Bioactive α-Pyrone Analogs from the Endophytic Fungus Diaporthe sp. CB10100: α-Glucosidase Inhibitory Activity, Molecular Docking, and Molecular Dynamics Studies

Two α-pyrone analogs were isolated from the endophytic fungus Diaporthe sp. CB10100, which is derived from the medicinal plant Sinomenium acutum. These analogs included a new compound, diaporpyrone F (3), and a known compound, diaporpyrone D (4). The structure of 3 was identified by a comprehensive examination of HRESIMS, 1D and 2D NMR spectroscopic data. Bioinformatics analysis revealed that biosynthetic gene clusters for α-pyrone analogs are common in fungi of Diaporthe species. The in vitro α-glucosidase inhibitory activity and antibacterial assay of 4 revealed that it has a 46.40% inhibitory effect on α-glucosidase at 800 μM, while no antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Mycolicibacterium (Mycobacterium) smegmatis or Klebsiella pneumoniae at 64 μg/mL. Molecular docking and molecular dynamics simulations of 4 with α-glucosidase further suggested that the compounds are potential α-glucosidase inhibitors. Therefore, α-pyrone analogs can be used as lead compounds for α-glucosidase inhibitors in more in-depth studies.

α-Pyrone, an aromatic unsaturated lactone, is present as a substructure in a diverse range of natural substances that exhibit noteworthy biological activities [14].Many αpyrones with anti-diabetes effects have been discovered from Diaporthe species [10,15,16].For example, cytospone E (Figure 1A, 1) derived from the endophytic fungus Cytospora rhizophorae A761 showed a 41.0%rate of α-glucosidase inhibition at 100 μM; in contrast, acarbose inhibited α-glucosidase by 34.5% at the same concentration [15].Alternolide C (Figure 1A, 2), isolated from the marine-derived fungus Alternaria alternata LW37, was demonstrated to exhibit inhibitory effects against α-glucosidase, with an IC50 value of 451.25 ± 6.95 μM [16].As part of our ongoing inquiry into the secondary metabolites produced by endophytic fungi, we discovered a number of intriguing natural products in Diaporthe sp.CB10100 that was isolated from Sinomenium acutum.These products consist of ellagic acid B, a dibenzo-α-pyrone derivative, and four α-pyrones, diaporpyrones A-D (S1-S4) [17].In the present work, further chemical studies were performed with this fungus to identify novel bioactive chemicals.In this study, an undescribed α-pyrone derivative named diaporpyrone F (Figure 1B, 3) was isolated from the endophytic Diaporthe sp.CB10100, as well as a known compound, diaporpyrone D (Figure 1B, 4).The structures of the new natural compounds were established with full confidence using NMR and HRESIMS.An in vitro α-glucosidase inhibitory activity and antibacterial assay revealed that 4 exhibits a 46.40% inhibitory effect on α-glucosidase at 800 µM but shows no antibacterial activity against MRSA, Mycolicibacterium (Mycobacterium) smegmatis or Klebsiella pneumoniae at 64 µg/mL.Molecular docking and molecular dynamics In the present work, further chemical studies were performed with this fungus to identify novel bioactive chemicals.In this study, an undescribed α-pyrone derivative named diaporpyrone F (Figure 1B, 3) was isolated from the endophytic Diaporthe sp.CB10100, as well as a known compound, diaporpyrone D (Figure 1B, 4).The structures of the new natural compounds were established with full confidence using NMR and HRESIMS.An in vitro α-glucosidase inhibitory activity and antibacterial assay revealed that 4 exhibits a 46.40% inhibitory effect on α-glucosidase at 800 µM but shows no antibacterial activity against MRSA, Mycolicibacterium (Mycobacterium) smegmatis or Klebsiella pneumoniae at 64 µg/mL.Molecular docking and molecular dynamics simulations of 4 with α-glucosidase further suggested that 4 is a potential α-glucosidase inhibitor.Our investigation indicated that compound 4, together with the commonly used α-pyrone scaffold, shows potential as a powerful inhibitor of α-glucosidase.

