Exploration of Baicalein-Core Derivatives as Potent Antifungal Agents: SAR and Mechanism Insights

Highlights What are the main findings? Baicalein-Core Derivatives were designed and synthesized as Potent Anti-Fluconazole-resistant fungal Agents. O-dihydroxyls and vic-trihydroxy groups on either the A ring or B ring of flavones play a crucial role. MoA: Inhibit hypha formation; Little effect on ergosterol biosynthesis; Weak inhibitory effect on Eno1. Potential targets: 1,3-β-d-glucan synthase catalytic subunit, 1,3-β-d-glucan-UDP glucosyltransferase, and glycosyl-phosphatidylinositol protein by in-silico assay. What is the implication of the main finding? Provide potential synergistic antifungals with new MoA. Abstract Baicalein (BE), the major component of Scutellaria Baicalensis, exhibited potently antifungal activity against drug-resistant Candida albicans, and strong inhibition on biofilm formation. Therefore, a series of baicalein-core derivatives were designed and synthesized to find more potent compounds and investigate structure–activity relationship (SAR) and mode of action (MoA). Results demonstrate that A4 and B5 exert a more potent antifungal effect (MIC80 = 0.125 μg/mL) than BE (MIC80 = 4 μg/mL) when used in combination with fluconazole (FLC), while the MIC80 of FLC dropped from 128 μg/mL to 1 μg/mL. SAR analysis indicates that the presence of 5-OH is crucial for synergistic antifungal activities, while o-dihydroxyls and vic-trihydroxyls are an essential pharmacophore, whether they are located on the A ring or the B ring of flavonoids. The MoA demonstrated that these compounds exhibited potent antifungal effects by inhibiting hypha formation of C. albicans. However, sterol composition assay and enzymatic assay conducted in vitro indicated minimal impact of these compounds on sterol biosynthesis and Eno1. These findings were further confirmed by the results of the in-silico assay, which assessed the stability of the complexes. Moreover, the inhibition of hypha of this kind of compound could be attributed to their effect on the catalytic subunit of 1,3-β-d-glucan synthase, 1,3-β-d-glucan-UDP glucosyltransferase and glycosyl-phosphatidylinositol protein, rather than inhibiting ergosterol biosynthesis and Eno1 activity by Induced-Fit Docking and Molecular Dynamics Simulations. This study presents potential antifungal agents with synergistic effects that can effectively inhibit hypha formation. It also provides new insights into the MoA.


Introduction
Invasive fungal infections (IFIs) result in high levels of sickness and death among individuals with weakened immune systems, such as those with AIDS, receiving cancer chemotherapy, or undergoing immunosuppressive therapy [1].C. albicans is a common pathogen that causes life-threatening infections in these patients [2].The frontline treatment for systemic C. albicans infections is Fluconazole (FLC).Unfortunately, over the past few decades, traditional antifungal drugs have become less effective against drug-resistant fungi, creating a dire situation for humans [3,4].Consequently, the development of novel antifungal drugs is urgently needed.One promising approach to overcome azole resistance involves sensitizing C. albicans using small molecules, such as arthro-colins [5], 1,3,5triazines derivatives [6], and natural products [7][8][9][10][11].
C. albicans can form adherent biofilms on various surfaces in vitro, leading to devicerelated infections [12].The presence of hypha plays a crucial role in the biofilm formation of C. albicans.This unique structural feature allows the fungus to withstand the host's immune defense and enhances its resistance to antifungal drugs [13].Consequently, targeting biofilm is an ideal strategy for antifungal drugs.Echinocandin is a successful example, as it exhibits better selectivity and lower toxicity by specifically targeting fungal biofilms [14].
Flavonoids are a group of natural polyphenol substances widely distributed in plants, such as vegetables and fruits.in daily diet.A variety of biological activities have been reported, including anti-inflammatory, anti-tumor, hypolipidemic, anti-fungal and viral [15].Baicalein (BE, 5,6,7-trihydroxyflavone) is the major constituent in plants of Scutellaria baicalensis.The roots of S. baicalensis, known as Huang qin, have been widely used in traditional Chinese medicines for thousands of years to treat common colds, fever and other respiratory disorders.
Our previous study found that BE had strong antifungal activity against drug-resistance fungus when used alone or in combination with FLC.The MIC 80 of FLC decreases from >64 µg/mL when used alone to ≤0.125-2 µg/mL when used in combination with 2-16 µg/mL of BE.Additionally, it could be observed that the biofilm of C. albicans was inhibited in a dose dependent manner at concentrations between 4-32 µg/mL [16,17].Studies on mode of action (MoA) of BE revealed that BE could exert antifungal activity through inhibition of hypha formation and glycolysis disruption, which demonstrated that BE could be multi-target [18].
