Single letter amino acid sequence for
Abstract
In this study, we determined the 3D structure of Arabidopsis thaliana KAPAS by homology modeling. We then investigated the binding mode of compounds obtained from the in-house library using computational docking methods. From the flexible docking study, we achieved high dock scores for the active compounds denoted in this study as compound 3 and compound 4. Thus, we highlight the flexibility of specific residues, Lys 312 and Phe 172, when used in active sites.
Keywords
- KAPAS
- Herbicides
- Homology modeling
- Protein docking
1. Introduction
Agricultural research efforts for discovery of herbicides acting on new target sites are increasing due to demand from farmers and multinational companies in many countries. However, new modes of action have not been turned to commercial success for the past 10 years. We have recently reported 7-keto-8-aminopelargonic acid synthase (KAPAS, also known as 8-amino-7-oxononanoate synthase, ANONS) and have suggested the potential KAPAS inhibitor triphenyltin. Research on herbicides has advanced during the past 50 years to the point that herbicides can now protect crops and improve the quality and quantity of agricultural products. However, the successful development of herbicides has decreased recently owing to new environmental regulations and lack of discovery. To overcome this problem, there is an urgent need for new herbicidal targets and new techniques [1].
While the traditional approach to discover lead compounds heavily depends on serendipity given the poor understanding of biological modes of actions, the structure-based approach utilizes the structure of appropriate target proteins, which have well-known binding sites for possible rational designs. The introduction of new herbicides with either a new mode of action or of a novel chemical class has lingered. However, discovering a chemical structure that could enter the pest, be transported within it, inhibit a key target, get away from detoxification, and also be modified to allow it to fulfill increasing regulatory criteria with respect to environmental compatibility has been required. The structure-based approach uses only appropriate target proteins instead of the entire plant for
Several enzymes in plants are known to be essential enzymes, meaning that they are crucial for the plant’s survival. Disrupting a single essential enzyme leads to severe disorder of metabolic processes in the plant, ultimately causing a lethal outcome. The enzyme 7-keto-8-aminopelargonic acid synthase from
Although the physiological systems of humans and plants are different in various ways, the misuse of agricultural chemicals can be extremely harmful to humans. Therefore, use of herbicides must follow strict toxicity regulations that are in place to prevent harm to humans and other life. As mentioned above, the novel herbicidal target 7-keto-8-aminopelargonic acid synthase functions in the initial steps of the biosynthetic pathways of biotin (vitamin H) in plants and microorganisms. Because biosynthetic steps of biotin exist only in plants, we expect that the inhibition of the potent target
In this chapter, we aim to obtain potential
2. Building a homology model of AtKAPAS
To apply the structure-based drug design (SBDD) method using current knowledge of protein and drug interactions, a three-dimensional protein structure is necessary [4]. Because the known protein crystal structural information of 7-keto-8-aminopelargonic acid synthase from an experiment was absent, a homology model of
10 | 20 | 30 | 40 | 50 | 60 |
madhswdktv | eeavnvlesr | qilrslrpic | Msrqneeeiv | ksranggdgy | evfdglcqwd |
70 | 80 | 90 | 100 | 110 | 120 |
rtsvevsvsi | ptfqkwlhde | psngeeifsg | dalaecrkgr | fkklllfsgn | dylglsshpt |
130 | 140 | 150 | 160 | 170 | 180 |
isnaaanavk | eygmgpkgsa | licgyttyhr | Llesslaqlk | kkedclvcpt | gfaanmaamv |
190 | 200 | 210 | 220 | 230 | 240 |
aigsvaslla | asgkplknek | vaifsdalnh | Asiidgvrla | erqgnvevfv | yrhcdmyhln |
250 | 260 | 270 | 280 | 290 | 300 |
sllsnckmkr | kvvvtdslfs | mdgdfapmee | lsqlrkkygf | llviddahgt | fvcgengggv |
310 | 320 | 330 | 340 | 350 | 360 |
aeefnceadv | dlcvgtlska | agchggfiac | skkwkqliqs | rgrsfifsta | ipvpmaaaay |
370 | 380 | 390 | 400 | 410 | 420 |
aavvvarkei | wrrkaiwerv | kefkelsgvd | Isspiislvv | gnqekalkas | ryllksgfhv |
430 | 440 | 450 | 460 | 470 | 480 |
mairpptvpp | nscrlrvtls | aahttedvkk | Litalsscld | fdntathips | flfpkl |
To obtain the 3D-structure of
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|
|
1FC4 | 36.3 | 1.83 |
1DJE | 39.5 | 1.66 |
2BWO | 39.6 | 1.59 |
2WKA | 32.2 | 2.42 |
3KKI | 30.3 | 2.64 |
3DXV | 16.6 | 3.26 |
3DXW | 16.8 | 3.25 |
2OAT | 15.7 | 3.38 |
1GBN | 15.6 | 3.43 |
1MLY | 15.5 | 3.51 |
To identify the binding mode of
3. Rigid docking
As a structure-based drug design, we used an automated docking method [16]. To dock the compounds as shown in Table 3 into the protein active site, we used the rigid docking method implemented in Discovery Studio 3.1 (Accelrys, Inc.), which adopts a Monte-Carlo algorithm to generate ligand conformations and docks the generated ligands into the active site using a shape-based filtering method. The rigid docking process consists of two main steps: defining a binding cavity and docking ligands onto the defined cavity [17]. The
4. Flexible docking
Various docking methods are utilized by researchers. Each approach was developed by focusing on different aspects of docking. One of the factors determining the accuracy of docking is protein flexibility. Much emphasis has been placed on the conformational changes of protein binding sites, where different ligands form interactions. The Flexible Docking protocol of Discovery Studio 3.1 allows receptor flexibility during the docking of ligands [18-20]. To confirm the flexibility of the selected residues in the
5. In Vitro assay
Pimeloyl CoA was synthesized according to a previously described method [21].
