Integracides: Tetracyclic Triterpenoids from Fusarium sp.—Their 5-Lipoxygenase Inhibitory Potential and Structure–Activity Relation Using In Vitro and Molecular Docking Studies

Inflammation is a complicated disorder that is produced as a result of consecutive processes. 5-LOX (5-lipoxygenase) is accountable for various inflammation mediators and leukotrienes synthesis, and its inhibition is the target of anti-inflammation therapeutics. Fungi have acquired enormous attentiveness because of their capability to biosynthesize novel bio-metabolites that reveal diversified bio-activities. A new tetracyclic triterpenoid, integracide L (1), along with integracides B (2) and F (3), were separated from Mentha longifolia-associated Fusarium sp. (FS No. MAR2014). Their structures were verified utilizing varied spectral analyses. The isolated metabolites (1–3), alongside the earlier reported integracides G (4), H (5), and J (6), were inspected for 5-LOX inhibition capacity. Interestingly, 1–6 possessed marked 5-LOX inhibition potentials with IC50s ranging from 1.18 to 3.97 μM compared to zileuton (IC50 1.17 µM). Additionally, molecular docking was executed to examine the interaction among these metabolites and 5-LOX, as well as to validate the in vitro findings. The docking study revealed their inhibitory activity interactions in the binding pocket. These findings highlighted the potential of integracides as lead metabolites for anti-inflammation drug discovery.


5-LOX Inhibitory Activity
In the present investigation, integracides 1-6 reported from Fusarium sp. (FS No. MAR2014) were investigated to explore their 5-LOI capability. It is noteworthy that

Molecular Docking Studies
The docking study was performed with the Schrodinger program. Inregracides, zileuton, and NDGA (native co-crystallized-inhibitor) were prepared before docking using the "LigPrep" tool, where all ligand 2D structures were converted to 3D and energy minimized [36]. All the ligands' possible tautomeric states and ionization were created as well. The crystal structure of stable 5-LOX complexed with the NDGA inhibitor was downloaded from the protein-data-bank (PDB-ID: 6N2W), prepared, and energy minimized employing "Protein_Preparation_Wizard" [37,38]. A grid box was created around the active site of the stable 5-LOX (PDB: 6N2W) containing the co-crystallized inhibitor NDGA using Glide's "Receptor-Grid-Generation" tool in the Schrödinger suite. Finally, the "Ligand_Docking" tool was implemented for docking [39,40]. All the studied ligands were docked inside the grid box with XP (extra-precision) protocol [41]. The docking method was validated by redocking NDGA (native co-crystallized-inhibitor) in the prepared protein active site. When it was superimposed over the original co-crystallized in-  (1)(2)(3)(4)(5)(6). * Compared to control group; # compared to indomethacin group (one-way ANOVA followed by Tukey-Kramer).

Molecular Docking Studies
The docking study was performed with the Schrodinger program. Inregracides, zileuton, and NDGA (native co-crystallized-inhibitor) were prepared before docking using the "LigPrep" tool, where all ligand 2D structures were converted to 3D and energy minimized [36]. All the ligands' possible tautomeric states and ionization were created as well. The crystal structure of stable 5-LOX complexed with the NDGA inhibitor was downloaded from the protein-data-bank (PDB-ID: 6N2W), prepared, and energy minimized employing "Protein_Preparation_Wizard" [37,38]. A grid box was created around the active site of the stable 5-LOX (PDB: 6N2W) containing the co-crystallized inhibitor NDGA using Glide's "Receptor-Grid-Generation" tool in the Schrödinger suite. Finally, the "Ligand_Docking" tool was implemented for docking [39,40]. All the studied ligands were docked inside the grid box with XP (extra-precision) protocol [41]. The docking method was validated by redocking NDGA (native co-crystallized-inhibitor) in the prepared protein active site. When it was superimposed over the original co-crystallized inhibitor, it gave a 1.3132 Å RMSD (root-mean-square deviation) calculated value, indicating a valid docking method ( Figure 5). The chosen crystal structure of 5-LOX (PDB: 6N2W) is a protein-stabilized form in which stabilizing mutations are implemented, and membrane insertion loops are absent [37]. However, it still contains the canonical LOX fold, which is composed of the aminoterminal β-barrel membrane-binding domain and the α-helical domain in which the active site is located [38]. The active site of 5-LOX (where the arachidonic acid binds) forms a Ushape hydrophobic cavity, which contains a catalytic metal iron [37,42].
The docking findings performed with the XP mode for the energy-minimized 3D structures of integracides and zileuton are listed below in Table 2. The docked compounds are ranked based on their gscores related to the free energy of the binding; the more negative scores imply better binding. The generated scores are the gscore (ranks different metabolites), emodel (ranks different conformers), and XP gscore. Glide employs emodel scoring to choose the docked compounds' best poses; then, it ranks the best poses relying on the given gscores. The XP gscore ranks the Glide XP mode-created poses. In general, Glide uses the gscore to sort and rank the docked metabolites.