Therefore, BE is an ideal lead compound used as a sensitizer for drug-resistant fungus in combination with FLC.However, as far as we know, there is a lack of systemic study on BE or flavonoids regarding their antifungal activity and structure-activity relationship.Inspired by the unique vic-trihydroxy-4H-chromen-4-one structure of BE, a series of natural flavonoids were screened for their alone and synergistic antifungal activity with FLC, and based on these results a series of BE derivatives were further designed, synthesized and screened for their antifungal activities, alone and in combination with FLC.Their possible MoA was also investigated.The SAR study based on the structure of BE was carried out according to the strategy shown in Figure 1.Based on these findings, our objective is to identify potential compounds that possess strong synergistic antifungal effects, particularly in inhibiting hypha formation.Furthermore, we aim to investigate the possible mechanisms of action (MoA) underlying these effects.

Molecular Design of BE-Core Derivatives
This study examined the initial structural modification carried on the A ring of BE, aiming to evaluate the impact of its distinctive vic-trihydroxyls on its antifungal activities.The in vitro synergistic antifungal activities of BE analogues were tested using the micro broth dilution method, according to the standards of the National Committee Clinical and Laboratory Standards Institute, USA [22].When FLC-resistant C. albicans was treated with FLC or BE analogues, the individual MIC 80 (the lowest concentration of the agents that inhibited growth by 80%) values were determined.Furthermore, the fractional inhibitory concentration index (FICI) of each agent was calculated by determining the ratio of the interaction of MIC 80 /individual MIC 80 .The interaction modes, either synergistic or indifferent, were defined according to FICI values of ≤0.5 or ≥1, respectively [23].The MIC 80 of FLC against the FLC-resistant C. albicans (clinical isolate 103) was determined to be 128.0µg/mL.In this assay, the final concentration of FLC (FICI, in Tables 1-4) in each well was fixed at a single value (8.0 µg/mL), whereas BE analogues were diluted into a series of gradient concentrations (0.25-64.0 µg/mL).important pharmacophore [9,24].Furthermore, bromo, methoxy, nitro or amino group were introduced to position 6 and 8 of BE to give A18-A20 to investigate their effects on the antifungal activity.A18 and A19, with bromo or nitro group linked to position 6 or 8, showed similar activity to BE. A20, with only 5,7-dimethoxyl, and 8-nitro group and A21 with only methoxyls showed poor or no activity.Thus, introducing of bromo, nitro, amino or methoxy groups to the A ring of flavone could not increase the synergic antifungal activity.Based on modification on ring A, the effect of substituents on ring B on antifungal activity was investigated.The results are shown in Table 2.We firstly retained the structure of the A ring of BE, then introduced nitro group, bromo, amino group or hydroxyl to 3′ or 4′ position of the B ring to obtain B2, B4, B5 and B7-B9.It was found that, except for B9 (4′-hydroxybaicalein), all the other five flavones (B2, B4, B5, B7 and B8) showed superior or comparable anti-fungal activity to BE.Even compounds B4 (4′bromobaicalein), B5 (4′-aminobaicalein) and B8 (3′-aminobaicalein) showed stronger antifungal activity alone than BE (MIC80 = 32, 16 and 16 μg/mL for B4, B5 and B6).However, methylation of all the hydroxyl groups of the A-ring resulted in complete loss of activities (B1, B3 and B6, MIC > 64 μg/mL).Changing 5,6,7-trihydroxyl into the 5hydroxyl or 5,7-dihydroxyl of the A ring and introducing 4′-OH (B13) or 3′,4′-dihydroxyl (B10 and B11) or 3′-hydroxyl-4′-methoxyl (B12) to the B ring led to weak or no activity compared to the A series of flavones and BE.This suggested that 5,6,7-trihydroxy moiety on the A ring of BE is important for the antifungal activities if introducing hydroxyls into the B ring of flavone.However, if 5-OH is retained and an aromatic heterocyclic ring is used instead of the benzene of the B ring, the compound (B17) has strong synergistic activities (MIC80 = 8.0 μg/mL), whereas methylation of 5-OH of the B series of compounds caused a complete loss of activities (B14-B16).These results were consistent with that of the A series of compounds.on the A ring of BE is important for the antifungal activities if introducing hydroxyls into the B ring of flavone.However, if 5-OH is retained and an aromatic heterocyclic ring is used instead of the benzene of the B ring, the compound (B17) has strong synergistic activities (MIC80 = 8.0 μg/mL), whereas methylation of 5-OH of the B series of compounds caused a complete loss of activities (B14-B16).These results were consistent with that of the A series of compounds.The SAR study of series A and B guided us to design C and D series of flavonols to study the impact of 3-OH or 3-substituted oxy group on the antifungal effect.The results are presented in Table 3.In the A and B series of flavones, compounds with 5,7-dihydroxyl (A5, B11-13) showed weak or no activities.However, in the C series of flavones, compounds with 5,7-dihydroxyl (C1-C4, C6-C7) showed moderate or potent activities.This demonstrated that 3-hydroxyl is favored for synergic antifungal effect.One exception, i.e., that C9 (5,7-dihydroxy-4′-methoxy-8-isopentenylflavonol) has no antifungal activity, could be due to the large hindrance of the 8-position isopentenyl position of C9.Additionally, similar results for the importance of 5-hydroxy was also observed in the C series of flavones.Most of the compounds with 5-OMe or 5-H in the C series of flavones were inactive, and the methylated flavones at positions 5 and 7 (C10-C11 and D1) have no or only weak activity.When o-dihydroxy, or even vic-trihydroxy moiety, was introduced to the B-ring to afford C2 (Quercetin), C5 (Robinetin), C7 (Myricetin), C14 (3′,4′-dihydroxyflavonol) and C16 (Vincetoxicoside B), regardless of whether there is a 5-hydroxy group in the A-ring, all these compounds demonstrated potent synergic antifungal activity (MIC80 value ranging from 2.0 μg/mL to 4 μg/mL).Nevertheless, fisetin (C12, 7,3′,4′-trihydroxyflavonol) exhibited weak synergistic antifungal activity (MIC80 = 32 μg/mL) as an exception, compared with the structures of C2, C7 and C16, C12, lacking the 5-OH on the A ring; whereas compared with the structure of C14, C12 has 7-OH on the A ring, while C14 does not have any substituents on the A ring.Thus, it could be concluded that 7-OH is not necessary, and even not conducive to improving antifungal activity.