Analysis of Secondary Metabolite Biosynthetic Potential
Five α-pyrone derivatives, including diaporpyrone F (3), have been isolated from Diaporthe sp.CB10100; thus, we were curious whether similar backbone compounds could be found in other fungi of the genus Diaporthe [17].Whole-genome sequencing has been performed for 18 strains of the genus Diaporthe according to the NCBI database [10].According to the antiSMASH 7.1.0database, all eighteen of these whole-genome sequences included pyrone biosynthetic gene clusters (Table 2).Surprisingly, the full gene sequence of Diaporthe sp.HANT25 contained four clusters of genes for the biosynthesis of α-pyrone analogs.This information will set the stage for further studies on the biosynthesis and synthetic biology of α-pyrone analogs.

Antibacterial Assay
Using the microbroth dilution method, the antibacterial activities of 3 and 4 against MRSA, Mycolicibacterium (Mycobacterium) smegmatis and Klebsiella pneumoniae were determined (Figure S21).The MICs of these compounds were greater than 64 µg/mL, and no significant inhibitory activity was observed (Table 3).

Molecular Docking
To investigate the molecular interactions between 4 and α-glucosidase, a molecular docking study was performed using the program AutoDock Vina 1.1.2.The molecular docking models of 4 are illustrated in Figure 2. The docking results revealed that 4 formed a hydrogen bond and a hydrophobic interaction with Tyr-299, a hydrophobic interaction with Trp-406 and one salt bridge with the His-600 residue (Figure 2).Furthermore, the affinities of the aforementioned inhibitors were calculated, revealing that acarbose has a binding energy of 5.5 kcal/mol and that 4 has a binding energy of 5.4 kcal/mol; therefore, these compounds may stably bind to α-glucosidase (Table S1).The results obtained for the docking energy and α-glucosidase inhibitory activity experiments corresponded well.As shown in Figure S20, more hydrogen bonds and hydrophobic bonds formed between acarbose and α-glucosidase than between compound 4 and α-glucosidase.This docking experiment may provide insight into mechanisms by which α-pyrone and α-glucosidase bind since different intermolecular interactions may exert varying inhibitory effects.

Molecular Dynamics Simulations
Subsequently, a molecular dynamics simulation was run under physiologica simulated conditions to clarify the binding pattern, stability, and molecular interact mode of 4 with the α-glucosidase protein complex.Root-mean-square deviation (RMS root-mean-square fluctuation (RMSF), and hydrogen bond studies were utilized investigate the dynamic changes and stability of complex systems.Structural stability often characterized by low RMSD and RMSF values [18].As shown in Figure 3A, RMSD of the two systems, α-glucosidase/acarbose and α-glucosidase/diaporpyrone D are plotted in the RMSD variation graphs during the simulation.The two syste gradually converge in the first 5 ns of the simulation and maintain very stable fluctuati in the subsequent simulations, with the RMSD maintaining fluctuations within 1-2 Based on the stable fluctuations of the two systems, the systems are stable in combinati As shown in Figure 3B, the RMSF of all proteins after binding different small molecu was low, which indicates that the core structure of the proteins has good rigid Therefore, these proteins are more rigid as binding small molecules, and these sm molecules have an inhibitory effect.Notably, the red line and the blue line have a h degree of superposition, indicating that the two small molecules have similar effects the proteins.The radius of gyration (RoG) reflects the embodied compactness and reflect the degree of densification of the system.In Figure 3C, we can observe that glucosidase/acarbose and α-glucosidase/diaporpyrone D (4) fluctuate similarly, and b systems exhibit similar binding effects.A detailed analysis revealed that the RoG of glucosidase/acarbose was mostly smaller during the simulation, implying that the syst became more compact, corresponding to relatively stronger binding.

