A Novel Scaffold for Developing Specific or Broad-Spectrum Chitinase

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A Novel Scaffold for Developing Specific or Broad-spectrum Chitinase Inhibitors Xi Jiang, Ashutosh Kumar, Tian Liu, Kam Y.J. Zhang, and Qing Yang J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00615 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 17, 2016

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Journal of Chemical Information and Modeling

A Novel Scaffold for Developing Specific or Broadspectrum Chitinase Inhibitors Xi Jiang,†, ‡,⊥ Ashutosh Kumar,§,⊥ Tian Liu,†,‡ Kam Y. J. Zhang,*,§ and Qing Yang*,†,‡

†

State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China

‡

School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China

§

Structural Bioinformatics Team, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan

1 ACS Paragon Plus Environment


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ABSTRACT

Chitinases play important roles in pathogen invasion, arthropod molting, plant defense and human inflammation. Inhibition of the activity of a typical chitinase by small molecules is of significance in drug development and biological research. Based on a recent reported crystal structure of OfChtI, the insect chitinase derived from the pest Ostrinia furnacalis, we have computationally identified 17 compounds from a library of over 4 million compounds by two rounds virtual screening. Among these compounds, 3 compounds from one chemical class inhibited the activity of OfChtI with single-digit micromolar IC50 values and 1 compound from another chemical class exhibited a broad inhibitory activity not only toward OfChtI but also toward bacterial, fungi and human chitinases. A new scaffold was discovered and the structureinhibitory activity relationship was proposed. This work may provide a novel starting point for developing specific or broad-spectrum chitinase inhibitors.


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INTRODUCTION Chitinase (EC 3.2.1.14) is an enzyme that catalyzes the degradation of chitin, a β-1,4-linked N-acetylglucosamine (GlcNAc) polymer, which is a main structural component of exoskeletons in arthropods and fungal cell walls.1,

2

Chitinases have various physiological functions in

different species. Bacterial chitinases are linked to metabolism and antifungal activity.3, Protozoan pathogens use chitinases as virulence enhancers against parasitic hosts.5, chitinases act in defense against biotic or abiotic stress.7,

8

6

4

Plant

In insects, chitinases are the key

enzymes for remodeling chitin-containing tissues, including the cuticle, trachea and peritrophic membrane.9 RNA interference-mediated suppression of insect chitinases resulted in severe molting defects and lethality.10 Even though human beings cannot synthesize chitin, we have two chitinases, acidic mammalian chitinase and macrophage chitotriosidase, which are involved in the pathogenesis of asthma, pulmonary mycoses and many other immunologic responses and disorders.11-13 Chitinase inhibitors have considerable interest for use as potential insecticides, fungicides and medicines to fight microbial infections.14-17 Although several small molecule chitinase inhibitors have been reported, such as xanthine and purine derivatives as the fungal chitinase AfChiB1 inhibitors and acetazolamide as an AfChiA1 inhibitor, their inhibitory activities are relatively weak.18-21 Most potent chitinase inhibitors are derived from natural products, such as cyclic peptides (argifin22 and argadin,23 cycle-(L-Arg-D-Pro),24 styloguanidine25 and psammaplin26) and pseudotrisaccharide allosamidin.27 These inhibitors and their derivatives are hampered from wide-spread use due to their complexity of synthesis.28 Thus, a novel, easily synthesizable scaffold will be of great value.



