In Silico Evaluation of Synthetic Hydrophobic Fluorescent NBD- and DANSYL-Derivatives as Potential Inhibitors of Insect Chitinases ^†

Abstract

Docking calculations in semi-automatic virtual screening mode have been performed using AutoDock Vina (5 × 5 × 5 nm grid box, centered on the chain A) and FYTdock helper software. N-hexanoyl ciprofloxacin has been found to bind with chitinases from Ostrinia furnacalis (pdb codes: 7vrg, 6jaw, 6jay, 6jmn, 5y2b; energy of bindings (Ebind) −10.2…−9.7). N-hexanoyl-N’-NBD-piperazine, and NpipHex bind in silico with the enzyme less effectively (pdb codes: 6jaw, 5y2b, 6jay, 5y2c, 3wkz; Ebind −9.3…−8.9). Lipid-like N-NBD-oleylamine and N-Dansyl-oleylamine demonstrated quite similarly, but smaller affinity (Ebind −8.6…−8.0). Examples of interactions close to the active sites of the chitinases were found for all compounds. These results provide new insights into insect biochemistry of chitinases showing new molecular scaffolds suitable as prototypes of tools for pest control or fluorescence-based screening.

1. Introduction

Chitinases (EC 3.2.1.14, 1,4-beta-poly-N-acetylglucosaminidases) are enzymes catalyzing hydrolytic cleavage of glyosidic β-1→4-bonds in chitin (poly(β-(1→4)-N-acetyl-d-glucosamine). They are found in plants [1], yeast and fungi [2], bacteria [3], algae, crustaceans and insects [4,5] as well as in humans [6] and other organisms. In insects, including pests and diseases vectors, the enzymes play important roles during ecdysis and, for some species, feeding. Thus, insect chitinases are promising as molecular targets for new insecticides development aiming crop protection and healthcare purposes.

A number of 3D structures for insect chitinases have been solved and presented in Protein data Bank (https://www.rcsb.org/) online repository, e.g., from moth Asia corn borer Ostrinia furnacalis. The circumstance opens a good option to use in silico approaches to select possible new affine ligands of the enzymes [7,8] from vast number of compounds in chemical space to rationalize further in vitro tests. Notably, some chitinases have a lot of hydrophobic amino acids residues close to catalytic sites, resulting in a selection criterion for possible inhibitors. Using the circumstance, a fact about fatty acid blocks in structures of novel azamacrolide chitinase inhibitor [9] and the repurposing approach, we decided to evaluate in silico a few lipophilic molecules from our lab, including two previously reported fluorescent N-hexanoyl-piperazine derivatives based on 7-nitrobenzofurazan-4-yl- (NBD-) and ciprofloxacin (CPF), in aspects of cytochrome P450 inhibition and biocompatible polymerization photo-initiator [10,11]. In this work we provide an insight that such compounds cold be prototypes for insect chitinase inhibitors.

2. Materials and Methods

The chosen structures for docking were as follows: N-hexanoyl-ciprofloxacin (CPFHex), N-hexanoyl-N′-(NBD-)piperazine (NpipHex), N-hexanoyl-N′-(NBD-)ethylenediamine (NedaHex), N-NBD-oleylamine (NOLA) and N-dansyl-oleylamine (DOLA). Their structures are depicted on Figure 1.

Docking calculations in semi-automatic virtual screening mode have been carried out using AutoDock Vina v. 1.1.2 and FYTdock v. 1 helper software based on Python scripts and Microsoft Excel tables [12] to organize, run and analyze results. Chains A only, 5 × 5 × 5 nm grid box (centered on a protein), exhaustiveness of 15 were used. The PDB codes for 3D structures of proteins from Ostrinia furnacalis used were as follow: 3wmc, 9g3q, 3wmb, 7vrg, 5gpr, 3wqw, 3wl1, 3wqv, 5wv9, 5y2a, 5y2c, 5wvb, 5wus, 5wvg, 5wvf, 5wvh, 5gqb, 6jmn, 3w4r, 6jax, 6jay, 5wup, 5wv8, 3wkz, 3wl0, 6jav, 5y2b, 5y29, 6jaw, 6jmb, 6jm8, 6jm7. Values for docking scores (energies of binding, Ebind, kcal/mol) and amino acid surroundings predicted for the compounds were tabulated. Only the top 5 docking simulations results based on Ebind values are mentioned in the article. Figures were prepared using Biovia Discovery Studio software v. 16.1.0.

3. Results

Results of docking simulations of aforesaid chitinase and compounds are given in

Table 1. Top 5 of docking simulations results of interactions of insect chitinase with CPFHex.