Discussion
Inflammation is a complicated response that is produced as a result of consecutive processes, one of which is arachidonic acid's metabolism, which commences with oxida- The chosen crystal structure of 5-LOX (PDB: 6N2W) is a protein-stabilized form in which stabilizing mutations are implemented, and membrane insertion loops are absent [37]. However, it still contains the canonical LOX fold, which is composed of the amino-terminal β-barrel membrane-binding domain and the α-helical domain in which the active site is located [38]. The active site of 5-LOX (where the arachidonic acid binds) forms a U-shape hydrophobic cavity, which contains a catalytic metal iron [37,42].
The docking findings performed with the XP mode for the energy-minimized 3D structures of integracides and zileuton are listed below in Table 2. The docked compounds are ranked based on their gscores related to the free energy of the binding; the more negative scores imply better binding. The generated scores are the gscore (ranks different metabolites), emodel (ranks different conformers), and XP gscore. Glide employs emodel scoring to choose the docked compounds' best poses; then, it ranks the best poses relying on the given gscores. The XP gscore ranks the Glide XP mode-created poses. In general, Glide uses the gscore to sort and rank the docked metabolites.

Discussion
Inflammation is a complicated response that is produced as a result of consecutive processes, one of which is arachidonic acid's metabolism, which commences with oxidation by 5-LOX. It is proved that 5-LOX has a leading function in inflammation through the synthesis of various inflammation mediators and LTs (leukotrienes). 5-LOX has protruded as a prospective target for inflammation-linked disorders, including rheumatoid arthritis and asthma. Many of the available 5-LOX inhibitors are of synthetic origin and reveal untoward aftereffects, such as zileuton, that have the hepato-toxic potential [8,9]. Hence, finding out safe and efficacious anti-inflammation agents that modulate LT production is an imperious demand.
Fungi possess a miracle capability to produce unrivaled metabolites, and the varied bioactivities among them are terpenoids, including sesqui-, di-, mero-, and tri-terpenoids. Several reports stated the anti-inflammation potential of fungal terpenoids through varied mechanisms [43,44].
Among the reported fungal terpenoids, integracides are an uncommon class of triterpenoids that report mainly from Fusarium sp. (FS No. MAR2014). This class of metabolites displayed varied bioactivities.
In the current study, a new metabolite belonging to this class, along with the known ones, was purified and characterized utilizing various tools. Their in vitro anti-inflammation potencies, as assessed by their attenuation of 5-LOX, demonstrated the powerful 5-LOI capability of these metabolites. It was noted that compounds 4, 5, and 6 were the most potent compounds that had IC 50 values that were comparable to that of the positive control, zileuton.
It is noteworthy that there were many studies that proved the anti-inflammatory effectiveness of triterpenoids via inhibition of 5-LOX [45][46][47].
In the molecular docking studies of this work, the data showed a better understanding of each inhibitor's potency as well as their correlation with the bioassay throughout the binding and mode of interactions. For instance, the docking results of 4 were consistent with its biochemical inhibitory results, where it produced the lowest IC 50 (1.18 µM) similar to that of zileuton ( Figure 4).
The XP gscore ranking of integracide F (3), H (5), and J (6) showed minimal correlation to their in vitro inhibitory effectiveness, although the scores were close to each other. On the other hand, integracide B (2) and L (1) were ranked the least in terms of their gscores and IC 50 values as well.
The general schematic structure of integracides contains a cyclic hydrophobic skeleton composed of four rings (A, B, C, and D), a hydrophobic R group on C17 of ring D, an acetyloxy group on C12 of ring C, a hydroxyl group on C11 of ring C, a hydroxyl group on C2 of ring A (which may be acetylated) and a hydroxyl group on C3 of ring A (which is usually esterified by different groups) ( Figure 6). The presence and type of the esterified groups on C3 could influence the binding affinity to the 5-LOX active site. synthesis of various inflammation mediators and LTs (leukotrienes). 5-LOX has protruded as a prospective target for inflammation-linked disorders, including rheumatoid arthritis and asthma. Many of the available 5-LOX inhibitors are of synthetic origin and reveal untoward aftereffects, such as zileuton, that have the hepato-toxic potential [8,9]. Hence, finding out safe and efficacious anti-inflammation agents that modulate LT production is an imperious demand. Fungi possess a miracle capability to produce unrivaled metabolites, and the varied bioactivities among them are terpenoids, including sesqui-, di-, mero-, and tri-terpenoids. Several reports stated the anti-inflammation potential of fungal terpenoids through varied mechanisms [43,44].