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In silico analysis is an alternative approach that has been used to obtain chitinase inhibitors. Based on the crystal structures of xanthine derivatives obtained from high-throughput screening, Schüttelkopf. et al designed a fungal chitinase inhibitor, bisdionin C, with a submicromolar IC50 value.29 They then solved the crystal structure of AfChiB1 complexed with bisdionin C to guide future modifications. Due to the increasing number of crystal structures being reported, structure based virtual screening (SBVS) has become an efficient and reliable way to obtain lead compounds.30-36 Here we utilized the recently reported crystal structure of OfChtI37 in complex with a bound ligand (GlcNAc)3 to obtain potent small molecule inhibitors. OfChtI is a chitinase derived from the seriously destructive crop pest, Ostrinia furnacalis, also known as Asian corn borer. Homologs to OfChtI in various insect species are indispensable for molting. By using SBVS combined with bioactivity assay, a new scaffold of chitinase inhibitors, 1-thia-7-aza-2indenecarboxamide (TAI), has been discovered. Subsequently, we have evolved this scaffold into two chemical series of chitinase inhibitors, furo[2,3-b]quinoline-2-carboxamide (FQ) and 5,6,7,8-tetrahydrothieno[2,3-b][1,6]naphthyridin-6-ium-2-carboxamide (TP) series. FQ series specifically inhibit OfChtI, and TP series have a broad inhibitory activity toward chitinases from insect, human, fungi and bacterium. This work provides a starting point for the future development of novel chemicals against chitinase-related disease and pathogens.

MATERIALS AND METHODS Chemicals. Compounds TP1-14 and FQ1-3 were purchased from ChemDiv (USA). The purity of these compounds was verified by the vendor to be more than 95 %. All compounds were maintained as DMSO stock solution.


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Protein Expression and Purification. The catalytic domain of OfChtI (OfChtI-CAD) and human chitotriosidase (HsCht)38 were expressed in the yeast strain Pichia pastoris and purified using metal-chelating chromatography as described previously.39 Bacterial chitinases SmChiA and SmChiB40 derived from Serratia marcenses and the fungal chitinase AfChiB41 from Aspergillus fumigatus were expressed in the Escherichia coli strain BL21(DE3) and also purified using metal-chelating chromatography. All of the expressed proteins were quantified using a BCA protein assay kit (TaKaRa Biotech, China) and their purities were determined by SDSPAGE. Enzymatic Assay. Enzymatic activity was determined using 4-methylumbelliferyl-N,N'diacetyl-β-D-chitobioside (MU-β-(GlcNAc)2) (provided by Prof. Jianjun Zhang at China Agricultural University) as a substrate. The reaction mixtures used for inhibitor screening consisted of 100 µl of 10 nM enzyme, 50 µM MU-β-(GlcNAc)2, 100 µM inhibitors and 2% DMSO in a 20 mM sodium phosphate buffer (pH 6.5). The reaction in the absence of inhibitors was used as a positive control. After incubating at 30 °C for 30 minutes, 0.5 M sodium carbonate was added to the reaction mixture, and the fluorescence produced by the released 4-MU was quantified using a Varioskan Flash microplate reader (Thermo Fisher Scientific) using excitation and emission wavelengths of 360 and 450 nm, respectively. Experiments were performed in triplicate. For IC50 determination, the inhibitor concentrations were varied in the above reaction and the amount of 4-MU released was quantified as above. IC50 values were obtained by curve fitting using Prism software (GraphPad). Shape similarity calculations. Molecular conformations for shape similarity calculations were generated using Omega program.42, 43 A maximum of 200 conformations per compound were generated using 0.5 Å root mean square distance cutoff. ROCS program44, 45 was employed



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to perform the ligand three-dimensional (3D) shape similarity calculations. Initially, all conformers of compounds in the screening library were aligned onto the query molecule. Chemical features encoded by “colors” in ROCS were then used to refine the shape-based superpositions. Shape overlaps between two molecules were determined using TanimotoCombo similarity metric, which is a combined Tanimoto coefficient46 for shape and chemical feature overlap. The conformer with the highest TanimotoCombo score for a particular compound was selected and all the compounds were further ranked based on their TanimotoCombo scores in a descending order. Electrostatic potential similarity calculations. ROCS calculations were performed with “eon_input” flag to prepare input files for electrostatic potential similarity calculations using EON program.47 The similarity calculations were performed only on selected ROCS hits and their conformational ensemble in pre-aligned conformation with the query molecule was used. Partial charges were added to molecules based on MMFF forcefield.48, 49 ET_Combo score, a combined score for shape and electrostatic overlap was used to rank order compounds. Molecular docking. Protein structures for molecular docking calculation were prepared using the protein preparation utility in Maestro.50 OfChtI crystal structure in complex with (GlcNAc)3 (PDB code 3WL1) was used for molecular docking calculations.37 To prepare protein structure, hydrogen atoms were added, bond orders were assigned and the protonation states of all charged residues were determined. All water molecules except those within 4.5 Å vicinity of (GlcNAc)3 were removed. Ligands for molecular docking were prepared using a collection of tools integrated within LigPrep program.51 Hydrogen atoms were added while the ionization and tautomeric states of ligands at a target pH of 7.0 ± 2.0 were generated using Epik.52, 53 The 3D geometry of each compound was optimized using OPLS-2005 forcefield.54 Shape and properties


