Synthesis and Structure-Activity Relationship Studies of Hydrazide-Hydrazones as Inhibitors of Laccase from Trametes versicolor

A series of hydrazide-hydrazones 1–3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM with the predicted and desirable competitive type of inhibition. The structure–activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.


Introduction
The discovery of new eco-friendly pesticides, having readily biodegradable natural units, is an essential need of the agrochemical industry [1]. The global human population is constantly growing, therefore, food production needs to be still enhanced. Synthetic pesticides, which usually act indirectly or non-specifically, cause considerable contamination of the environment. Furthermore, crop losses result from the rapid evolution of pest resistance to these chemicals. These problems lead to a high demand for new cultivation approaches and less toxic plant protection products [2,3]. Commonly occurring and troublesome pathogens of crop plants are filamentous fungi that belong to the Sclerotniaceae family, for instance, gray mold (Botrytis cinerea) which attacks flowers, green tissues of crop plants [4] and fruits such grapes [5]. The increasing problem is the abundancy of wood-decay fungi that attack young and healthy but also old vintage trees in forestry, fruit orchard culture, horticulture, and in the old parks [6]. Insects e.g., aphids, Drosophila species, termites, etc., also cause horticulture, and in the old parks [6]. Insects e.g., aphids, Drosophila species, termites, etc., also cause plant damage which influences the crop production, vitality and aesthetic values of gardens and domestic ornamental plants [7]. Therefore, new natural and natural product-derived plant protection agents with an easily biodegradable structure and specific unique mechanisms of action are highly desirable [8]. A common feature of the fungi and insects mentioned above is the production of laccase, which is specifically involved in various survival and physiological processes [9,10]. White-root fungi, for instance, use laccase to destroy the lignin that is the physical barrier in trees to prevent the degradation of cellulose [6]. Laccases of some molds neutralize phenolic antibiotics, such as phytoanticipins or phytoalexins, produced by the plant immune system as a response to a pathogen attack [11,12]. Laccase together with tyrosinase also play a crucial role in the sclerotia formation and virulence of Sclerotinia sclerotiorum [13,14] as well as hardening cuticle in insects [15]. Thus, regulation of the activity of this enzyme might weaken pathogen activity and provide time to strengthen the plant defense system.
Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that is widely distributed in nature. The enzyme catalyzes the radical reduction of atmospheric oxygen to water with simultaneous oxidation of electron-rich aromatic compounds such as polyphenols and anilines [16]. The broad spectrum of substrates that can be converted, mostly by laccases of fungal origin, is due to the high redox potential of a specific copper atom in the enzyme active site [17]. Laccase from Trametes versicolor is a typical "blue" oxidase which contains a cluster of four copper atoms categorized as type 1, 2 and 3 [18]. Type 1 is a paramagnetic blue copper and a site of a substrate molecule binding. One copper atom of type 2 and two copper atoms of type 3, form a trinuclear center which binds and reduces dioxygen. The electron transfer from oxidized substrate follows from type 1 Cu center through His458-Cys453-His452 tripeptide to the trinuclear-copper cluster of 2 and 3 types where it is used for the reduction of dioxygen to water molecules [19]. The catalytic cycle includes oxidation of four single-center substrates to four radical products with simultaneous reduction of one molecule of O2 to two molecules of H2O. The oxidation of a substrate results in the active radicals, which either could be mediators in the radical oxidation reactions or could be non-enzymatically converted to the final oxidation products (Scheme 1).