In addition, when the 3-OH of flavonol was glycosylated, the corresponding compounds D2-D4 lost their antifungal activity.Furthermore, the only structural difference between B10 (5,3′,4′-trihydroxyflavone) and C14 (3′,4′-dihydroxyflavonol), and The SAR study of series A and B guided us to design C and D series of flavonols to study the impact of 3-OH or 3-substituted oxy group on the antifungal effect.The results are presented in Table 3.In the A and B series of flavones, compounds with 5,7-dihydroxyl (A5, B11-13) showed weak or no activities.However, in the C series of flavones, compounds with 5,7-dihydroxyl (C1-C4, C6-C7) showed moderate or potent activities.This demonstrated that 3-hydroxyl is favored for synergic antifungal effect.One exception, i.e., that C9 (5,7-dihydroxy-4′-methoxy-8-isopentenylflavonol) has no antifungal activity, could be due to the large hindrance of the 8-position isopentenyl position of C9.Additionally, similar results for the importance of 5-hydroxy was also observed in the C series of flavones.Most of the compounds with 5-OMe or 5-H in the C series of flavones were inactive, and the methylated flavones at positions 5 and 7 (C10-C11 and D1) have no or only weak activity.When o-dihydroxy, or even vic-trihydroxy moiety, was introduced to the B-ring to afford C2 (Quercetin), C5 (Robinetin), C7 (Myricetin), C14 (3′,4′-dihydroxyflavonol) and C16 (Vincetoxicoside B), regardless of whether there is a 5-hydroxy group in the A-ring, all these compounds demonstrated potent synergic antifungal activity (MIC80 value ranging from 2.0 μg/mL to 4 μg/mL).Nevertheless, fisetin (C12, 7,3′,4′-trihydroxyflavonol) exhibited weak synergistic antifungal activity (MIC80 = 32 μg/mL) as an exception, compared with the structures of C2, C7 and C16, C12, lacking the 5-OH on the A ring; whereas compared with the structure of C14, C12 has 7-OH on the A ring, while C14 does not have any substituents on the A ring.Thus, it could be concluded that 7-OH is not necessary, and even not conducive to improving antifungal activity.
In addition, when the 3-OH of flavonol was glycosylated, the corresponding compounds D2-D4 lost their antifungal activity.Furthermore, the only structural difference between B10 (5,3′,4′-trihydroxyflavone) and C14 (3′,4′-dihydroxyflavonol), and The SAR study of series A and B guided us to design C and D series of flavonols to study the impact of 3-OH or 3-substituted oxy group on the antifungal effect.The results are presented in Table 3.In the A and B series of flavones, compounds with 5,7-dihydroxyl (A5, B11-13) showed weak or no activities.However, in the C series of flavones, compounds with 5,7-dihydroxyl (C1-C4, C6-C7) showed moderate or potent activities.This demonstrated that 3-hydroxyl is favored for synergic antifungal effect.One exception, i.e., that C9 (5,7-dihydroxy-4′-methoxy-8-isopentenylflavonol) has no antifungal activity, could be due to the large hindrance of the 8-position isopentenyl position of C9.Additionally, similar results for the importance of 5-hydroxy was also observed in the C series of flavones.Most of the compounds with 5-OMe or 5-H in the C series of flavones were inactive, and the methylated flavones at positions 5 and 7 (C10-C11 and D1) have no or only weak activity.When o-dihydroxy, or even vic-trihydroxy moiety, was introduced to the B-ring to afford C2 (Quercetin), C5 (Robinetin), C7 (Myricetin), C14 (3′,4′-dihydroxyflavonol) and C16 (Vincetoxicoside B), regardless of whether there is a 5-hydroxy group in the A-ring, all these compounds demonstrated potent synergic antifungal activity (MIC80 value ranging from 2.0 μg/mL to 4 μg/mL).Nevertheless, fisetin (C12, 7,3′,4′-trihydroxyflavonol) exhibited weak synergistic antifungal activity (MIC80 = 32 μg/mL) as an exception, compared with the structures of C2, C7 and C16, C12, lacking the 5-OH on the A ring; whereas compared with the structure of C14, C12 has 7-OH on the A ring, while C14 does not have any substituents on the A ring.Thus, it could be concluded that 7-OH is not necessary, and even not conducive to improving antifungal activity.