Among the reported fungal terpenoids, integracides are an uncommon class of triterpenoids that report mainly from Fusarium sp. (FS No. MAR2014). This class of metabolites displayed varied bioactivities.
In the current study, a new metabolite belonging to this class, along with the known ones, was purified and characterized utilizing various tools. Their in vitro anti-inflammation potencies, as assessed by their attenuation of 5-LOX, demonstrated the powerful 5-LOI capability of these metabolites. It was noted that compounds 4, 5, and 6 were the most potent compounds that had IC50 values that were comparable to that of the positive control, zileuton.
It is noteworthy that there were many studies that proved the anti-inflammatory effectiveness of triterpenoids via inhibition of 5-LOX [45][46][47].
In the molecular docking studies of this work, the data showed a better understanding of each inhibitor's potency as well as their correlation with the bioassay throughout the binding and mode of interactions. For instance, the docking results of 4 were consistent with its biochemical inhibitory results, where it produced the lowest IC50 (1.18 μM) similar to that of zileuton ( Figure 4).
The XP gscore ranking of integracide F (3), H (5), and J (6) showed minimal correlation to their in vitro inhibitory effectiveness, although the scores were close to each other. On the other hand, integracide B (2) and L (1) were ranked the least in terms of their gscores and IC50 values as well.
The general schematic structure of integracides contains a cyclic hydrophobic skeleton composed of four rings (A, B, C, and D), a hydrophobic R group on C17 of ring D, an acetyloxy group on C12 of ring C, a hydroxyl group on C11 of ring C, a hydroxyl group on C2 of ring A (which may be acetylated) and a hydroxyl group on C3 of ring A (which is usually esterified by different groups) ( Figure 6). The presence and type of the esterified groups on C3 could influence the binding affinity to the 5-LOX active site. Integracide G (4) was the top-scored metabolite with a gscore of −6.708 kcal/mol among the other derivatives and was closer in score to NDGA (co-crystallized inhibitor) ( Table 2).  Integracide G (4) was the top-scored metabolite with a gscore of −6.708 kcal/mol among the other derivatives and was closer in score to NDGA (co-crystallized inhibitor) ( Table 2).
The aliphatic substitution on the cyclopentene ring (ring D) involved in hydrophobic interactions with a hydrophobic pocket contained mainly Ile, Leu, and Ala residues. Both the OH group on C11 and the carbonyl oxygen of the acetyloxy group on ring C formed aromatic-hydrogen and H-bond interactions, respectively, with His432. The molecule has a hydroxy decanoate chain (substituted on C3 of ring A) where part of it was exposed to the solvent, and the rest entered a small groove in the active site, adding extra binding interactions (Figure 7). The aliphatic substitution on the cyclopentene ring (ring D) involved in hydrophobic interactions with a hydrophobic pocket contained mainly Ile, Leu, and Ala residues. Both the OH group on C11 and the carbonyl oxygen of the acetyloxy group on ring C formed aromatic-hydrogen and H-bond interactions, respectively, with His432. The molecule has a hydroxy decanoate chain (substituted on C3 of ring A) where part of it was exposed to the solvent, and the rest entered a small groove in the active site, adding extra binding interactions (Figure 7).  Integracide H (5) contains two acetyloxy substituents on C2 and C3 of ring A and is involved in H-bond interactions with Arg596. However, the two groups were partially solvent-exposed (Figure 8). Integracide H (5) contains two acetyloxy substituents on C2 and C3 of ring A and is involved in H-bond interactions with Arg596. However, the two groups were partially solvent-exposed (Figure 8).  For integracide J (6), it has a p-hydroxybenzoyloxy substitution on C3 of ring A instead of the long chain present in 4. The C=O of the acetyloxy group on C12 and -OH on C2 interacted with His432 through H-bonds. Moreover, the benzoyl C=O formed an H-bond with Arg596; however, the phenolic OH group was exposed to the solvent and did not involve any type of binding with the protein (Figure 9). For integracide J (6), it has a p-hydroxybenzoyloxy substitution on C3 of ring A instead of the long chain present in 4. The C=O of the acetyloxy group on C12 and -OH on C2 interacted with His432 through H-bonds. Moreover, the benzoyl C=O formed an Hbond with Arg596; however, the phenolic OH group was exposed to the solvent and did not involve any type of binding with the protein (Figure 9).  