Isolation of bioactive natural compounds from plant sources and microorganisms for agricultural applications is usually a challenge due to the small amount of the active ingredient(s), availability of the biomass and cost of the process. Therefore, in our studies, we focused on the use of commonly available natural salicylic aldehydes and carboxylic acids combined with hydrazine linkers to form natural product-derived compounds such hydrazide-hydrazones. Furthermore, we aimed at discovering a new group of organic low-molecular-weight compounds which activity that weakens a pest's activity and consequently provides conditions for improving the plant defense system. The imines 1-3 were synthesized as part of a program to discover low-molecular-weight compounds that were both the inhibitors of essential enzymes overexpressed during disease development [25][26][27] and act directly against pathogenic microorganisms [28,29]. A common polypore fungus Trametes versicolor, which is an abundant tree parasite causing a white-rot of wood, was chosen as a model organism. Infection by T. versicolor results in a degradation of lignin by the laccase mediator system, and ultimately to decomposition of the wood structure.

Syntheses and Characterizations
The various twenty-three imine derivatives 1-3 were synthesized using 4-hydroxybenzoic acid hydrazide (4a) and diverse aldehydes 5-7 having a benzene ring. In two cases, 4-methoxy-and 3-hydroxybenzoic acid hydrazide 4b and 4c were used for the preparation of hydrazide-hydrazones 2h and 3g, respectively. Among them, fourteen products are new. The hydrazide-hydrazones listed in Table 1 were thus prepared as pure compounds in good to quantitative (76-100%) yields.
Generally, in their Fourier-transform infrared spectroscopy (FT-IR) spectra, the salicylaldehyde derivatives 2a-c and 3a-b, which were functionalized in the neighborhood of the hydroxyl group, had a very wide absorption band at 2500-3300 cm −1 , caused by hydrogen bonds in the crystal lattice.  [22,24]). Both 4-hydroxybenzyl alcohol and 4-hydroxybenzoic acid (4-HBA) are naturally occurring and are recognized as useful mediators in laccase catalyzed reactions. These simple compounds are also readily available. Moreover, the 4-HBA is only needed in 0.1 mM concentration to significantly increase the oxidation efficacy of resistant compounds in laccase-catalyzed reactions [22]. It seems that such high affinity of the 4-HBA for the substrate pocket in laccase and the presence of an easily chemically modified free carboxyl group would enable us to prepare a collection of 4-HBA derivatives with better fitting to the enzyme cavity and control of their inhibition potency.
Isolation of bioactive natural compounds from plant sources and microorganisms for agricultural applications is usually a challenge due to the small amount of the active ingredient(s), availability of the biomass and cost of the process. Therefore, in our studies, we focused on the use of commonly available natural salicylic aldehydes and carboxylic acids combined with hydrazine linkers to form natural product-derived compounds such hydrazide-hydrazones. Furthermore, we aimed at discovering a new group of organic low-molecular-weight compounds which activity that weakens a pest's activity and consequently provides conditions for improving the plant defense system. The imines 1-3 were synthesized as part of a program to discover low-molecular-weight compounds that were both the inhibitors of essential enzymes overexpressed during disease development [25][26][27] and act directly against pathogenic microorganisms [28,29]. A common polypore fungus Trametes versicolor, which is an abundant tree parasite causing a white-rot of wood, was chosen as a model organism. Infection by T. versicolor results in a degradation of lignin by the laccase mediator system, and ultimately to decomposition of the wood structure.

Syntheses and Characterizations
The various twenty-three imine derivatives 1-3 were synthesized using 4-hydroxybenzoic acid hydrazide (4a) and diverse aldehydes 5-7 having a benzene ring. In two cases, 4-methoxy-and 3-hydroxybenzoic acid hydrazide 4b and 4c were used for the preparation of hydrazide-hydrazones 2h and 3g, respectively. Among them, fourteen products are new. The hydrazide-hydrazones listed in Table 1 were thus prepared as pure compounds in good to quantitative (76-100%) yields. Generally, in their Fourier-transform infrared spectroscopy (FT-IR) spectra, the salicylaldehyde derivatives 2a-c and 3a-b, which were functionalized in the neighborhood of the hydroxyl group, had a very wide absorption band at 2500-3300 cm −1 , caused by hydrogen bonds in the crystal lattice.