Molecular Dynamics Simulations
Subsequently, a molecular dynamics simulation was run under physiologically simulated conditions to clarify the binding pattern, stability, and molecular interaction mode of 4 with the α-glucosidase protein complex.Root-mean-square deviation (RMSD), rootmean-square fluctuation (RMSF), and hydrogen bond studies were utilized to investigate the dynamic changes and stability of complex systems.Structural stability is often characterized by low RMSD and RMSF values [18].As shown in Figure 3A, the RMSD of the two systems, α-glucosidase/acarbose and α-glucosidase/diaporpyrone D (4), are plotted in the RMSD variation graphs during the simulation.The two systems gradually converge in the first 5 ns of the simulation and maintain very stable fluctuations in the subsequent simulations, with the RMSD maintaining fluctuations within 1-2 Å.Based on the stable fluctuations of the two systems, the systems are stable in combination.As shown in Figure 3B, the RMSF of all proteins after binding different small molecules was low, which indicates that the core structure of the proteins has good rigidity.Therefore, these proteins are more rigid as binding small molecules, and these small molecules have an inhibitory effect.Notably, the red line and the blue line have a high degree of superposition, indicating that the two small molecules have similar effects on the proteins.The radius of gyration (RoG) reflects the embodied compactness and can reflect the degree of densification of the system.In Figure 3C, we can observe that α-glucosidase/acarbose and α-glucosidase/diaporpyrone D (4) fluctuate similarly, and both systems exhibit similar binding effects.A detailed analysis revealed that the RoG of α-glucosidase/acarbose was mostly smaller during the simulation, implying that the system became more compact, corresponding to relatively stronger binding.
Based on the trajectories of the molecular dynamic simulations, we calculated the binding energies using the MM-GBSA method, which can more accurately reflect the binding modes of small molecules and target proteins.The binding energies of the αglucosidase/acarbose and α-glucosidase/diaporpyrone D (4) complexes were −36.59 ± 3.30 and −23.06 ± 3.77 kcal/mol, respectively (Table S2), and negative values indicate that the two molecules have the potential to bind to the target proteins, while lower values indicate stronger binding.The lower the value is, the stronger the binding.Our calculations show that compared to diaporpyrone D (4), α-glucosidase/acarbose binds better and has a value slightly lower.For the α-glucosidase/acarbose complex, the binding energy is mainly contributed by electrostatic energy and van der Waals energy; for α-glucosidase/diaporpyrone D (4), the binding energy is mainly contributed by van der Waals energy.The nonpolar solvation free energy contributes weakly to both complexes.Hydrogen bonding is among the strongest noncovalent binding interactions, and a greater number of hydrogen bonds indicates better binding.Figure 3D shows that the number of hydrogen bonds of αglucosidase/acarbose was maintained at 2-9 and mostly fluctuated around 5, which implies that hydrogen bonding plays an important role in the stabilization of acarbose binding.In contrast, the number of hydrogen bonds in the α-glucosidase/diaporpyrone D (4) complex fluctuated more during the simulation period (0-5), and the number of hydrogen bonds was greater in the presimulation period (2)(3)(4); the number of hydrogen bonds was also lower in the middle of the simulation period (0-2) and late simulation period, (0-4).This finding implies that hydrogen bonding contributes weakly to α-glucosidase/diaporpyrone D (4) binding.Based on the trajectories of the molecular dynamic simulations, we calculated the binding energies using the MM-GBSA method, which can more accurately reflect the binding modes of small molecules and target proteins.The binding energies of the αglucosidase/acarbose and α-glucosidase/diaporpyrone D (4) complexes were −36.59 ± 3.30 and −23.06 ± 3.77 kcal/mol, respectively (Table S2), and negative values indicate that the two molecules have the potential to bind to the target proteins, while lower values indicate stronger binding.The lower the value is, the stronger the binding.Our calculations show that compared to diaporpyrone D (4), α-glucosidase/acarbose binds better and has a value slightly lower.For the α-glucosidase/acarbose complex, the binding energy is mainly contributed by electrostatic energy and van der Waals energy; for αglucosidase/diaporpyrone D (4), the binding energy is mainly contributed by van der Waals energy.The nonpolar solvation free energy contributes weakly to both complexes.Hydrogen bonding is among the strongest noncovalent binding interactions, and a greater number of hydrogen bonds indicates better binding.Figure 3D shows that the number of hydrogen bonds of α-glucosidase/acarbose was maintained at 2-9 and mostly fluctuated around 5, which implies that hydrogen bonding plays an important role in the stabilization of acarbose binding.In contrast, the number of hydrogen bonds in the αglucosidase/diaporpyrone D (4) complex fluctuated more during the simulation period (0-5), and the number of hydrogen bonds was greater in the presimulation period (2-4);