PDB Code	Ebind	Amino Acids Surrounding 1
7vrg	−10.2	Ser357, Gly359, Lys362, Tyr411, Ala358, Phe309, Met381, Asp384, Glu308, Trp268, Trp389, Arg439, Trp532, Tyr437, Val469, Glu533, Phe385
6jav	−9.9	Val1740, Asn1778, Trp1809, Trp1691, Asp1804, Pro1777, Tyr1803, Tyr1734, Gln1858, Phe1899, Trp1961, Tyr1856, Asp1731, Glu1733, Met1801
6jaw	−9.9	Val1740, Asn1778, Trp1809, Trp1691, Asp1804, Tyr1734, Gln1858, Tyr1856, Phe1899, Trp1961, Tyr1803, Glu1733, Asp1731
6jay	−9.9	Trp1809, Val1740, Trp1691, Asp1804, Tyr1734, Gln1858, Tyr1856, Phe1899, Trp1961, Tyr1803, Glu1733, Phe1648
6jmn	−9.9	Ser357, Gly359, Lys362, Tyr411, Ala358, Phe309, Asp384, Met381, Trp268, Glu308, Trp389, Arg439, Trp532, Trp160, Phe184, Thr269, Phe385

¹ The active site residues are typed using italic.

,

Table 2. Top 5 of docking simulations results of interactions of insect chitinase with NpipHex ¹.

PDB Code	Ebind	Amino Acids Surrounding
6jaw	−9.3	Trp1621, Trp1961, Phe1899, Ala1896, Arg1625, Gln1858, Asp1804, Trp1691, Trp1809
5y2b	−9.1	Phe1648, Asn1692, Asp1693, Gly1689, Gly1690, Trp1691, Trp1621, His1660, Trp1961, Asp1804, Gln1858, Tyr1856, Phe1899, Leu1965
6jay	−9.1	Trp1621, Trp1961, Phe1899, Ala1896, Arg1625, Thr1894, Gln1858, Asp1804, Trp1691, Trp1809, Val1740
5y2c	−9	Phe2094, Gly2136, Asp2139, Asp2140, Gly2137, Trp2138, Trp2067, His2107, Trp2398, Phe2336, Tyr2303, Gln2305, Leu2402
3wkz	−8.9	Tyr217, Tyr272, Trp372, Asp218, Gln148, Trp107, Phe61, Met215, Asp146, Trp34, Phe309, Arg274, Ala306, Arg38, Thr304

¹ The active site residues are typed using italics.

,

Table 3. Top 5 of docking simulations results of interactions of insect chitinase with NedaHex.

PDB Code	Ebind	Amino Acids Surrounding 1
5gpr	−9	Trp268, Asp384, Arg439, Phe309, Tyr383, Ala358, Met381, Ser357, Gly359, Lys362, Phe385, Tyr411, Glu308, Trp532, Phe184, Tyr156, Asp306, Ala355
6jmn	−9	Trp268, Asp384, Arg439, Phe309, Met381, Ala358, Tyr383, Ser357, Gly359, Lys362, Tyr411, Glu308, Trp532, Tyr437, Tyr156, Phe184, Asp306
7vrg	−9	Met381, Tyr383, Asp384, Ala358, Phe309, Lys362, Ser357, Tyr411, Gly359, Glu308, Trp268, Arg439, Trp389
3nsn 2	−9	Glu328, Trp483, Trp490, Glu526, Val327, Val484, Asn489, Arg220, Trp524, Tyr475, Glu368, Trp448, Asp367, Trp424
3ozp 2	−8.9	Glu328, Trp483, Trp490, Glu526, Val327, Asn489, Asp477, Arg220, Trp524, Glu368, Asp367, Trp448, Tyr475, Trp424

¹ The active site residues are typed using italics. ² The structures are for OfHex1 enzyme.

,

Table 4. Top 5 of docking simulations results of interactions of insect chitinase with NOLA.

PDB Code	Ebind	Amino Acids Surrounding 1
6jmn	−8.6	Lys362, Tyr411, Ser357, Gly359, Ala358, Phe309, Asp384, Trp268, Trp389, Arg439, Glu308, Phe184, Trp532, Tyr383, Tyr156, Met381, Asp306
6jm8	−8.4	Trp33, Arg37, Tyr303, Trp365, Glu368, Ser366, Tyr270, Glu300, Phe60, Trp105, Ser106, Tyr213, Asp214, Met211, Arg272, Trp219, Thr369
5gqb	−8.2	Ser357, Ala358, Gly359, Tyr411, Phe385, Met381, Tyr383, Phe309, Asp384, Lys362, Trp389, Trp268, Arg439, Trp532, Phe184, Glu308, Asp306, Tyr156
7vrg	−8.1	Ser357, Ala358, Gly359, Lys362, Tyr411, Met381, Tyr383, Phe309, Trp389, Trp268, Asp384, Arg439, Glu308, Phe184, Trp532
6jmb	−8	Tyr147, Arg150, Ser186, Val188, Trp219, Asp214, Trp105, Arg272, Phe60, Trp365, Glu146, Met211, Tyr213, Tyr270, Ala187, Ala191, Met215

¹ The active site residues are typed using italics.

and

Table 5. Top 5 of docking simulations results of interactions of insect chitinase with DOLA.