For all of the remaining hydrazide-hydrazones 1-3, the wavenumbers corresponding to the stretching vibration of an amide single bond N-H was around 3016-3307 cm −1 , the carbonyl group (C=O) bands were at 1580-1644 cm −1 , and the imine bond (C=N) at 1538-1589 cm −1 .
Determination of the kinetic parameters for laccase and inhibition constants (K i ) for the tested hydrazide-hydrazones was performed in aqueous or aqueous-organic solution at 25 • C and pH 5.3. An organic solvent, CH 3 OH or dimethyl sulfoxide (DMSO), in 6.25% v/v concentration was necessary to enhance the solubility of the tested compounds since the experiments in buffer solution gave inaccurate results. At such concentrations, methanol influenced laccase stability to a negligible extent when compared to DMSO solvent, which significantly decreased the activity of the enzyme [75] (see Supplementary Materials). The kinetic parameters in 6.25% v/v of methanol solution were estimated using Michaels-Menten model [76] as follows: K m = 4.276 µM, k 3 = 4.343 µmol/min/mg of protein, the coefficients for the goodness of fit were R 2 : 0.9745, sum of squared errors(SSE): 1.6767, root mean squared error (RMSE): 0.0984. Fifteen derivatives showed no effect on the tested oxidoreductase for an arbitrarily assumed criterion of K i ≥ 1000 µM. Eleven compounds had inhibition potency at the level of K i values between 939 µM and 17.9 µM. The predominant mode of interaction of 4-HBAH derivatives 1c, 1e, 1i, 2a-b, 2g, 3d with laccase was the competitive type of inhibition. The hydrazide-hydrazones of the first group containing the only one biogenic substituent in different positions of aldehyde fragment ( Table 2) appeared to be weak or moderate inhibitors. Therefore, for these compounds, the enzyme activity was measured for an extended range of K i concentrations (up to 2400 µM). Table 2. Structures of the hydrazide-hydrazones 1a-j, 2a-h, 3a-g and their inhibition constants (K i ) determined for laccase from Trametes versicolor, and the skeleton numbering system. Significant inhibition is marked in bold. The activity of the reference compounds, 4-hydroxybutylacrylate (4-HBA), and NaN 3 (a dioxygen reduction centre inhibitor), are given.
for 1e, 1g, and 1i, respectively. Interestingly, among alkylated ones, the best Ki = 251 µ M was observed for compound 1c having a small methyl substituent at position 4 of the benzylidene unit. This is in agreement with general trends for enzymes for which alkyl groups provided better filling of the hydrophobic area in substrate binding pocket [77]. Consequently, for further studies, generally, we concentrated on C or O alkylated the 2-and/or 4-hydroxy-and/or 3-methoxybenzaldehyde derivatives 2a-h and 3a-g. Table 2. Structures of the hydrazide-hydrazones 1a-j, 2a-h, 3a-g and their inhibition constants (Ki) determined for laccase from Trametes versicolor, and the skeleton numbering system. Significant inhibition is marked in bold. The activity of the reference compounds, 4-hydroxybutylacrylate (4-HBA), and NaN3 (a dioxygen reduction centre inhibitor), are given.
