Combining CBP Pharmacophore Construction and Molecular 1 Docking to Search for Potential Competitive Inhibitors of Chitin 2 Deacetylase 3

: Chitin deacetylase (CDA) is a key enzyme for plant pathogens to evade host defense 14 recognition. However, in the study of CDA inhibitors, only chitin deacetylase from colletotrichum 15 lindemuthianum (ClCDA) was found to participate in the reverse hydrolysis reaction in sodium acetate 16 to acetylate free amino sugar residues into N-acetylated forms. Based on this, we selected 10,632 small 17 molecules from the DrugBank database for computer virtual screening to find new potential CDA 18 inhibitors. First, we use the CBP model with ROC = 0.800 to coarsely screen small molecules. Then 19 we use the LibDock and CDOCKER programs in Discovery Studio 2016 (DS 2016) to dock the best- 20 matched small molecules to identify interactions with key residues on the active site of ClCDA. Finally, 21 we found two potential compounds with good adaptability, high docking score and important 22 interactions with protein active sites. And we confirm that their structures are stable and have multiple 23 non-bonding interactions with important amino acid sites such as ASP50, TYR145, HIS206 and 24 ZN1255 by MD simulations.


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In the long struggle between plants and pathogens, plants have evolved a highly efficient and 30 complex immune system, and pattern recognition receptors located on plant cell membrane epitopes play 31 an important role in sensing the presence of pathogens and activating immunity. Chitin is one of the 32 important components of fungal cell wall, and chitin released by pathogenic fungi in the process of 33 infecting host will be recognized by host membrane receptors to induce immune response [1][2][3]. However 34 there is cumulative evidence that fungi evade plant defense mechanisms by partially deacetylating either 35 their exposed cell wall chitin [4][5][6]. In this cases, the resulting partially deacetylated oligomers are not 36 well recognized by the specific plant receptors reducing or preventing the elicitation of the defense 37 responses [7]. Currently, antifungals targeting cell walls include β-D-glucan synthase inhibitor, chitin 38 synthase inhibitors and glycosyl-phosphatidyl Inositol (GPI) anchor pathway inhibitor [8,9]. Thus, CDA 39 represents a promising target for antifungals.

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Chitin deacetylase is one of the members of Carbohydrate esterase 4 superfamily, which can 41 hydrolyze acetyl groups of N-acetylglucosamine units of chitin and chitin oligosaccharides, thus 42 producing acetic acid and chitosan, the poor substrates of chitinase [10]. As important enzyme catalyzing 43 the conversion to chitin to chitosan, chitin deacetylase plays a very important role in agriculture and drug 44 discovery. We can seek chitin deacetylase inhibitors to block the deacetylation modification of chitin by 45 chitin deacetylase, tear apart the cunning camouflage of pathogenic fungi, expose their true state, and 46 make the organisms play a therapeutic role in their own prevention and treatment.

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Colletotrichum is a genus of soil-borne plant fungi widely distributed in tropical, subtropical and 48 temperate regions, which often infects crops and induces serious economic losses [11]. In a vote organized 49 by Molecular Plant Pathology magazine in 2012, the pathogenic fungi of the genus Anthrax were 50 promoted as the eighth most important phytopathogenic fungi in the world according to their scientific 51 significance and economic importance [12]. Among the numerous studies on chitin deacetylase, some 52 researchers have found that in 3.0 mol/L sodium acetate, ClCDA can participate in the reverse hydrolysis 53 reaction, acetylate the free amino sugar residues into N-acetylated form, and some studies have confirmed 54 that acetate plays a competitive inhibitory role in this process. This makes it possible to control plant 55 pathogens by inhibiting chitin deacetylase [13][14][15]. 56 3 With the development of computer-aided drug design, structural biology, protein crystallization and 57 resolution technology (X-ray diffraction, nuclear magnetic resonance). The use of computational 58 techniques in drug discovery and development has become the most effective method. Ligand-based 59 virtual screening of drugs can efficiently screen potential compounds from a large number of compounds 60 through the interaction between proteins and small molecule compounds, avoiding blind screening, 61 thereby reducing human, financial and time costs [16]. This study will use DS2016 software to further 62 explore competitive inhibitors of chitin deacetylase on the basis of docking acetate and other molecules 63 with chitin deacetylase. The virtual screening flow chart is shown in Fig. 1  distance was set to5.5 Å and the maximum hydrogen bond distance was set to 3.0 Å. Other parameters 87 such as 'mode' and 'docking' were set as 'fast ' and 'rigid', respectively[18]. In order to verify the 88 selectivity of the obtained pharmacophore model, ACT, NAG and NAG3 were used as active ligands of 89 CBP for model validation, and 38 compounds in the ClCDA-bait set were randomly selected as inactive 90 ligands. The optimal model was selected and virtual screening was performed using the Drugbank 91 database to identify new potential chitin deacetylase inhibitors [16,19].

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ClCDA is a member of the Carbohydrate esterase 4 superfamily, whose members share a conserved 129 region in their primary structure that is recognized as a catalytic subunit [22]. It is known that the ClCDA 130 catalytic subunit is generated from a zinc-binding triad consisting of two histidines (His104, His108) and 131 aspartate (Asp50) [22]. Therefore, we used the zinc-binding triplet as the active center for LibDock 132 docking with the substrates of ClCDA (NAG, NAG2, NAG3) and competitive inhibitors (ACT). At the 133 same time, in order to intuitively demonstrate the mechanism of action of the inhibitors, we docked the 134 substrates again while retaining the docking of the inhibitors. The docking results are shown in Table 1 135 and 2 below.  The key characteristic of a good docking program is its ability to reproduce the experimental 206 binding modes of ligands. To test this, a ligand is taken out of the X-ray structure of its protein-ligand 207 complex and docked back into its binding site. The docked binding mode is then compared with the 208 experimental binding mode, and a root-mean-square distance (RMSD) between the two is calculated; a 209 prediction of a binding mode is considered successful if the RMSD is below a certain value (

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Then, the chemical structures of the 7 compounds were analyzed by molecular structure inspection.

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The 7 complexes that ClCDA combined with 7 candidacies have more chemical bonds than ACT (show 284 in Fig. 6), which again indicates that these 7 compounds could bind with ClCDA at active site more 285 stably. In addition, DB02470 and DB03227 can interact with all the active groups ASP50, TYR145, 286 His206, Zn1255, which again demonstrates the reliability of again demonstrates the reliability of the 287 above suspected active sites, and they may contribute to competitive inhibition of activity of ClCDA.

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Finally, their stability in the natural environment were assessed performing molecular dynamics 289 simulation, and it is computational results showed that RMSD, potential energy and total energy of these 290 ligand-ClCDA complexes tend to stability with time (show in Fig. 7, 8 and 9), which suggested that these 291 2 complexes could exist in the natural environment stably. Molecular dynamics module computation 292 confirmed that RMSD of DB03227 and DB02470 were obviously lower than the reference ligand ACT, 293 which demonstrates these 2 compounds may have a higher stability with ClCDA compared with ACT.