PDB Code	Ebind	Amino Acids Surrounding 1
6jav	−8.7	Glu1733, Met1801, Tyr1734, Tyr1803, Asp1804, Val1740, Pro1777, Asn1778, Trp1809, Trp1691, Asp1731, Trp1961, Phe1648, Trp1621, Phe1899
5y2b	−8.5	Trp1809, Gln1858, Phe1899, Tyr1856, Asp1804, Trp1961, Leu1965, Trp1621, Trp1691, Tyr1803, Phe1648, Glu1733, Tyr1734, Val1740
7vrg	−8.5	Trp268, Arg439, Trp532, Phe184, Glu308, Tyr383, Asp384, Met381, Phe309, Ala358, Trp389, Lys362
3wqv	−8.4	Trp34, Phe61, Trp107, Trp372, Glu148, Tyr217, Tyr272, Asp218, Arg274, Phe309, Ala306, Arg38, Thr304, Ala108, Gly106, Glu109, Ile74, Asn33
5gpr	−8.4	Asp384, Trp389, Phe309, Trp268, Glu308, Arg439, Trp532, Phe184, Tyr383, Tyr156, Asp306, Ala355, Met381, Lys362

¹ The active site residues are typed using italics.

.

Localization details of the most affine interaction in the set is shown in Figure 2.

4. Discussion

The new in silico data concerning potential affinities of aforementioned fluorescent hydrophobic compound to chitinases of O. furnacalis were reported for the first time. The best affinity (the lowest Ebind) was obtained for CPFHex, an acylated derivative of the well-known FDA-approved antibacterial compound ciprofloxacin, and a structure of OfChiH (PDB 7vrg). The active site of the chitinase include aa acidic catalytic triad (Asp304, Asp306 and Glu308) of DXDXE motif as well as substrate-binding cavity built of solvent-exposed aromatic residues of domain I (Trp27 and Trp63) and domain II (Trp160, Tyr163, Trp225, Trp238, Trp268, Trp389, and Trp532) [13]. In the predicted complex (Table 1) CPFHex was found to be surrounded by a set of residues, including Glu308, Trp268, Trp389 and Trp532, showing a partial overlap with the active site. Similarly, for other chitinase structures equivalent residues are Glu1733 and Asp1729 as well as Trp1621, Tyr1624, Trp1663, Trp1691, Trp1809 and Trp1961 [14]. As it can be seen from Table 1, Table 2, Table 3, Table 4 and Table 5 that all ligands tested demonstrate at least partial overlap with catalytic and substrate-binding centers of the chitinases structures.

It should be noted that till the publication, to the best of our knowledge, such compounds were not estimated as possible inhibitors of chitinases. Else, we found only one publication concerning a fluorescent substrate for acidic mammalian chitinase (AMCase) based on fluoresceinated chitin oligomers [15] and the very recent one concerning NBD-based C-glycoside as fluorescent inhibitor of insect chitinases OfChtII and OfHex1 acting as insecticides [16]. In a broader way, it can be noted that there are a lack of data concerning NBD-, DANSYL-, fluoroquinolone and other common fluorophore derivatives with chitinases of both mammals and insects in spite of the potential of such derivatives as molecular probes and screening tools for the enzymes and growing interest to the proteins as potential targets for drugs [17,18] or new types of insecticides [8]. These circumstances seem to highlight the novelty of our virtual screening data and could encourage researchers to create new molecular tools, drugs or insecticides.

Notably, because pros and cons of ciprofloxacin for human health are considered to be studied well, itself and some its derivatives seem to be promising as a template of safe insecticides. On the other hand, our NBD-based and DANSYL-based structures represent original ones and, thus, their biosafety level is unknown, but broad use of the fluorophores in various biophysical and biochemical studies also provide a chance to tune selective toxicity against insects. Also, notably, there are a small number of reports concerning fluorescent compounds’ interactions with insect proteins, even at in silico level, and cells, in comparison with fluorescent probes usage for mammalian cells, yeasts and bacteria.

Our virtual screen was set in inverse mode using multiple chitinases structures. Such an approach is good because it allows for taking into account various differences in crystal structures of the same proteins, providing a base for more weighted prognosis in spite of rigid docking limitations. Our helper software FYTdock is designed for such inverse virtual screening mode and also provides an option for broader searches to discover new affine off-target interactions and new plausible targets, like in this report. It is interesting to note that due to availability of recently developed novel technology Alphafold [19], model structures of chitinases for other insects can be obtained and taken in virtual screening mode to obtain insight on possible successes or failures of future in vitro tests.

5. Conclusions

Here we report about virtual screening results that involved 30 PDB chitinase structures and some original hydrophobic fluorescent compounds with 7-nitrobenzoxadiazol (NBD) and dansyl scaffolds. N-hexanoyl ciprofloxacin has been found to bind with chitinases from Ostrinia furnacalis (the best result with Ebind −10.2). N-hexanoyl-N′-NBD-piperazine, NpipHex, and its open-ring analogue of NpipHex, N-hexanoyl-N′-NBD-ethylenediamine, has demonstrated quite similar affinity. Also lipid-like N-NBD-oleylamine and N-Dansyl-oleylamine demonstrated less affine binding affinity. These results open opportunity for the corresponding in vitro tests with insects or purified chitinases as well as for wider in silico screen using Alphafold options to predict 3D structural models for others insect chitinases as well as other chitinases from various species to address healthcare and agricultural challenges.