Ph In the second group, the combination of the hydroxy and methoxy substituents, in the para and meta positions of vanillic aldehyde, resulted in an approximately two-fold improvement in the Ki value to 251 µ M for compound 2g compared to related monosubstituted benzaldehyde derivatives 1g and 1i. This compound is a derivative of naturally occurring vanillic aldehyde, commonly found in vegetation. Therefore, the constant inhibition and biodegradable scaffold components of vanillic derivative 2g seemed acceptable for the protection of plants, especially ornamental plants. An opposite effect to the derivative 2g was observed for isomer 2e when the hydroxy and methoxy groups were present in the ortho and ortho' positions in the benzaldehyde framework. Similarly, the pairs of two methoxy and two hydroxy groups in the ortho, ortho' and ortho, para positions in 2f and 2c, respectively, were practically inactive towards laccase. The hydrazide-hydrazone derivatives of 2,5-dihydroxybenzaldehyde (gensinealdehyde) acted as laccase inhibitors when they were linked to coumarin with an ether bond [34]. In our studies, the hydrazide-hydrazone derivative of this aldehyde with 4-HBAH displayed intense pigmentation. Furthermore, the reaction results were Among the most interesting potent oxa substituents, the inhibition constants ranged between 416 µM and 674 µM for hydroxy and methoxy groups localized at the ortho and para or meta position for 1e, 1g, and 1i, respectively. Interestingly, among alkylated ones, the best K i = 251 µM was observed for compound 1c having a small methyl substituent at position 4 of the benzylidene unit. This is in agreement with general trends for enzymes for which alkyl groups provided better filling of the hydrophobic area in substrate binding pocket [77]. Consequently, for further studies, generally, we concentrated on C or O alkylated the 2-and/or 4-hydroxy-and/or 3-methoxy-benzaldehyde derivatives 2a-h and 3a-g.
In the second group, the combination of the hydroxy and methoxy substituents, in the para and meta positions of vanillic aldehyde, resulted in an approximately two-fold improvement in the K i value to 251 µM for compound 2g compared to related monosubstituted benzaldehyde derivatives 1g and 1i. This compound is a derivative of naturally occurring vanillic aldehyde, commonly found in vegetation. Therefore, the constant inhibition and biodegradable scaffold components of vanillic derivative 2g seemed acceptable for the protection of plants, especially ornamental plants. An opposite effect to the derivative 2g was observed for isomer 2e when the hydroxy and methoxy groups were present in the ortho and ortho' positions in the benzaldehyde framework. Similarly, the pairs of two methoxy and two hydroxy groups in the ortho, ortho' and ortho, para positions in 2f and 2c, respectively, were practically inactive towards laccase. The hydrazide-hydrazone derivatives of 2,5-dihydroxybenzaldehyde (gensinealdehyde) acted as laccase inhibitors when they were linked to coumarin with an ether bond [34]. In our studies, the hydrazide-hydrazone derivative of this aldehyde with 4-HBAH displayed intense pigmentation. Furthermore, the reaction results were inconclusive (unpublished data) because gensinaldehyde as the hydroquinone was known to be a good laccase substrate. Generally, O-methylation of phenolic hydroxy groups in the Ar-2-OH, Ar-4-OH or 4- OH  compounds 1e, 1g, and 2a to 1h, 1j, and 2h, respectively, caused the loss of inhibition potency (K i ≥ 1000 µM). Interestingly, the introduction of phenyl and tert-butyl substituents at position 3 of salicylic aldehyde fragments, in the hydrazide-hydrazones 2a and 2b, provided the expected competitive type of inhibition with K i values of 25.3 µM and 24.0 µM, respectively (Table 2).
In the third group, the salicylic aldehyde fragment was modified with alkyls on purpose to provide better interaction with the hydrophobic area of the enzyme-substrate cavity ( Table 2). The introduction of hydroxymethyl and hydroxy groups in positions 3 and 5 in the aldehyde framework of 3a or conversely in positions 5 and 3 of 3b caused a total decrease in the activity with K i ≥ 1000 µM. Even the presence of the smallest alkyl unit, the methyl group, at position 6 and an isopropyl group in position 3 in 3e compound, was also not desirable. On the other hand, the transfer of the methyl group to position 5, in a structurally similar molecule, 3d, maintained K i = 26.4 µM at a comparable level to 2a and 2b. Analogous modification in position 5 with the bulky tert-butyl group, in the case of hydrazide-hydrazone 3c, resulted in improvement to the best K i value to 17.9 µM with a simultaneous change in the enzyme-inhibitor interaction. Finally, the transfer of the hydroxy group to the meta position in the hydrazide decoy fragment in 3g, in line with our expectations, changed the mechanism of action to non-competitive with quite good K i = 32.0 µM.