General Methods
As previously reported, various instruments (including those used for MS and NMR) and standard reagents for chemical isolation and biological evaluation were utilized [17].The details are provided in the Supplementary Information.

Fermentation of Diaporthe sp. CB10100
The fermentation and extraction processes for Diaporthe sp.CB10100 were identical to those reported in previous work [17].

α-Glucosidase Inhibition Assay
The inhibitory activity of compounds 3 and 4 against α-glucosidase [Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China, Product No. G5003] was examined using the Worawalai technique with minor modifications [19].The in vitro α-glucosidase inhibitory activity test was performed spectrophotometrically by detecting the α-glucosidase levels at 405 nm.The reaction system is described in the Supplementary Information.

Antibacterial Assay
The broth dilution technique was used to determine the MICs [20].The specifics are included in the Supplementary Information.

Conclusions
In conclusion, a new α-pyrone, diaporpyrone F (3), together with one known compound, diaporpyrone D (4), was isolated from the endophytic fungus Diaporthe sp.CB10100.NMR and HRESIMS spectra were used to establish the structures of 3 and 4. Bioinformatics analysis revealed that biosynthetic gene clusters for α-pyrone analogs are common in fungi of Diaporthe species.These compounds were evaluated for their inhibitory activity against α-glucosidase, and 4 showed a 46.40% inhibitory effect against α-glucosidase at 800 µM.Evaluations of the inhibitory activity against MRSA, M. smegmatis and K. pneumoniae revealed that the MICs of these compounds were greater than 64 µg/mL.Molecular docking and molecular dynamics simulations of 4 with α-glucosidase further suggested that 4 is a potential α-glucosidase inhibitor.In view of the above results, α-pyrone skeletons can be further investigated as lead compounds for α-glucoside inhibitors.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29081768/s1,Table S1: Logarithms of free binding energies (FBE, kcal/mol) of diaporpyrone D (4) and acarbose to the active cavities of α-glucosidase (PDB ID: 2QMJ) and targeting residues of the binding site located on the mobile flap; Table S2: Binding free energies and energy components predicted by MM/GBSA (kcal/mol); Figure S1:

Molecules 2024, 29 , 1768 6 of 10 Figure 3 .
Figure 3. Molecular dynamics of acarbose and 4 with residues in the active pocket of α-glucosidase.(A) RMSD values of the complex and protein backbone systems in the dynamic simulation.(B) RMSF change profiles of binding site residues in the free protein and complex systems.(C) The gyration radius of the four systems during the molecular dynamics simulation.(D) The number of hydrogen bonds in the molecular dynamics simulation.

Figure 3 .
Figure 3. Molecular dynamics of acarbose and 4 with residues in the active pocket of α-glucosidase.(A) RMSD values of the complex and protein backbone systems in the dynamic simulation.(B) RMSF change profiles of binding site residues in the free protein and complex systems.(C) The gyration radius of the four systems during the molecular dynamics simulation.(D) The number of hydrogen bonds in the molecular dynamics simulation.