In addition, when the 3-OH of flavonol was glycosylated, the corresponding compounds D2-D4 lost their antifungal activity.Furthermore, the only structural difference between B10 (5,3′,4′-trihydroxyflavone) and C14 (3′,4′-dihydroxyflavonol), and  To further study the antifungal activity, the most potent compounds A4, B4, B5, C5  As shown in Table 1, BE showed potent synergistic activity in combination with FLC (MIC 80 = 8.0 µg/mL, FICI = 0.313).Flavonoids of series A were designed to investigate the effects of the type, number, and position of the substituents on the A ring on the activity.The results demonstrated that, except for A5 and A22, all other flavones with 5-OH (A1, A4, A8-A10, A12-13, A16, A18-19, and A25) showed synergistic antifungal activity when used in combination with 8 µg/mL of FLC.Among them, the MIC values of A1, A4, A8, A9, A12-13, A16 and A18 were all less than or equal to that of BE.Compounds A5 (5,7-dihydroxyflavone) and A22 (8-amino-5-hydroxy-7-methoxyflavone) did not show antifungal activity, which could be due to meta-disubstituted (5,7-dihydroxy), whereas, if 5-OH was changed into 5-OMe or H, all the compounds (A2, A3, A14, A15, A17, A20, A21 and A23) showed weak or no antifungal activity.Interestingly, compounds A6, A7 and A24, from which 5-OH was absent, also exerted comparable antifungal activity on BE.An obvious feature of the structures of A6 and A7 is that they all possess the o-dihydroxy group.A8, another O-dihydroxy flavone (5,6-dihydroxy-7-methoxyflaonve), which was obtained by methylation of 7-OH of BE, also showed equivalent activity to BE.Thus, 5-OH or o-dihydroxyl moiety on the A ring of flavone is favored for synergistic antifungal activity.Our previous studies also demonstrated that 5-OH or o-dihydroxy moiety were important pharmacophore [9,24].Furthermore, bromo, methoxy, nitro or amino group were introduced to position 6 and 8 of BE to give A18-A20 to investigate their effects on the antifungal activity.A18 and A19, with bromo or nitro group linked to position 6 or 8, showed similar activity to BE. A20, with only 5,7-dimethoxyl, and 8-nitro group and A21 with only methoxyls showed poor or no activity.Thus, introducing of bromo, nitro, amino or methoxy groups to the A ring of flavone could not increase the synergic antifungal activity.
Based on modification on ring A, the effect of substituents on ring B on antifungal activity was investigated.The results are shown in Table 2.We firstly retained the structure of the A ring of BE, then introduced nitro group, bromo, amino group or hydroxyl to 3 or 4 position of the B ring to obtain B2, B4, B5 and B7-B9.It was found that, except for B9 (4 -hydroxybaicalein), all the other five flavones (B2, B4, B5, B7 and B8) showed superior or comparable anti-fungal activity to BE.Even compounds B4 (4 -bromobaicalein), B5 (4 -aminobaicalein) and B8 (3 -aminobaicalein) showed stronger antifungal activity alone than BE (MIC 80 = 32, 16 and 16 µg/mL for B4, B5 and B6).However, methylation of all the hydroxyl groups of the A-ring resulted in complete loss of activities (B1, B3 and B6, MIC > 64 µg/mL).Changing 5,6,7-trihydroxyl into the 5-hydroxyl or 5,7-dihydroxyl of the A ring and introducing 4 -OH (B13) or 3 ,4 -dihydroxyl (B10 and B11) or 3 -hydroxyl-4methoxyl (B12) to the B ring led to weak or no activity compared to the A series of flavones and BE.This suggested that 5,6,7-trihydroxy moiety on the A ring of BE is important for the antifungal activities if introducing hydroxyls into the B ring of flavone.However, if 5-OH is retained and an aromatic heterocyclic ring is used instead of the benzene of the B ring, the compound (B17) has strong synergistic activities (MIC 80 = 8.0 µg/mL), whereas methylation of 5-OH of the B series of compounds caused a complete loss of activities (B14-B16).These results were consistent with that of the A series of compounds.