Integracide L (1) has a saturated cyclopenty ring and OH group on C17, which are not present in any other integracide analogs. These modifications may not be favored, directing the R group away from the hydrophobic pocket and being more exposed to the solvent. This change may influence the binding affinity and increase the gscore and IC 50 for this compound (Table 2 and Figure 4). The acetyloxy groups on rings A and C formed H-bonds with Arg596 and His432, respectively ( Figure 11).   Integracide L (1) has a saturated cyclopenty ring and OH group on C17, which are not present in any other integracide analogs. These modifications may not be favored, directing the R group away from the hydrophobic pocket and being more exposed to the solvent. This change may influence the binding affinity and increase the gscore and IC50 for this compound (Table 2 and Figure 4). The acetyloxy groups on rings A and C formed H-bonds with Arg596 and His432, respectively ( Figure 11).  Integracide B (2) was bound to the active site in a different mode, opposite to what has been noted with other integracides (Figure 12). The presence of free OH groups on ring A could contribute to its low binding affinity, illustrated by the gscore of −5.051 kcal/mol and IC 50 value of 3.97 µM, among other integracides (Table 2). color within the active site of 5-LOX. The H-bond and aromatic-hydrogen interactions are in low and purple dotted lines, respectively. Integracide B (2) was bound to the active site in a different mode, opposite to has been noted with other integracides (Figure 12). The presence of free OH group ring A could contribute to its low binding affinity, illustrated by the gscore of − kcal/mol and IC50 value of 3.97 μM, among other integracides (Table 2).  The positive control inhibitor, zileuton, was also docked in the 5-LOX active site to investigate its binding mode. The amide-NH 2 formed an H-bond with Gln363, whereas the thiophene ring formed a π-π interaction with the imidazole ring in His372 (Figure 13), similar to what was observed for NDGA ( Figure 14). Although zileuton produced the highest potency in the biochemical assay (IC 50 1.17 µM), little correlation was observed with its gscore (−4.766 kcal/mol), and it ranked last among the other tested compounds ( Table 2).
The positive control inhibitor, zileuton, was also docked in the 5-LOX active site to investigate its binding mode. The amide-NH2 formed an H-bond with Gln363, whereas the thiophene ring formed a π-π interaction with the imidazole ring in His372 (Figure 13), similar to what was observed for NDGA ( Figure 14). Although zileuton produced the highest potency in the biochemical assay (IC50 1.17 μM), little correlation was observed with its gscore (−4.766 kcal/mol), and it ranked last among the other tested compounds ( Table 2).  Accordingly, it was noted that the substitution pattern of the intergracides' terpenoid framework might influence the efficacy. Substitution at C-3 by long-chain fatty acid acyl as in 4 or p-hydroxy benzoyl as in 6 was found to elevate the activity. Additionally, increasing the number of acetyl groups raised the activity, as well as the conjugated C8-C9-C14-C15, which may have a role in the activity ( Figure 15). Further, the lacking a C14-C15  Accordingly, it was noted that the substitution pattern of the intergracides' terpenoid framework might influence the efficacy. Substitution at C-3 by long-chain fatty acid acyl as in 4 or p-hydroxy benzoyl as in 6 was found to elevate the activity. Additionally, increasing the number of acetyl groups raised the activity, as well as the conjugated C 8 -C 9 -C 14 -C 15, which may have a role in the activity ( Figure 15). Further, the lacking a C14-C15 double bond and the presence of the C17-OH group substitution minimized the efficacy. Accordingly, it was noted that the substitution pattern of the intergracides' terpenoid framework might influence the efficacy. Substitution at C-3 by long-chain fatty acid acyl as in 4 or p-hydroxy benzoyl as in 6 was found to elevate the activity. Additionally, increasing the number of acetyl groups raised the activity, as well as the conjugated C8-C9-C14-C15, which may have a role in the activity ( Figure 15). Further, the lacking a C14-C15 double bond and the presence of the C17-OH group substitution minimized the efficacy.