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of protein binding pocket were mapped on a grid using Maestro’s receptor grid preparation utility. The grid box dimensions were determined based on the coordinates of a bound ligand (GlcNAc)3 in OfChtI crystal structure. Inner grid box of 10×10×10 Å and outer grid box of 27×27×27 Å was used. Grid was generated without enforcing any constraints. Virtual screening workflow of Glide program55-58 was used to dock hits obtained after shape and electrostatic similarity calculations. Molecular docking using Glide was performed in hierarchical manner. Initially, Glide in standard precision (SP) mode was used to generate several poses/states (ionization and tautomeric state) per compound and all the high scoring poses/states were retained. These poses/states were then redocked using Glide in extra precision (XP) mode and only the best scoring pose/state per compound was retained. Compounds were ranked based on Glide-XP scoring function.

RESULTS AND DISCUSSION TP and FQ series obtained by structure-based virtual screening. In order to discover small molecule inhibitors, we used two rounds of virtual screening (Figure 1). A subset of over 4 million commercially available small molecules from ZINC screening database59, 60 including compounds from ChemDiv, Chembridge, Enamine, Matrix and Maybridge were used for virtual screening. This subset was selected because of their on-shelf availability from a local distributor. Moreover, this subset was found to possess similar physicochemical properties as chitinase inhibitors obtained from ChEMBL database61 (Supporting information Figure S1). The virtual screening protocol involved the identification of small molecules that had similar shape and electrostatic properties to β-1,4-linked N-acetylglucosamine (GlcNAc)3 units, the shortest oligosaccharide substrate a chitinase can recognize. The bioactive conformation of (GlcNAc)3,



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which is the co-crystallized ligand in OfChtI crystal structure (PDB code 3WL1), was utilized for the shape and electrostatic comparisons (Supporting information Figure S2A). ROCS program was used to perform the shape matching calculations and compounds were rank-ordered based on TanimotoCombo scores. The top ranking 1000 compounds displayed TanimotoCombo scores ranging from 0.907 to 0.730. Although, the overall TanimotoCombo scores were found to be low for these 1000 top ranking hits but the shape overlap (ShapeTanimoto) was consistently higher (more than 0.6 for most of the compounds) (Supporting information Figure S3). Low chemical feature overlap (ColorTanimoto) between the sugar moiety in the query and screening compounds dragged the TanimotoCombo down. However, there was reasonable chemical feature overlap for some of the compounds and hence we relied on TanimotoCombo scores to rank-order compounds in the screening library. It was interesting to see that by employing shape similarity we were able to obtain not only sugar-based compounds but also compounds that were chemically different from the query molecule. The three-dimensional overlay of a few topranking hits with significantly different chemical structure but reasonable shape overlap has been given as supporting information Figure S2. To complement shape similarity calculations, the highest ranking 1,000 compounds were then subjected to electrostatic comparison using EON program. This hierarchical filtering of virtual screening library has been successfully used previously.32, 33, 62 Compounds were sorted based on decreasing ET_Combo scores and the top ranking 500 compounds with ET_Combo scores were selected for further analysis. The ET_Combo score of the highest rank compound was found to be 1.255 that demonstrates reasonable electrostatic similarity. To further prioritize these 500 hits for the evaluation of chitinase inhibitory activity, molecular docking was used to analyze the hits for their energetic and geometric complementarity with the OfChtI active site.