The SAR study of series A and B guided us to design C and D series of flavonols to study the impact of 3-OH or 3-substituted oxy group on the antifungal effect.The results are presented in Table 3.In the A and B series of flavones, compounds with 5,7-dihydroxyl (A5, B11-13) showed weak or no activities.However, in the C series of flavones, compounds with 5,7-dihydroxyl (C1-C4, C6-C7) showed moderate or potent activities.This demonstrated that 3-hydroxyl is favored for synergic antifungal effect.One exception, i.e., that C9 (5,7-dihydroxy-4 -methoxy-8-isopentenylflavonol) has no antifungal activity, could be due to the large hindrance of the 8-position isopentenyl position of C9.Additionally, similar results for the importance of 5-hydroxy was also observed in the C series of flavones.Most of the compounds with 5-OMe or 5-H in the C series of flavones were inactive, and the methylated flavones at positions 5 and 7 (C10-C11 and D1) have no or only weak activity.When o-dihydroxy, or even vic-trihydroxy moiety, was introduced to the B-ring to afford C2 (Quercetin), C5 (Robinetin), C7 (Myricetin), C14 (3 ,4dihydroxyflavonol) and C16 (Vincetoxicoside B), regardless of whether there is a 5-hydroxy group in the A-ring, all these compounds demonstrated potent synergic antifungal activity (MIC 80 value ranging from 2.0 µg/mL to 4 µg/mL).Nevertheless, fisetin (C12, 7,3 ,4trihydroxyflavonol) exhibited weak synergistic antifungal activity (MIC 80 = 32 µg/mL) as an exception, compared with the structures of C2, C7 and C16, C12, lacking the 5-OH on the A ring; whereas compared with the structure of C14, C12 has 7-OH on the A ring, while C14 does not have any substituents on the A ring.Thus, it could be concluded that 7-OH is not necessary, and even not conducive to improving antifungal activity.
To further study the antifungal activity, the most potent compounds A4, B4, B5, C5 and C14 were evaluated for their antifungal acidity by a checkerboard microdilution assay.As shown in Table 4, all tested compounds showed potent synergic antifungal activities with MIC 80 values ranging from 0.125 µg/mL to 8 µg/mL when used in combination with 0.25-1 µg/mL of FLC against FLC-resistant C. albicans.C5 and C14, at concentrations of 4-8 µg/mL, were found to decrease the MIC 80 value of FLC from 128 µg/mL to 0.25 µg/mL.Even at a concentration of 0.125 µg/mL, A4 and B5 were able to significantly decrease the MIC 80 value of FLC from 128 µg/mL to 1 µg/mL.Additionally, B4 showed more potent antifungal activity when used alone, with a MIC 80 value of 16 µg/mL, which was four times more potent than BE (MIC 80 = 16 µg/mL, alone).In summary, while C14 (FICI = 0.0645) and B4 (FICI = 0.0703) exhibited comparable FICI values to BE (FICI = 0.0645), A4 (FICI = 0.0088), B5 (FICI = 0.0088) and C5 (FICI = 0.0332) demonstrated significantly greater synergistic activity than BE.To further confirm their synergistic antifungal activity, A4 and B5 were selected for testing of their antifungal effects against two additional FLC-resistant clinical isolates: C. albicans 938 and 939.The results showed that A4 and B5 exhibited significant synergistic antifungal effects against C. albicans 938 and C. albicans 939 (with a MIC 80 of 0.25 µg/mL when combined with FLC at 8 µg/mL).These findings indicate that these potent compounds possess broad-spectrum and potent synergistic antifungal activities.
In conclusion, 5-OH or o-dihydroxy moiety on the A ring of flavone is favored for synergistic antifungal activity.Methylation of hydroxyl decreased the antifungal activity.When 5-OH is present, introduction of the 3-OH could significantly improve antifungal activity.Overall, the presence of o-dihydroxy or vic-trihydroxy moiety on either the A or B ring has been found to be favorable to antifungal activity.Introduction of Br or NH 2 to the B ring of BE could enhance antifungal activity when used alone.Thus, it was clear that appropriate structural modification of BE could obtain higher active flavonoids with synergistic antifungal activity against FLC-resistant C. albicans.

In Vitro Hyphal Formation Assay
In a previous study, BE exhibited significant inhibition of fungal biofilm formation [16].Thereby, compounds A4, B4 and B5 with the best antifungal activity were selected for in vitro hypha formation assay.The results are presented in Figures 2 and 3. Apparently, FLC did not inhibit the biofilm formation of clinical FLC-resistant C. albicans 901 and 904 at the concentrations of 2-16 µg/mL (Figure 2A-E).However, compared to FLC, A4 and B4 alone at 8 µg/mL significantly reduced the density of the C. albicans (Figure 3B-D).Furthermore, the combination of FLC with compounds A4, B4 and B5 remarkably inhibited the hypha formation (Figure 3E-H).The density of fungus and the length of hypha were intensely suppressed by compounds A4, B4 and B5 when used in combination with FLC.This mechanism may contribute to the destruction of the defense of FLC-resistant C. albicans and provide an advantageous opportunity for FLC to attack the fungus.
In conclusion, 5-OH or o-dihydroxy moiety on the A ring of flavone is favored for synergistic antifungal activity.Methylation of hydroxyl decreased the antifungal activity.When 5-OH is present, introduction of the 3-OH could significantly improve antifungal activity.Overall, the presence of o-dihydroxy or vic-trihydroxy moiety on either the A or B ring has been found to be favorable to antifungal activity.Introduction of Br or NH2 to the B ring of BE could enhance antifungal activity when used alone.Thus, it was clear that appropriate structural modification of BE could obtain higher active flavonoids with synergistic antifungal activity against FLC-resistant C. albicans.