The representative Lineweaver-Burk plots for laccase from T. versicolor in the presence of competitive 2b and uncompetitive 2c inhibitors are depicted in Figure 2. Further results of our research, that is focused on naturally-derived arenecarboxylic acid hydrazide-hydrazones, will be the subject of our future publications. inconclusive (unpublished data) because gensinaldehyde as the hydroquinone was known to be a good laccase substrate. Generally, O-methylation of phenolic hydroxy groups in the Ar-2-OH, Ar-4-OH or 4- OH compounds 1e, 1g, and 2a to 1h, 1j, and 2h, respectively, caused the loss of inhibition potency (Ki ≥ 1000 µM). Interestingly, the introduction of phenyl and tert-butyl substituents at position 3 of salicylic aldehyde fragments, in the hydrazide-hydrazones 2a and 2b, provided the expected competitive type of inhibition with Ki values of 25.3 µ M and 24.0 µ M, respectively (Table 2). In the third group, the salicylic aldehyde fragment was modified with alkyls on purpose to provide better interaction with the hydrophobic area of the enzyme-substrate cavity ( Table 2). The introduction of hydroxymethyl and hydroxy groups in positions 3 and 5 in the aldehyde framework of 3a or conversely in positions 5 and 3 of 3b caused a total decrease in the activity with Ki ≥ 1000 µM. Even the presence of the smallest alkyl unit, the methyl group, at position 6 and an isopropyl group in position 3 in 3e compound, was also not desirable. On the other hand, the transfer of the methyl group to position 5, in a structurally similar molecule, 3d, maintained Ki = 26.4 µ M at a comparable level to 2a and 2b. Analogous modification in position 5 with the bulky tert-butyl group, in the case of hydrazide-hydrazone 3c, resulted in improvement to the best Ki value to 17.9 µ M with a simultaneous change in the enzyme-inhibitor interaction. Finally, the transfer of the hydroxy group to the meta position in the hydrazide decoy fragment in 3g, in line with our expectations, changed the mechanism of action to non-competitive with quite good Ki = 32.0 µ M.
The representative Lineweaver-Burk plots for laccase from T. versicolor in the presence of competitive 2b and uncompetitive 2c inhibitors are depicted in Figure 2. Further results of our research, that is focused on naturally-derived arenecarboxylic acid hydrazide-hydrazones, will be the subject of our future publications.

Structure-Activity Relationship (SAR)
The resulting hydrazide-hydrazones contain two terminal benzene units combined with a hydrazide-hydrazone linker (-(CO)-NH-N=CH-). The compounds act as aza derivatives of naturally occurring 4-hydroxybenzoic acid (4-HBA) or related arenecarboxylic acids which aromatic fragments were found in plants, e.g., in the lignin structural units such as para-coumaryl, coniferyl, and sinapyl alcohols [78]. 4-HBA is also a mediator of laccase [22]. Furthermore, this acid presented in a hydrazide fragment is a naturally occurring motif found in the shikimate biosynthesis pathway. Thus, this part of the molecule was a natural substrate for laccase and serves as a decoy. The second terminal aromatic ring might act similarly, especially if it contains a hydroxy group. The amide group in the linker connected with an azomethine group with a nitrogen-nitrogen single bond (-(CO)-NH-N=CH-) is known to form coordination bonds with metals, i.e., copper cations [79][80][81].