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The docking was performed at a non-reducing end, specifically, at -3 to -1 subsites of OfChtI. Glide-XP scoring functions were used to rank-order these hits and the top 10% compounds were analyzed for chemical diversity and their interactions with OfChtI active site. Hierarchical clustering of docking prioritized hits using MACCS structural keys resulted in 15 clusters. The largest cluster contained 12 molecules of thienopyridine-based chemical scaffold (Supporting information Table S1). It was interesting to see that seven of these molecules were identified in the top 10 docking solutions. The enrichment of this scaffold was not due to the quality of our screening library as a substructure search for this thienopyridine-based scaffold could retrieve only 0.02 % compounds from the screening library. The most probable reason other than shape and electrostatic similarity for the enrichment of this scaffold would be hydrophobic and stacking interactions formed by the three rings in thienopyridine-based scaffold with several aromatic residues (Tyr30, Trp34, Phe61, Trp107, Tyr217, Try272 and Trp372) in OfChtI active site. Finally, eight compounds including the two highest scoring compounds from the largest cluster and six additional compounds representing other clusters were selected for biological validation (Supporting information Table S2). Evaluation of OfChtI inhibitory activity of the selected hits resulted in the identification of TP1 and TP2 compounds representing a novel scaffold with weak OfChtI activity. Both compounds displayed inhibition rates between 20-30 % at 100 μM compound concentration (Table 1 and Supporting information Table S2). Although this weak inhibition was unappealing but thienopyridine-based scaffold represents a novel compound class with weak OfChtI activity and to the best of our knowledge it has never been previously described to possess activity against any chitinase. To identify compounds with improved OfChtI inhibition, another round of virtual screening was carried out following a similar protocol. This time we were only interested



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in identifying compounds structurally similar with the thienopyridine scaffold, hence TP2 that possesses slightly better activity than TP1 was used as a query for shape and electrostatic similarity calculations. Compound prioritization using molecular docking followed by similarity calculations enabled us to select another set of 15 compounds (TP3-TP14 and FQ1-3). These compounds can be classified into two series of compounds, TP and FQ. The major structural difference between TP and FQ series lies in part I, in which TP series possesses a piperidine ring while FQ possesses a benzene ring (Figure 2). These compounds were then tested for inhibition of OfChtI activity. Their inhibition rates were determined from four independent experiments and are presented as mean ± SD in Table 1. It can be seen that most compounds within TP series displayed a similar level of activity except compound TP3 for which around 50 % OfChtI inhibition rate at 100 µM concentration was observed. However, the OfChtI inhibitory activity of TP3 is decreased to 8 % at 20 µM concentration of TP3 (Table 1). Structural comparison among the TP series compounds indicated no substantial changes in the activity due to substitutions at R2 position. Unlike TP series compounds, all the three FQ compounds (FQ1-3) inhibited over 90% enzymatic activity of OfChtI at a compound concentration of 100 µM. FQ3 exhibited the highest activity, with an inhibition rate of over 97.8% and 88.6% at compound concentrations of 100 µM and 20 µM, respectively. The inhibitory activities of TP3 and FQ1-3 were confirmed by testing them at varying concentrations (Table 1 and Figure 3). The IC50 values of TP3, FQ1, FQ2 and FQ3 toward OfChtI were determined to be 101±14.9 µM (Figure 3A), 10.3±1.4 µM (Figure 3B), 9.1±0.7 µM (Figure 3C) and 6.4±0.5 µM (Figure 3D), respectively. Taken together, FQ series compounds are most active in inhibiting the activity of OfChtI. Inhibitory specificities of TP and FQ series of compounds against diverse chitinases. To evaluate the inhibitory specificity of TP and TQ series, we tested their potency toward chitinases