In Vitro Hyphal Formation Assay
In a previous study, BE exhibited significant inhibition of fungal biofilm formation [16].Thereby, compounds A4, B4 and B5 with the best antifungal activity were selected for in vitro hypha formation assay.The results are presented in Figures 2 and 3. Apparently, FLC did not inhibit the biofilm formation of clinical FLC-resistant C. albicans 901 and 904 at the concentrations of 2-16 μg/mL (Figure 2A-E).However, compared to FLC, A4 and B4 alone at 8 μg/mL significantly reduced the density of the C. albicans (Figure 3B-D).Furthermore, the combination of FLC with compounds A4, B4 and B5 remarkably inhibited the hypha formation (Figure 3E-H).The density of fungus and the length of hypha were intensely suppressed by compounds A4, B4 and B5 when used in combination with FLC.This mechanism may contribute to the destruction of the defense of FLC-resistant C. albicans and provide an advantageous opportunity for FLC to attack the fungus.

In Vitro Sterol Composition Assay
The potent compound A4 was evaluated for its impact on the sterol composition in the cell membrane of C. albicans through a gas chromatography-mass spectrometry method.Results demonstrated that treatment with FLC at a concentration of 8 μg/mL resulted in a significant reduction of ergosterol in C. albicans, while showing elevated levels of eburicol, obtusifoliol and fecosterol.Similarly, in combination with FLC (8 μg/mL) and A4 (8 μg/mL), the ergosterol decreased, and the eburicol and obtusifoliol increased (Figure 4).However, compound A4 (8 μg/mL) alone for C. albicans did not significantly alter the levels of ergosterol compared to the control group, nor did it increase the levels of eburicol, obstusifoliol, and fecosterol.These findings suggest that compound A4 did not induce any notable changes in the sterol biosynthesis, indicating that these BE derivatives have no impact on the sterol biosynthesis pathway.

In Vitro Sterol Composition Assay
The potent compound A4 was evaluated for its impact on the sterol composition in the cell membrane of C. albicans through a gas chromatography-mass spectrometry method.Results demonstrated that treatment with FLC at a concentration of 8 µg/mL resulted in a significant reduction of ergosterol in C. albicans, while showing elevated levels of eburicol, obtusifoliol and fecosterol.Similarly, in combination with FLC (8 µg/mL) and A4 (8 µg/mL), the ergosterol decreased, and the eburicol and obtusifoliol increased (Figure 4).However, compound A4 (8 µg/mL) alone for C. albicans did not significantly alter the levels of ergosterol compared to the control group, nor did it increase the levels of eburicol, obstusifoliol, and fecosterol.These findings suggest that compound A4 did not induce any notable changes in the sterol biosynthesis, indicating that these BE derivatives have no impact on the sterol biosynthesis pathway.
The potent compound A4 was evaluated for its impact on the sterol compositio the cell membrane of C. albicans through a gas chromatography-mass spectrom method.Results demonstrated that treatment with FLC at a concentration of 8 μg resulted in a significant reduction of ergosterol in C. albicans, while showing elev levels of eburicol, obtusifoliol and fecosterol.Similarly, in combination with FL μg/mL) and A4 (8 μg/mL), the ergosterol decreased, and the eburicol and obtusif increased (Figure 4).However, compound A4 (8 μg/mL) alone for C. albicans did significantly alter the levels of ergosterol compared to the control group, nor d increase the levels of eburicol, obstusifoliol, and fecosterol.These findings suggest compound A4 did not induce any notable changes in the sterol biosynthesis, indica that these BE derivatives have no impact on the sterol biosynthesis pathway.

In Vitro Eno1 Enzymatic Assay
In our previous work, we found that Eno1 is one of the targets of BE [18].Thus, we selected potent compounds A4, B4, B5, C5 and C7 for in vitro enzymatic assay.The results are shown in Figure 5. Unexpectedly, these compounds did not exhibit stronger enzyme inhibitory activity than BE (IC 50 = 62.9 ± 2.2 µM).The observed enzyme inhibition activity did not correspond to their in vitro antifungal activity.These results imply the presence of an alternative target for these compounds.Thus, these potent active compounds are worthy of further study on MoA.

In Vitro Eno1 Enzymatic Assay
In our previous work, we found that Eno1 is one of the targets of BE [18].Thus, we selected potent compounds A4, B4, B5, C5 and C7 for in vitro enzymatic assay.The results are shown in Figure 5. Unexpectedly, these compounds did not exhibit stronger enzyme inhibitory activity than BE (IC50 = 62.9 ± 2.2 μM).The observed enzyme inhibition activity did not correspond to their in vitro antifungal activity.These results imply the presence of an alternative target for these compounds.Thus, these potent active compounds are worthy of further study on MoA.