Structure-Activity Relationship (SAR)
The resulting hydrazide-hydrazones contain two terminal benzene units combined with a hydrazide-hydrazone linker (-(CO)-NH-N=CH-). The compounds act as aza derivatives of naturally occurring 4-hydroxybenzoic acid (4-HBA) or related arenecarboxylic acids which aromatic fragments were found in plants, e.g., in the lignin structural units such as para-coumaryl, coniferyl, and sinapyl alcohols [78]. 4-HBA is also a mediator of laccase [22]. Furthermore, this acid presented in a hydrazide fragment is a naturally occurring motif found in the shikimate biosynthesis pathway. Thus, this part of the molecule was a natural substrate for laccase and serves as a decoy. The second terminal aromatic ring might act similarly, especially if it contains a hydroxy group. The amide group in the linker connected with an azomethine group with a nitrogen-nitrogen single bond (-(CO)-NH-N=CH-) is known to form coordination bonds with metals, i.e., copper cations [79][80][81]. We assumed that these three elements would provide the necessary interactions with the laccase substrate-binding pocket. As presented in Figure 3, all highly active compounds contained a mono-or disubstituted salicylic aldehyde framework. Single tert-butyl or phenyl substituenta (R 1 ) located directly at position 3 show K i = 24-25 µM, while disubstituted salicylic aldehydes having a tert-butyl R 1 and methyl or a second tert-butyl substituents R 2 at positions 3 and 5, respectively, provided a comparable inhibition effect with K i = 18-26 µM. comparable inhibition effect with Ki = 18-26 µ M.
The compounds 4-HBAH and 4-HBA had no inhibitory potency towards the target protein. The results were in agreement with the literature data in which the 4-HBA was tested as a natural mediator in laccase-catalyzed reactions [22]. To the best of our knowledge, only humic acids are proved to be potent natural inhibitors of laccase from Panus tigrinus, with an inhibition constant Ki = 3-25 mg/dm 3 (Ki = 0.23-1.62 µ M) [71]. Unfortunately, these soil acids were a mixture of high molecular organic compounds with a variable composition depending on the organic matter they originated from [82]. Also, the mechanism of action was not specific since humic acids have been found recently to be polymerized efficiently by laccase from Streptomyces anulatus [83]. A guanidinium alkaloid isolated from marine organisms, ptilomycalin A, is another example of an organic laccase inhibitor reported in the literature so far [72]. Just like the aforementioned humic acids, they seem unsuitable for the needs of the agricultural industry. The azide ion is a well-known and the most potent inorganic inhibitor of laccases. Furthermore, it is not specific only for laccase, and its inhibition efficacy for other metalloenzymes is generally recognized [84]. Therefore, it was used in our study as a positive reference compound. Until now, the Ki values and mechanism of inhibition were determined simultaneously for laccase from two Pleurotus species using ABTS as substrate. The laccase from Pleurotus eryngii was inhibited by azide ion via a mixed type of inhibition with Kic = 17.6 µ M and Kiu = 10.6 µ M constants [85]. The latter studies performed by Patel and co-workers for laccase from closely related species, Pleurotus ostreatus, showed a similar value of constant inhibition Ki = 16.5 µ M, nonetheless, with a completely different non-competitive type of inhibition [86]. Taking into consideration a discrepancy between these mechanisms of inhibition for NaN3 determined by using ABTS, we carried out measurements with syringaldazine. For this substrate, we obtained data similar to the latter [86], a non-competitive mechanism of inhibition towards laccase from Trametes versicolor. The determined constant is six-fold higher ( Table 2) than the corresponding Ki value measured for laccase from Pleurotus ostreatus.

The Docking Studies
The crystal structures of laccases are widely available in databases. Therefore, the choice of high-resolution structure allowed for careful consideration of the interaction of inhibitors with the enzyme. The laccase from Trametes versicolor PDB: 1GYC [87] was used for SAR studies.
The inhibitors and syringaldazine were docked in the active sites of laccase. It is known that the substrates take part in electron transfer by the interactions with at least one of the following amino acids: Asp206, Asn264 and His458 [88]. The histidine stabilizes one of the copper ions catalyzing the reaction (Figure 4). The compounds 4-HBAH and 4-HBA had no inhibitory potency towards the target protein.
The results were in agreement with the literature data in which the 4-HBA was tested as a natural mediator in laccase-catalyzed reactions [22]. To the best of our knowledge, only humic acids are proved to be potent natural inhibitors of laccase from Panus tigrinus, with an inhibition constant K i = 3-25 mg/dm 3 (K i = 0.23-1.62 µM) [71].