6. Results and discussion
Interesting results were obtained from the docking processes undertaken in this study. The rigid docking output scores of the in-house compounds are shown in Table 4. The most active compounds, in this case compound 3 and compound 4, obtained high dock scores of 104.21 and 105.47, respectively. Moreover, active ligands which have IC50 (μM) values 1.07 and 1.26 (compound 15 and compound 16, respectively) formed a stable docking pose (Fig. 2) with high dock scores 106.03 and 72.5, respectively. However, other active compounds, specifically compound 1, compound 9, and compound 10, showed rather low dock scores, as shown in Table 3.
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1 | 40.40 | 5.48 | 10 | 45.79 | 4.95 |
2 | 67.64 | 5.36 | 11 | 49.91 | 4.38 |
3 | 104.21 | 6.23 | 12 | 63.13 | 5.68 |
4 | 105.47 | 6.20 | 13 | 52.84 | 5.02 |
5 | 55.73 | 4.25 | 14 | 52.99 | 5.36 |
6 | 42.30 | 4.27 | 15 | 106.03 | 5.97 |
7 | 50.67 | 4.39 | 16 | 72.51 | 5.90 |
8 | 51.67 | 4.33 | 17 | 52.31 | 5.36 |
9 | 58.72 | 4.95 |
To obtain a better docking result, we used a flexible docking strategy, as mentioned in the experimental section. In the result for flexible docking 1, which stipulated that the Lys 312 residue of the
According to the docking result, the diverse conformation of Lys 312 directly affects the pose of the ligands and the related activity. The Lys 312 residue forms a hydrogen bond or undergoes the π-cation interaction mostly with the oxygen moiety of the ligand by flexibly moving through the protein cavity (Fig. 2). Interestingly, we obtained relatively high dock scores for compound 1, compound 9, and compound 10. This was unobtainable with the rigid docking process. The correlation coefficient between pIC50 and the dock score for the flexible docking 1 set was 0.72, as shown in Fig. 3.
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1 | 74.00 | 5.48 |
2 | 83.14 | 5.36 |
3 | 134.03 | 6.23 |
4 | 125.27 | 6.20 |
5 | 53.40 | 4.25 |
6 | 50.83 | 4.27 |
7 | 59.46 | 4.39 |
8 | 54.40 | 4.33 |
9 | 105.54 | 4.95 |
10 | 76.74 | 4.95 |
11 | 74.36 | 4.38 |
12 | 83.56 | 5.68 |
13 | 76.92 | 5.02 |
14 | 80.04 | 5.36 |
15 | 131.67 | 5.97 |
16 | 96.88 | 5.90 |
17 | 75.41 | 5.36 |
By flexibly moving the residue Phe 172 while the flexible docking 2 process was underway, a better result than the flexible docking 1 process was obtained (Table 6). The flexibility of the Phe 172 residue has a significant effect on the ligand binding at the
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|
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1 | 75.60 | 5.48 |
2 | 85.84 | 5.36 |
3 | 119.05 | 6.23 |
4 | 113.06 | 6.20 |
5 | 48.11 | 4.25 |
6 | 44.80 | 4.27 |
7 | 53.49 | 4.39 |
8 | 49.60 | 4.33 |
9 | 72.97 | 4.95 |
10 | 74.43 | 4.95 |
11 | 57.66 | 4.38 |
12 | 85.24 | 5.68 |
13 | 79.99 | 5.02 |
14 | 67.73 | 5.36 |
15 | 113.29 | 5.97 |
16 | 90.84 | 5.90 |
17 | 65.49 | 5.36 |
The correlation coefficient between pIC50 and the dock score for the flexible docking 2 set was 0.85, as shown in Fig. 4.
The two specific
|
|
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1 | 71.47 | 5.48 |
2 | 95.12 | 5.36 |
3 | 120.48 | 6.23 |
4 | 119.46 | 6.20 |
5 | 40.92 | 4.25 |
6 | 40.03 | 4.27 |
7 | 54.90 | 4.39 |
8 | 61.54 | 4.33 |
9 | 80.87 | 4.95 |
10 | 72.13 | 4.95 |
11 | 75.00 | 4.38 |
12 | 78.83 | 5.68 |
13 | 74.82 | 5.02 |
14 | 73.04 | 5.36 |
15 | 124.28 | 5.97 |
16 | 91.92 | 5.90 |
17 | 70.70 | 5.36 |
7. Summary
In this study, we determined the 3D-structure of
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