In Silico Studies 2.7.1. Molecular Dynamic Simulation
To understand the reason for the limited affinity of potent compounds binding to Eno1, we chose compound B5 to perform Molecular Dynamics Simulations (MDs) in conjunction with CaEno1 (PDB ID: 7V67) by virtual docking.Figures 6-9 are presented for the obtained results.Comparison of BE with compound B5 indicated that there were distinctive characteristics of hydrogen bond formation between the complex and B5.Specifically, the interaction between 5-OH and LYS221 formed a hydrogen bond, while 7-OH formed an ionic bond with LYS273 in B5 complex rather than a hydrogen bond in BE complex.Notably, the absence of a hydrogen bond between 6-OH and SER269 was observed in Figure 6, which diminished the stability of the B5 complex.Examination of the binding pocket potential diagram revealed electrostatic repulsion between the 4 -NH 2 group of B5 and LYS273, LYS221.Consequently, the B ring of B5 could not fit within the cavity formed by LYS273 and LYS221 (Figure 7).Moreover, due to steric hindrance, the anticipated hydrogen bond formation between the 5,6,7-trihydroxy group and SER269, as well as ASP263, was disrupted in comparison to BE complex.Thus, the number of hydrogen bonds decreased from three in BE complex to one in B5 complex, potentially decreasing the capacity for enzyme inhibition of B5.During MDs, the RMSD values of the complexes exhibited fluctuations ranging from 1.38614 (conformation 55) to 1.71876 (conformation 24), as depicted in Figure 8, with a variation range of 0.33 Å.The average RMSD value was 1.54786.These outcomes suggested that the complexes remained relatively stable during the simulation, which aligned with the fact that B5 possessed weaker enzyme inhibitory activity.To investigate the variation of amino acids within the complexes, the RMSF values for all amino acids were calculated (Figure 9).Notably, LYS 55 (RMSF 1.63966), GLN 205 (RMSF 1.45114) and GLU 268 (RMSF 2.03006) exhibited significant fluctuations, indicating higher flexibility at these positions.However, these positions surrounded key residues ASP 263, SER 269 and LYS 273, which was detrimental for maintaining complex stability.The hydrogen heat map (Figure 10) highlights the hydrogen bond distribution within the conformations.The hydrogen bonds between LYS 221-B5 and LYS 273-B5 were observed in nearly all conformations, signifying their persistence and stability.This finding was consistent with the docking results.These findings suggest that the enzyme inhibitory ability of B5 may be compromised due to the reduction in the number of hydrogen bonds.However, the presence of residual hydrogen bonds helped to maintain the stability of B5 complex.As a result, B5 exhibited some inhibitory ability in in vitro enzyme inhibition assay, although it was weaker than BE.During MDs, the RMSD values of the complexes exhibited fluctuations ranging from 1.38614 (conformation 55) to 1.71876 (conformation 24), as depicted in Figure 8, with a variation range of 0.33 Å.The average RMSD value was 1.54786.These outcomes suggested that the complexes remained relatively stable during the simulation, which aligned with the fact that B5 possessed weaker enzyme inhibitory activity.To investigate the variation of amino acids within the complexes, the RMSF values for all amino acids were calculated (Figure 9).Notably, LYS 55 (RMSF 1.63966), GLN 205 (RMSF 1.45114) and GLU 268 (RMSF 2.03006) exhibited significant fluctuations, indicating higher flexibility at these positions.However, these positions surrounded key residues ASP 263, SER 269 and LYS 273, which was detrimental for maintaining complex stability.The hydrogen heat map (Figure 10) highlights the hydrogen bond distribution within the conformations.The hydrogen bonds between LYS 221-B5 and LYS 273-B5 were observed in nearly all conformations, signifying their persistence and stability.This finding was consistent with the docking results.These findings suggest that the enzyme inhibitory ability of B5 may be compromised due to the reduction in the number of hydrogen bonds.However, the presence of residual hydrogen bonds helped to maintain the stability of B5 complex.As a result, B5 exhibited some inhibitory ability in in vitro enzyme inhibition assay, although it was weaker than BE.

Exploration of Possible Targets
These findings of the in-silico analysis suggest that our derivatives may influence multiple targets.Subsequently, we utilized Induced-Fit Docking (IFD) to screen proteins that have been reported to be closely associated with hypha and possess potential

Exploration of Possible Targets
These findings of the in-silico analysis suggest that our derivatives may influence multiple targets.Subsequently, we utilized Induced-Fit Docking (IFD) to screen proteins that have been reported to be closely associated with hypha and possess potential antifungal effects.The results were further assessed by MDs, and the outcomes are summarized in Table 5.The identified proteins include 1,3-β-D-glucan synthase catalytic subunit [25,26], 1,3-β-D-glucan-UDP glucosyltransferase [27,28], chitin synthase [29][30][31][32], δsterol 5-desaturase [33], 14-α demethylase [34], glycosyl-phosphatidylinositol protein [35][36][37], and agglutinin-like protein 3 [12,38].The binding free energy of enzyme protein receptor and ligand small molecule complexes is calculated by Molecular Mechanics/Generalized Born Surface Area (mmG-BSA) [39].This algorithm calculates the average binding free energy by extracting the architecture of a certain time interval from the MDs' trajectory of the complex and solving complex interactions between complex molecules by decomposing and calculating the parts that constitute the binding free energy.Then, the obtained free binding energy can be used to react to the stability of the complexes.