Unfortunately, these soil acids were a mixture of high molecular organic compounds with a variable composition depending on the organic matter they originated from [82]. Also, the mechanism of action was not specific since humic acids have been found recently to be polymerized efficiently by laccase from Streptomyces anulatus [83]. A guanidinium alkaloid isolated from marine organisms, ptilomycalin A, is another example of an organic laccase inhibitor reported in the literature so far [72]. Just like the aforementioned humic acids, they seem unsuitable for the needs of the agricultural industry. The azide ion is a well-known and the most potent inorganic inhibitor of laccases. Furthermore, it is not specific only for laccase, and its inhibition efficacy for other metalloenzymes is generally recognized [84]. Therefore, it was used in our study as a positive reference compound. Until now, the K i values and mechanism of inhibition were determined simultaneously for laccase from two Pleurotus species using ABTS as substrate. The laccase from Pleurotus eryngii was inhibited by azide ion via a mixed type of inhibition with K ic = 17.6 µM and K iu = 10.6 µM constants [85]. The latter studies performed by Patel and co-workers for laccase from closely related species, Pleurotus ostreatus, showed a similar value of constant inhibition K i = 16.5 µM, nonetheless, with a completely different non-competitive type of inhibition [86]. Taking into consideration a discrepancy between these mechanisms of inhibition for NaN 3 determined by using ABTS, we carried out measurements with syringaldazine. For this substrate, we obtained data similar to the latter [86], a non-competitive mechanism of inhibition towards laccase from Trametes versicolor. The determined constant is six-fold higher (Table 2) than the corresponding K i value measured for laccase from Pleurotus ostreatus.

The Docking Studies
The crystal structures of laccases are widely available in databases. Therefore, the choice of high-resolution structure allowed for careful consideration of the interaction of inhibitors with the enzyme. The laccase from Trametes versicolor PDB: 1GYC [87] was used for SAR studies.
The inhibitors and syringaldazine were docked in the active sites of laccase. It is known that the substrates take part in electron transfer by the interactions with at least one of the following amino acids: Asp206, Asn264 and His458 [88]. The histidine stabilizes one of the copper ions catalyzing the reaction (Figure 4). of the methyl group and the addition of the second lipophilic substituent near the hydroxy group ( Figure 5C). We thought that Leu164 might play a significant role, but the removal of the methyl group did not change the Ki value markedly. The inhibitor 2b ( Figure 5B) interacted with amino acid Pro391 by positioning the aromatic ring similar to Leu164. Therefore, we concluded that beneficial changes could be caused by increasing the hydrophobicity of the aldehyde ring and possible OH···N intramolecular interactions that stiffened the structure. Another case concerns compound 3g, which showed non-competitive inhibition. It could bind to the enzyme at the substrate centre, at any allosteric centre, or also to the enzyme-substrate complex. Its behaviour might mimic the natural ligand ( Figure 5D). Although it did not bind in precisely the same way as syringaldazine, as it interacted with His458 as well as the carbonyl oxygen atom as the H-bond acceptor. For compounds 3g and 3c, we also conducted theoretical studies of binding at other places on the enzyme surface ( Figure 6). The position of the inhibitors near the oxygen-binding site worked like a cork ( Figure 6A). Access to the copper ions was blocked. However, the position in a different place on the surface was preferable. Comparing the energy of The alkylated hydrazide-hydrazones 1c, 2b, and 3d presented a competitive type of inhibition which was determined experimentally ( Table 2). The similarity in their structures might allow interactions with the amino acids in the active centre ( Figure 5A-C) affecting the measured inhibition constant values. The substrate cavity was divided into two parts. The first was a deep pocket made of amino acids readily forming hydrogen bonds with Asp206, Asn208, Asn264; but also those stabilizing the position of the aromatic ring, characteristic for the substrate: Phe265, Pro394, Ile455. The second part had a completely different, hydrophobic character with an ample space likely to pre-capture the substrate. The methylated compound 1c ( Figure 5A) fitted this system perfectly. However, the inhibition constant was beneficially lowered for molecule 3d by the following changes: the addition of a hydroxy group near the imine group, the change of the position of the methyl group and the addition of the second lipophilic substituent near the hydroxy group ( Figure 5C). We thought that Leu164 might play a significant role, but the removal of the methyl group did not change the K i value markedly. The inhibitor 2b ( Figure 5B) interacted with amino acid Pro391 by positioning the aromatic ring similar to Leu164. Therefore, we concluded that beneficial changes could be caused by increasing the hydrophobicity of the aldehyde ring and possible OH···N intramolecular interactions that stiffened the structure.