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from diverse species. Four representative chitinases were chosen, which included SmChiA and SmChiB from the bacterium Serratia marcescens, AfChiB1 from the fungi Aspergillus funigatus and HsCht from human. The percent sequence identities between OfChtI, SmChiA, SmChiB, AfChiB1 and HsCht are given as supporting information Table S3. As shown in Figure 4, unlike their moderate activities against OfChtI, TP series exhibited much higher activity toward all the four tested enzymes. A majority of compounds in the TP series exhibited over 80% inhibitory rate toward SmChiB, suggesting SmChiB is sensitive to compounds with a TP scaffold structure. Among TP series, TP11 showed the highest activity toward all of the four enzymes, SmChiA (63%), SmChiB (94%), AfChiB1 (83%) and HsCht (68%), most likely due to a flexible phenethyl group at the R2 position facilitating inhibitor binding to the active pocket. The structural comparison between TP3 and TP6 indicated that benzene is better than cyclohexane for R2. Structural comparison among TP series compounds indicated that changes in chirality of R1 or property of R2 did not impair inhibitory ability towards these enzymes. Surprisingly, FQ series compounds showed lower activities toward the four enzymes with inhibitory rates between 2050%, meaning the four tested enzymes were not sensitive to compounds with a FQ scaffold. The IC50 values of TP3 and TP11 against SmChiA, SmChiB, AfChiB and HsCht were determined and compared with IC50 values for OfChtI (Table 2), which revealed FQ3 with an IC50 value of 6.4 µM was the most potent compound to inhibit OfChtI and both TP3 and TP11 with an IC50 value of 11.3 µM were efficient inhibitors against SmChiB. Taken together, FQ series showed a high specificity to inhibit OfChtI, while TP series showed broad activities toward all tested chitinases including OfChtI. Structure-function relationship analysis. In order to gain insights into the differences in inhibitory activity and specificity of TP and FQ series against various chitinases, structure-



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activity relationship and docking predicted binding modes in OfChtI, SmChiA, SmChiB, AfChiB and HsCht active site were analyzed. According to glycosyl hydrolase nomenclature, the active site in various chitinases has been labeled into different subsites from −n to +n.63 The –n represents the non-reducing end while reducing end is represented by +n subsites. The nonreducing end of substrate binds through –n subsites. Cleavage of substrate always takes place between the −1 and +1 subsites and processed substrate is expelled from +n subsites. The target region of our molecular docking calculations was based on (GlcNAc)3 position in OfChtI crystal structure (PDB code 3WL1) that occupies -3 to -1 subsites. Among the TP series compounds, only TP3 could exhibit moderate inhibition of OfChtI enzymatic activity. TP3 possessed isopropyl group at R1 position that seems a preferred functional group as compounds with methyl and ethyl substitutions at this position were weak inhibitors (TP1, TP2 and TP4-TP14). Predicted binding mode of compound TP3 suggests a binding mode where the three ring thienopyridinebased scaffold is held in the active site via stacking and hydrophobic interactions with several aromatic residues including Tyr30, Trp34, Phe61, Trp107, Tyr272, Phe309 and Trp372 (Figure 5A). The isopropyl group at R1 position is placed on top of the deep subpocket at -1 subsite where piperidine nitrogen forms a hydrogen bond with a conserved residue Glu148. No changes in OfChtI inhibitory activity due to substitutions at R2 position in TP series suggest the exposure of R2 towards solvent, which is in accordance with the predicted binding mode. All TP series compounds on the other hand possessed greater inhibitory activity against SmChiB. Sequence alignment of OfChtI, SmChiA, SmChiB, AfChiB and HsCht proteins suggest that there is less variability in amino acids at -n subsites as compared to +n subsites (Supporting information Figure S4 and S5). Significant differences in activity of TP series compounds between OfChtI and SmChiB with minimum variability suggest an alternative binding mode for


