Discovery of Kasugamycin as a Potent Inhibitor of Glycoside Hydrolase Family 18 Chitinases

Kasugamycin, a well-known aminoglycoside antibiotic, has been used widely in agriculture and medicine to combat microbial pathogens by binding the ribosome to inhibit translation. Here, kasugamycin was discovered to be a competitive inhibitor of glycoside hydrolase family 18 (GH18) chitinases from three different organisms (bacterium, insect and human). Results from tryptophan fluorescence spectroscopy and molecular docking revealed that kasugamycin binds to the substrate-binding clefts in a similar mode as the substrate. An electrostatic interaction between the amino group of kasugamycin and the carboxyl group of a conserved aspartate in GH18 chitinase (one of the catalytic triad residues) was found to be vital for the inhibitory activity. This paper not only reports new molecular targets of kasugamycin, but also expands our thinking about GH inhibitor design by using a scaffold unrelated to the substrate.


INTRODUCTION
The glycoside hydrolase family 18 (GH18) chitinases (EC 3.2.1.14) are key enzymes that biodegrade chitin, which is the most abundant aminopolysaccharide in nature and composed of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc). Chitinases are universally distributed in organisms from viruses to mammals and have diverse functions that include tissue degradation and remodeling, nutrition uptake, pathogen invasion and immune response regulation (Chen et al., 2020). For example, fungi and insects use chitinases to remodel their chitin-containing extracellular matrices (cell wall of fungi and exoskeleton of insects) (Zhu et al., 2016), whereas plants and humans, which do not contain chitin, use chitinases to degrade the chitin-containing shield of pathogens and create chitooligosaccharides (CHOS) to elicit immune responses (Kumar and Zhang, 2019).
Because GH18 chitinases play important roles in fungal pathogenesis, insect molting and human diseases, their inhibitors have potential applications as agrochemicals and human drugs (Chen et al., 2020). A core concept in the design of GH inhibitors involves simulating the structures of the substrate, transition state or reaction intermediate (Asano, 2009). Most carbohydrate-based inhibitors that target GH18 chitinases are based on these scaffolds. Allosamidin, the most studied GH18 chitinase inhibitor, is a pseudotrisaccharide composed of one N-acetyl-Dallosamine moiety and two GlcNAc moieties (Sakuda et al., 1986) ( Figure 1A). The inhibitory mechanism involves the N-acetyl-D-allosamine moiety occupying the -1 subsite of the substratebinding clefts (SBCs) and mimicking the conformation of the reaction intermediate (Pantoom et al., 2011). Chitotriose thiazolines are designed GH18 chitinase inhibitors with similar structures and inhibitory mechanisms as allosamidin (Macdonald et al., 2010) ( Figure 1A). The only structural difference is the N-acetyl-Dallosamine moiety in allosamidin is replaced by a GlcNActhiazoline. In addition to reaction intermediate mimics, various substrate mimics such as deacetylated CHOS (Chen et al., 2014) (Figure 1A), diacetylchitobiosyl amides (Rottmann et al., 1999) and N,N′-diacetylchitoxime-N-phenylcarbamate (Vaaje-Kolstad et al., 2004) have been discovered as GH18 chitinase inhibitors.
Kasugamycin is an aminoglycoside agrochemical that has been used widely to treat plant bacterial and fungal diseases. Kasugamycin kills the target microorganisms by interfering with the interaction between mRNA and the 30S subunit of the ribosome (Schluenzen et al., 2006). In this study, kasugamycin was discovered for the first time to inhibit bacterial, insect and human GH18 chitinases. However, the structure of kasugamycin is totally different from that of CHOS. Furthermore, tryptophan fluorescence spectroscopy and molecular docking were used to study the mechanisms of potent inhibition as well as the relative selectivity among GH18 chitinases.