Another case concerns compound 3g, which showed non-competitive inhibition. It could bind to the enzyme at the substrate centre, at any allosteric centre, or also to the enzyme-substrate complex. Its behaviour might mimic the natural ligand ( Figure 5D). Although it did not bind in precisely the same way as syringaldazine, as it interacted with His458 as well as the carbonyl oxygen atom as the H-bond acceptor. For compounds 3g and 3c, we also conducted theoretical studies of binding at other places on the enzyme surface ( Figure 6). The position of the inhibitors near the oxygen-binding site worked like a cork ( Figure 6A). Access to the copper ions was blocked. However, the position in a different place on the surface was preferable. Comparing the energy of the binding for near the oxygen surface to the energy of the binding to unspecific position on the surface for 3c (−33.93 kcal/mol vs −38.91 kcal/mol) and 3g (−36.21 kcal/mol vs −44.51 kcal/mol) revealed that their affinity to a non-oxygen cavity was higher. The oxygen tunnel did not change the original form then ( Figure 6B).

Molecular Docking
A crystal structure of an enzyme was obtained from the RCSB Protein Data Bank (PDB). The 1GYC [87] was prepared using Schrödinger's Protein Preparation Wizard module [100]. Water molecules and ligands were deleted and then hydrogen atoms were added and set states generated using the Epik with consideration of experimental pH. The next step was the optimization of structure with the use of PROPKA3 [101] with the experimental pH as well and final minimization with OPLS3e force field. All ligands were prepared and optimized with the OPLS3e force field in LigPrep Schrödinger's module [102]. The stereochemistry of the imine carbon atom was considered. Inhibitors were docked with the Induced Fit Docking Protocole [103]. The copper atoms were selected as centroids of the box with a size of 20 Å. Glide docking and prime refinement were followed as the recommended procedures when the XP precision was selected in the Glide redocking. All of the docked ligands were primed again with Prime MM-GBSA protocol [104] and the compounds with the lower ∆G Bind energy for each inhibitor were selected as the final results.

Conclusions
In this article, we identified a novel class of low-molecular-weight hydrazide-hydrazones, which contained naturally derived units of aldehydes having an aromatic motif and/or hydroxybenzoic acids. We presented the characteristics of 25 molecules, including 14 new ones, and their inhibition potency toward laccase of fungal origin. The results of the syringaldazine oxidation in the presence of the compounds indicated that they essentially acted as reversible competitive inhibitors, with some exceptions. The computational docking simulations showed that both aromatic fragments connected by a hydrazide linker in the molecules were crucial for their activity. The hydrazide fragment contained a para-hydroxybenzoic acid unit that served as a decoy, reminiscent of typical phenolic laccase substrates. In addition, salicylic aldehydes with bulky groups in the adjacent 3rd position provided strong interaction with the hydrophobic part of the substrate-binding centre. In this regard, the reversible competitive action on laccase of the tested hydrazide-hydrazones is beneficial for further design of inhibitors for bio-application.
Supplementary Materials: The following are available online, Figure S1-S354: NMR spectra and 2D experiments of selected compounds 1-8; Figure S355: The stability of laccase from T. versicolor in buffer-organic co-solvent media.