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these compounds. GlcNAc as well as chitinase inhibitors such as allosamidin are known to bind at both reducing and non-reducing end of chitinase active site. Docking of most potent TP series compounds (compound TP3, TP7 and TP11) to SmChiB indeed suggest the binding of these compounds occupying +1 and +2 subsites (Figure 5B). These compounds are predicted to bind through hydrophobic contacts with Phe190, Phe191, Tyr214, Leu216 and Leu265 (Phe194, Arg195, Tyr217, Leu219 and Leu246 in OfChtI). Compound TP11, which possesses a phenethyl group at the R2 position demonstrated highest activity against SmChiB. Due to flexible nature of this group, TP11 can form stacking interactions with Tyr240. Moreover, the substitution of cyclohexane is not preferred at R2 position and its substitution lead to decrease in the activity of compound TP6 because of the loss of hydrophobic contacts. Molecular docking predicted the placement of alkyl groups on the piperidine ring deep into -1 subsite that may be responsible for their higher activities in SmChiB as compared to OfChtI. An analysis of pocket size and volume of all crystal structures of OfChtI, SmChiA, SmChiB, AfChiB and HsCht proteins revealed that the active site in SmChiB is comparatively large and varies a lot than that of other four chitinases (Figure 5C). TP series compounds may fit better in the larger and more flexible SmChiB active site to block binding of substrate molecule. OfChtI active site pocket on the other hand is least flexible, which is also reflected in lower inhibitory potencies of TP series compounds. It was interesting to see that all FQ series compounds were selective for OfChtI while no activity was found against other chitinases. FQ series compounds (FQ1-3) possessed a benzene ring in place of piperidine ring in TP series compounds (Figure 2). As no hydrogen bond with Glu148 was possible in the absence of piperidine nitrogen, FQ series compounds (FQ3 shown here) adopted slightly different binding mode where the amide nitrogen formed a hydrogen bond with Glu148 sidechain (Figure 5D). In this position, FQ series compounds are predicted to occupy both -n



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and +n subsites, which may be responsible for their higher activity in comparison with TP series compounds. In contrast, similar binding mode could not be obtained for SmChiA, AfChiB1 and HsCht due to clashes with several variable residues at the reducing end of active site. For example, OfChtI Leu219 is replaced by Phe, Tyr and Phe in SmChiA, AfChiB1 and HsCht respectively while OfChtI Phe194 is changed to Asp in SmChiA and AfChiB1 and Thr in HsCht (Supporting information Figure S4). These subtle differences in active site may be responsible for the OfChtI selectivity of FQ series compounds. In comparison to other three chitinases, the active site of SmChiB is much more similar to OfChtI (Supporting information Figure S4), which may be responsible for some weak inhibitory activity of FQ series compounds against SmChiB (Figure 4).

CONCLUSION This work reported a novel chemical scaffold for designing specific or broad-spectrum chitinase inhibitors by structure-based virtual screening followed by enzymatic activity determination. The molecular docking further confirmed the structure-function relationship. Our shape and electrostatics matching strategy has been shown to be effective in the identification of small molecule inhibitors of OfChtI based on its crystal structure with a bound oligosaccharide substrate. The FQ series compounds are the first selective small molecule inhibitors of OfChtI reported to date. This work provides a new starting point for developing chitinase inhibitors.


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FIGURES

Figure 1. The virtual screening strategy of the first round was used to filter out TP2 and the second round was used to filter out compounds TP3-14 and FQ1-3. Programs ROCS and EON were used for shape matching and electrostatic comparison, respectively.



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Figure 2. The scaffold structures of TP series and FQ series.


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Figure 3. Determination of IC50 values of TP3, FQ1, FQ2 and FQ3 against OfChtI. (A) TP3. (B) FQ1. (C) FQ2. (D) FQ3. Error bars showed the standard deviations from three independent assays.



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Figure 4. Percent inhibition rate of compounds TP1-TP14, FQ1-FQ3 against five chitinases. All compounds were tested at 100 µM compound concentration.


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Figure 5. (A) Docking predicted binding mode of compound TP3 in OfChtI active site. (B) Docking predicted binding mode of compound TP3 (green), TP7 (magenta) and TP11 (yellow) in SmChiB active site. (C) Comparison of active site volumes in all available crystal structures of OfChtI, SmChiA, SmChiB, AfChiB1 and HsCht. (D) Docking predicted binding mode of compound FQ3 in OfChtI active site.



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TABLES.

Table 1. Inhibition rate of compounds TP1-TP15, FQ1-FQ3 against OfChtI.