Enzyme Preparation
The recombinant GH18 chitinases for inhibitory activity evaluation including OfChi-h and the catalytic domains of OfChtI from Ostrinia furnacalis (O. furnacalis), human HsCht and acidic mammalian chitinase (AMCase) were extracellularly expressed in Pichia pastoris GS115. SmChiA from Serratia marcescens was expressed in Escherichia coli BL21 (DE3). All the proteins were purified from the culture medium by immobilized metal affinity chromatography (IMAC) as described previously (Chen et al., 2014). The purities of the target proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining.

Inhibitory Activity Determination
Briefly, the reaction mixtures used for inhibitor screening had a final assay volume of 100 μL. 20 nM enzyme was incubated with 10 μL substrate [0.2 μM MU-β-(GlcNAc) 2 ] in 20 mM sodium phosphate buffer (pH 6.0 for OfChi-h, OfChtI, HsCht and SmChiA, and pH 5.2 for AMCase) containing 2 μL inhibitor at 30°C. The reaction in the absence of inhibitor and substrate was used as the control. After 30 min, an equal volume of 0.5 M Na 2 CO 3 was added to the reaction mixture to stop the reaction, and the fluorescence of the released MU was quantitated using an Infinite ® 200 PRO microplate reader (Tecan, Swiss) with excitation and emission wavelengths of 360 and 450 nm, respectively. Experiments were performed in triplicate. For the determination of the mode of inhibition and the inhibition constant (K i ) value, the reaction mixtures contained three MU-β-(GlcNAc) 2 concentrations (5, 10 and 20 μM for OfChi-h, AMCase and SmChiA; 5, 7.5 and 10 μM for HsCht; and 1, 2 and 4 μM for OfChtI). The inhibitor concentration varied according to the inhibitory activity. The Ki values and types of inhibition were determined by linear fitting of data in Dixon plots.

Tryptophan Fluorescence Spectroscopy
All tryptophan fluorescence spectroscopy experiments were performed using the Infinite ® 200 PRO microplate reader (Tecan, Swiss). The excitation wavelength was fixed to 285 nm and emission spectra were collected between 320 and 450 nm with a slit width of 2 nm. The temperature was maintained at 30°C. Fluorescence quenching experiments were performed in a 100 μL mixture containing 1 μM protein in the 20 mM sodium phosphate buffer (pH 6.0 for OfChi-h, OfChtI, HsCht and SmChiA, and pH 5.2 for AMCase), and by the successive addition of 1 μL compounds stock solution. The inhibitor

Molecular Docking
The PRODRG2 server was used to generate and optimize the initial structure of the compounds before docking (Schuttelkopf and Van Aalten, 2004