Cultivation of Fungal Material
The earlier separated and identified Fusarium sp. (FS No. MAR2014) was cultivated in 20 Erlenmeyer flasks (1 L each) as stated formerly [20,30].

Extraction and Isolation
The culture extraction was carried out utilizing EtOAc and was concentrated by a vacuum. Subsequently, mixing the extract with H 2 O (300 mL) and partitioning among n-hexane and MeOH (90%) were performed. The MeOH extract (6.9 g) was submitted to the Sephadex LH-20 column chromatography (CC, MeOH/CHCl 3

Statistical Analysis
IC 50 s were estimated by regression analysis (GraphPad-InStat 3, GraphPad Software, San Diego, CA, USA). The % 5-LOX inhibition of and IC 50 s are listed as mean-values ±SD. Statistical significance was analyzed among the samples by one-way-ANOVA and subsequently by Tukey-Kramer test (p < 0.005).

Ligand and Protein Preparation
The docking study was performed with the Schrodinger program (Schrödinger Release 2021-4: LigPrep, Schrödinger, LLC, New York, NY, USA, 2021). The crystal structure of stable 5-LOX complexed with the NDGA inhibitor was downloaded from the protein data bank (PDB; ID: 6N2W). The protein was prepared using the "Protein Preparation Wizard" tool in Maestro software, where the missing hydrogens were added to the residues, the metal ionization state was corrected, and the water molecules > 5 Å from protein residues were deleted. The protein contained two chains: A and B. Chain B was complexed with the inhibitor; therefore, it was chosen to perform the docking study. The protein was then refined by predicting the pKa of the ionizable residues using PROPKA and water molecules > 3 Å (not involved in the water bridge), which were removed [36]. Additionally, the proteins containing Fe 2+ coordinates with His residues were in the active site. The protein-Fe 2+ bonds were deleted, and finally, the minimization of the protein was applied using the OPLS4 force field. All ligands were prepared before docking using the "LigPrep" tool [53]. The 2D structures of the ligands were converted to 3D and energy-minimized using the OPLS3 force field. The hydrogens were added, and all possible ionization states and tautomeric forms were created at a pH of 7.0 ± 0.2 by Epik; a desalt option was also chosen. The H-bonds were optimized by predicting the pKa of ionizable groups using PROPKA [36].

Grid Generation and Molecular Docking
To perform the docking, a grid box was generated around the active site of stable 5-LOX (PDB: 6N2W) containing the co-crystallized inhibitor NDGA and using Glide's "Receptor-Grid-Generation" tool in Schrödinger suite [39]. All study ligands were docked inside the grid box with an extra precision (XP) protocol, and all other parameters were set to default [41]. The non-polar atoms were set for the VdW radii scaling factor by 1.0, and the partial charge cut-off was 0.25. Finally, the "Ligand Docking" tool was implemented for docking [40]. To validate the docking method, the co-crystallized inhibitor was re-docked inside the grid box and evaluated.

Conclusions
A new tetracyclic triterpenoid, integracide L (1) and two metabolites (2 and 3), were purified from Fusarium sp. (FS No. MAR2014). Their specification was achieved with the assistance of extensive spectral analyses. Compounds 1-6 demonstrated a noticeable 5-LOX inhibition potential. The docking study of integracides illustrated their binding mode in the active site with possible amino acid interactions, which could explain their inhibitory activity.
These findings may foster more inspection of the possible usage of integracides as 5-LOX inhibitors and draw further interest to the synthesis of structure-similar analogs with enhanced 5-LOX inhibition capacity. Moreover, additional in vivo and mechanistic investigations are needed.