Compounds

a

Inhibition rate (%)

IC50 ± SD

Structures (Mean ± SD) 100µM

(µM)

20µM

TP1 21.2 ± 2.9 1.5 ± 0.2

ND

25.5 ± 0.8 3.6 ± 0.1

ND

49.5 ± 1.1 8.3 ± 1.7

101 ± 14.9

21.8 ± 1.8 6.6 ± 1.8

ND

9.2 ± 2.4

0.0

ND

12.1 ± 2.7

0.0

ND

19.4 ± 1.0

0.0

ND

ZINC06737142

TP2 ZINC19852993 TP3 ZINC20289855 TP4 ZINC20289934 TP5 ZINC20289955 TP6 ZINC19828439 TP7 ZINC06737148 TP8 23.4 ± 1.2 3.1 ± 1.2 ZINC06697399


ND

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TP9 19.4 ± 0.7 1.8 ± 0.0

ND

26.6 ± 2.9 4.6 ± 0.4

ND

30.8 ± 2.3 1.5 ± 0.2

ND

26.2 ± 3.0 2.2 ± 1.7

ND

21.3 ± 0.3 2.8 ± 1.2

ND

28.3 ± 0.1 3.1 ± 1.2

ND

93.5 ± 0.666.0 ± 0.5

10.3 ± 1.4

94.8 ± 1.180.0 ± 0.6

9.1 ± 0.7

97.8 ± 1.188.6 ± 4.0

6.4 ± 0.5

ZINC12416777 TP10 ZINC20289886 TP11 ZINC06697323 TP12 ZINC20289911 TP13 ZINC06737147 TP14 ZINC19683213 FQ1 ZINC09528753 FQ2 ZINC04908670 FQ3 ZINC00429158 a

The ZINC number of TP series and FQ series.



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Table 2. IC50 values of TP3, TP11 and FQ3 for chitinases from different organisms.

IC50 (µM) Organism Enzyme

TP3

TP11

FQ3

Insect

OfChtI

101.9±14.9 ND

Human

HsCht

54.6±7.2

Fungi

AfChiB1 92.9±25.5

51.8±5.8 ND

Bacteria

SmChiA

69.0±13.6

45.9±5.9 ND

SmChiB

11.5±1.1

11.3±0.8 ND

6.4±0.5

67.6±8.0 ND

ND, not determined because the compound showed less than 50% inhibition at 100 µM.

ASSOCIATED CONTENT Supporting Information Five additional figures: (1) Comparison of physicochemical properties of the virtual screening library with known chitinase inhibitors. (2) A 3D shape overlay of query and a few top ranking hits. (3) A plot showing the ROCS scores for the top ranking 1000 hits. (4) Sequence alignment of insect (OfChtI), bacterial (SmChiA, SmChiB), fungal (AfChiB1) and human (HsCht) chitinases. (5) A surface plot of OfChtI active site colored by residue conservation. Three additional tables: (1) Structures, ranks and docking scores of thienopyridine-based compounds in the most populated cluster. (2) Inhibition rates of the initial screening hits against OfChtI. (3) Sequence percent identity between chitinases from different organisms. This material is available free of charge via the internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected] (K.Y.J.Z.) or [email protected] (Q.Y.).

Author Contributions

⊥These

authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Program for National Natural Science Funds for Distinguished Young Scholar (31425021) and the Program for Liaoning Excellent Talents in University (LJQ2014006). We thank Prof. Jianjun Zhang at China Agricultural University for providing MU-(GlcNAc)2 (synthesized in the NKT R&D Program of China, 2015BAK45B01, CAU). We thank Thomas Malott (Dalian University of Technology) for his contribution in the language editing of the manuscript. We acknowledge the Hokusai GreatWave supercomputer at RIKEN for the supercomputing resources used in this study. We acknowledge RIKEN Pioneering Project in Dynamic Structural Biology for funding.



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for Table of Contents use only

A Novel Scaffold for Developing Specific or Broadspectrum Chitinase Inhibitors Xi Jiang, Ashutosh Kumar, Tian Liu, Kam Y. J. Zhang and Qing Yang


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A Novel Scaffold for Developing Specific or Broad-Spectrum Chitinase

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