RESULTS AND DISCUSSION
In our preliminary screening of a natural products library, kasugamycin was found to display 92.54% inhibition against OfChtI, an insect GH18 chitinase from O. furnacalis, at 10 μM. In this research, the inhibition activities of kasugamycin against various GH18 chitinases including SmChiA from S. marcescens (Fuchs et al., 1986), OfChtI and OfChi-h from O. furnacalis (Liu et al., 2017), and HsCht and AMCase from Homo sapiens (Fusetti et al., 2002;Bussink et al., 2008) were studied. Inhibition kinetics demonstrated that kasugamycin inhibits all of the tested GH18 chitinases in a competitive mode ( Figure 1B; Supplementary Figure S1) with K i values varying from 0.25 to 29.00 μM ( Table 1).
Since the SBCs of GH18 chitinases usually contain several solvent-exposed tryptophan residues, tryptophan fluorescence quenching spectroscopy was used to determine the binding affinity of kasugamycin to GH18 chitinases. As shown in Figure 1C and Supplementary Figure S2, kasugamycin quenched the native tryptophan fluorescence of GH18 chitinases in a dose-dependent mode. The equilibrium dissociation constant (K d ) values of kasugamycin to GH18 chitinases varied from 0.92 to 34.11 μM ( Table 1). The tendency of the K d values is in good accordance with that of the K i values, although the values are not identical.
To further understand the inhibitory mechanism, kasugamycin was docked into the crystal structure of HsCht (Fusetti et al., 2002), which has the highest affinity toward kasugamycin. Although there is little structural similarity between kasugamycin and CHOS, kasugamycin bound the SBC of HsCht in a similar mode as (GlcNAc) 2 by forming CH-π interactions with the indole group of Trp 31 and Trp 358 (Figure 2A). The methylkasugaminide moiety occupied the -1 subsite of the SBC and formed hydrogen bonds with surrounding residues including Glu 140 , Tyr 141 and Asp 213 . The D-inositol moiety of kasugamycin occupied the -2 subsite of the SBC and formed a hydrogen bond with Asn 100 . Since the amino group of kasugamycin and the carboxyl group of Asp 138 (one of the catalytic triad residues) have opposite charges at pH 6.0, we hypothesized that the strong electrostatic interaction between them was a driving force for the inhibitory activity of kasugamycin against GH18 chitinases. To prove this hypothesis, we determined the K i value of kasugamycin against HsCht in a buffer containing 1.0 M NaCl to weaken the electrostatic interaction. Under these conditions, the K i value of kasugamycin against HsCht increased 6-fold to 1.62 μM ( Table 1; Supplementary Figure S3), demonstrating the importance of this electrostatic interaction in the binding affinity of kasugamycin to GH18 chitinases. Most of residues involved in binding were key residues for chitinase catalysis. Residues Asp 138 and Glu 140 are responsible for glycosidic bond breaking. Residue Asp 213 is involved in catalysis by stabilizing the −1 sugar in its distorted conformation (Synstad et al., 2004;Chen et al., 2020). Mutation of these residues in SmChiB yielded greatly reduction in catalytic activity (Synstad et al., 2004). Kasugamycin was first reported as a bacterial protein synthesis inhibitor, and the binding mechanism of kasugamycin to the 30S subunit of the bacterial ribosome has been studied by X-ray crystallography (Schluenzen et al., 2006). In this structure, kasugamycin binds the 16S ribosomal RNA within the messenger RNA channel. The electrostatic interaction formed between the amino group of kasugamycin and the backbone phosphate group of G1483 was also important for defining the binding affinity.
Kasugamycin was also docked into the crystal structures of OfChtI, OfChi-h, AMCase and SmChiA. The modeled complex-structures with the highest frequency were then superimposed to determine the molecular basis for the relative selectivity among different GH18 chitinases ( Figure 2B). The binding mode of the methylkasugaminide moiety was almost the same among the different GH18 chitinases whereas the binding mode of the D-inositol moiety differed. In OfChi-h and SmChiA, Tyr 141 in HsCht was replaced by a phenylalanine residue, which cannot participate in hydrogen bond formation ( Figure 2B; Supplementary Figure S4). The D-inositol moiety occupies the -2 subsite of the SBC of OfChtI but does not form a hydrogen bond with any residues (a hydrogen bond with Asn 100 was found in HsCht). As for OfChi-h, AMCase and SmChiA, which have much lower binding affinities to kasugamycin, the D-inositol moiety rotates about 28°away from the conserved tryptophan at the -2 subsite (Trp 31 in HsCht), which may result in the loss of the CH-π interaction with this residue ( Figure 2B; Supplementary Figure S4).
These results indicate that the inhibitory activity of kasugamycin against different GH18 chitinases can be optimized by modifying the D-inositol moiety to increase the number of intermolecular interactions with residues surrounding the -2 subsite of the SBC. After this article was accepted, we noticed that kasugamycin was reported by another research group as HsCht inhibitor, and its potential in the treatment of pulmonary fibrosis was also investigated (Lee et al., 2021).

CONCLUSION
In this work, kasugamycin was discovered as a competitive inhibitor of various GH18 chitinases. By tryptophan fluorescence spectroscopy and structure-based molecular docking, kasugamycin was revealed to bind SBCs of GH18 chitinases in a similar mode as the substrate through an electrostatic interaction, CH-π interactions and hydrogen bonds. This work does not reveal novel targets for kasugamycin but provides a new scaffold for designing novel GH inhibitors.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
Conceptualization, TL and QY; methodology, TL, HQ and XJ; investigation, HQ, XJ and YD; original draft preparation, TL, HQ and XJ; review and editing, TL and QY; project administration, TL and QY.