (a) (2) in Remark 1. In other words, several combinations, such as BB + FC (purple color), BB + FF (blue color), and BF + FF (orange color), may have larger r
test than those by others due to larger diversity scores. For the six prediction results, all of the 63 combinations were preformed and evaluated by its r
test, respectively, as shown in Figure 6. In Figure 6, for 15 pairs of two prediction results, the combinations BB + FB, BB + FC, and BB + FF have larger r
test than those by others. Moreover, the combination BB + FF has best r
test of value 0.816 among 15 combinations, even for 63 combinations. Besides, the average r
test by the combinations is larger than the individual prediction results. It means that the predictive accuracy for Chk2 inhibitors may be improved by considering the Besttrain and Fasttrain concurrently.Figure 5
The diversity rank/score graphs for 15 combinations of prediction results.Figure 6
Ther
test for all of 63 combinations from six prediction results.


## 3.4. Database Screen Results

The best PhModels, Besttrain and Fasttrain, were used to screen the NCI database with 260,071 compounds for new Chk2 inhibitor candidates by using HypoGen Best and Fast algorithms, respectively. The BesttrainBesttest and FasttrainFasttest prediction results for NCI database were combined in order to filter out possible false positive candidates. Of the 260,071 compounds, 191,505 passed the screening and best fitted to the chemical features in 3D space. 23 drug-like compounds that had an estimated IC50 value of less than 2 nM were studied in a molecular docking study (Figure 4).


## 3.5. Molecular Docking Results

23 drug-like compounds along with the training set compounds were docked into the active sites that were defined based on the bound inhibitor, PV1019, in a crystal structure of Chk2 (PDB: 2W7X). We used CDOCKER program to confirm that inhibitor candidates bind to the receptor. CDOCKER uses molecular dynamics (MD) in conjunction with the CHARMm force field to individually dock the compounds into the binding sites. The coordinates of Chk2 from the Chk2/PV1019 crystal structure were used after removing PV1019 and solvent molecules and adding protein hydrogen atoms. After docking each screened compound, its interaction energy value was calculated. The PV1019 was redocked into the Chk2 binding site by the CDOCKER program. Its-CDOCKER interaction energy was calculated by CDOCKER and determined to be 37.786 (kal/mol). The 23 drug-like compounds were docked into the Chk2 binding sites. Finally, there are 21 drug-like compounds with CDOCKER interaction energies greater than 37.786 (kal/mol). In addition, 11 drug-like compounds had high interaction value greater than 50 (kal/mol) (Figure4) and the top 2 are NSC136954 with 61.239 (kal/mol) and NSC70804 with 58.967 (kal/mol), respectively, kept for future characterization as inhibitors. The 21 drug-like compounds with their estimated IC50 values and CDOCKER interaction energy greater than 37.786 (kal/mol) were shown in Table 3.Table 3
The 21 drug-like compounds with their estimated IC50values and CDOCKER interaction energy greater than 37.786 (kal/mol).
Name
Estimated IC50(nM)
Interaction energy (kal/mol)
NSC 136954
1.989
61.239
NSC 70804
1.682
58.967
NSC 158029
1.885
57.944
NSC 603427
1.87
56.963
NSC 57782
1.6855
56.54
NSC 16739
1.5385
56.342
NSC 720227
1.914
55.839
NSC 618702
1.862
55.196
NSC 195178
1.7015
51.351
NSC 653142
1.557
51.19
NSC 653143
1.577
50.055
NSC 32200
1.901
49.439
NSC 342015
1.6515
47.327
NSC 343685
1.7615
46.436
NSC 205750
1.875
45.542
NSC 96538
1.705
44.344
NSC 210455
1.7935
42.258
NSC 314654
1.947
42.082
NSC 179894
1.6135
41.707
NSC 91710
1.701
40.533
NSC 370907
1.8785
40.502The structures and characteristics of the top 2 compounds are given in Table4, and we can find that some active site residues were identified from the Chk2 complex. The interaction sites of NSC136954 were Leu226, Val234, Ala247, Lys249, Ile251, Glu273, Ile274, Leu277, Ile286, Ile288, Ile299, Leu301, Leu303, Met304, Glu305, Gly306, Gly307, Glu308, Leu354, Thr367, Asp368, Phe369, and Gly370. On the other hand, the interaction sites of NSC70804 were Leu226, Leu227, Val234, Ala247, Ile248, Lys249, Ile251, Glu273, Leu277, Ile299, Leu301, Leu303, Met304, Gly307, Glu308, Glu351, Asn352, Leu354, Thr367, and Asp368. Several studies indicated that they are involved in hydrophobic interactions with Val234, Ile251, Leu354, Ile299, and the aliphatic portions of the side chains of Lys249, Thr367, and Asp368, in addition to several interactions of van der Waals or hydrophobic with Leu226, Val234, Leu303, Gly307, Leu354, and the aliphatic portions the side chains of Met304 and Glu308 [10, 11]. Furthermore, the quinazoline was sandwiched between the lipophilic side chains of Val234 and Leu354, with the side chains of Ala247, Leu301, and Leu303 also contributing to a hydrophobic surface surrounding the core and an interaction between the pyrazole and Lys249 is likely to account for the increase in Chk2 potency [12]. And residue Thr367 of Chk2 is a serine in Chk1. Portions of the glycine-rich P-loop in Chk2, which is located directly above the inhibitor, are disordered (residues 229–231), whereas this loop is well defined in the structure of Chk1, and Leu301 in Chk2 corresponds to the “gatekeeper” residue in many kinases, which has been found to form contacts with bound inhibitors and is poorly conserved [44].Table 4
The structures and characteristics of the top 2 compounds.
NSC 136954
NSC 70804
Structure
Superposition
Binding sites
Docking results


## 4. Conclusions

In this study, a novel design strategy for drug design was proposed to apply combinatorial fusion into PhModels and virtual screening techniques. 158 Chk2 inhibitors were divided into the training set and testing set, respectively. For 25 training set inhibitors, three best PhModels, Besttrain, Fasttrain, and Caseartrain, were generated at first, and then six prediction results for 133 testing set inhibitors were used for calculating 15 diversity rank/score functions. Finally, the combination BesttrainBesttest and FasttrainFasttest prediction results achieved the best r
test of value 0.816 among 63 combinations. Through these approaches, 23 potential Chk2 inhibitors with IC50 value less than 2 nM and interaction energy value larger than 37.786 (kal/mol) are retrieved from NCI database. This study can help medicinal chemists to identify or design new Chk2 inhibitors. Besides, the potential inhibitors of Chk2 retrieved in this work can be estimated by biologists for further study.



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*Source: 359494-2014-04-23.xml*