The MD results were presented in the form of a heat map (Figure 11), with the values indicating the binding free energy calculated by mmGBSA.Red color represents lower binding free energy, indicating a more stable binding state of the complex.It can be observed that the compounds exhibit greater sensitivity towards the 1,3-β-D-glucan synthase catalytic subunit, 1,3-β-D-glucan-UDP glucosyltransferase, and glycosyl-phosphatidylinositol protein.On the other hand, the interaction between the compounds and sterol biosynthesis pathway related proteins δ-sterol 5-desaturase and 14-α demethylase were weaker.These results are in line with the result of the in vitro sterol composition assay.It could be concluded that these compounds can potentially act on multiple targets.NA * indicates that the complex is unable to bind spontaneously in MDs.

Discussion
C. albicans is a predominant fungal pathogen, which poses a significant threat to individuals with compromised immune systems, including those with AIDS, tumors, leukemia, and undergoing organ transplants.The mortality rate associated with invasive candidiasis exceeds 40%.Moreover, the emergence of azoles and echinocandins-resistant strains has further complicated antifungal interventions.Consequently, there is an urgent imperative to develop highly effective and minimally toxic antifungal medications and

Discussion
C. albicans is a predominant fungal pathogen, which poses a significant threat to individuals with compromised immune systems, including those with AIDS, tumors, leukemia, and undergoing organ transplants.The mortality rate associated with invasive candidiasis exceeds 40%.Moreover, the emergence of azoles and echinocandins-resistant strains has further complicated antifungal interventions.Consequently, there is an urgent imperative to develop highly effective and minimally toxic antifungal medications and therapies [40,41].Exploring novel structurally based antifungal agents derived from natural sources represents a crucial avenue of research in this regard.
Several flavonoids have been reported to have antifungal activity, and most of these compounds are characterized by a polyhydroxy structure [42].However, there is a lack of systematic studies on the antifungal activity and structure-activity relationships of these compounds.In our previous study, BE showed significant antifungal activities [16,43].Inspired by flavonoids with polyhydroxy structures, such as BE, we designed and synthesized a series of baicalein-core derivatives and found that compounds A4, B4, B5, C5 and C7 exhibited higher synergistic antifungal activity than BE when used in combination with FLC.Among these compounds, A4 and B5 show the most potent synergistic antifungal activity with MIC 80 = 0.125 µg/mL (FICI = 0.0088).The SAR indicates that 5-OH is vital to the synergistic antifungal activity, and 3-OH improved this effect.Besides, o-dihydroxyls and vic-trihydroxy are an essential pharmacophore, either on the A ring or the B ring of these derivatives.
However, the relevant mechanisms are still unknown.Our previous work showed that BE displayed potent antifungal effect via hypha formation inhibition and glycolysis disruption [16,18].Hypha is a structural component of C. albicans' biofilm and plays a crucial role in its development and maintenance.Transitioning from yeast to the hypha morphotype and forming a biofilm greatly contributes to C. albicans' virulence.This impairs the eradication of immune cells and antifungal drugs, allowing C. albicans to withstand host immune defenses and increase resistance to antifungal drugs [13].This process is believed to be necessary for immune cells to escape through the induction of programmed cell death [44].To investigate the MoA, we initially conducted an in vitro hypha inhibition assay.Compounds A4, B4, and B5 all exhibited significant inhibition on formation and quantity of fungal hypha when used in combination with FLC.
Ergosterol, as the primary sterol in yeast, plays an essential role in maintaining the integrity and function of the cell membrane [45].The target of azole antifungal drugs is CYP51, which is encoded by the ERG11 gene in fungi.Azoles exert their antifungal activity by blocking ergosterol biosynthesis and causing the accumulation of toxic sterol intermediates [1,46].Some flavonoids, including Lichochalcone-A [47], 7,4 -dimethylapigenin [48], fisetin [49] and 5,6,8-trihydroxy-7,4 dimethoxy flavone [50], have an inhibitory effect on fungal ergosterol biosynthesis at the concentration of >51 µg/mL.Thus, the most potent compound A4 was tested for its effects on the in vitro fungal sterol composition.The results indicated that the combination of A4 and FLC did not result in significant alterations in the sterol biosynthesis pathway when compared to the use of FLC alone.
Eno1, encoded by the ENO1 gene, is vital for the survival, pathogenicity, and susceptibility of C. albicans to antifungal drugs.Eno1 exhibits enolase activity and is crucial for glycolysis in C. albicans.Absence of the ENO1 gene renders C. albicans unable to survive on glucose-based media.Additionally, deleting the ENO1 gene increases the susceptibility of C. albicans to frontline antifungal drugs, such as amphotericin B, FLC, miconazole, and voriconazole.Therefore, targeting Eno1 shows potential in treating C. albicans infections [51][52][53].BE may exert its antifungal effects through inhibition of Eno1 activity [18].Meanwhile, Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses demonstrate that Eno1 is highly expressed in fungal hypha and secreted through hyphal secretomes.In addition to its glycolytic function, Eno1 can help pathogens evade host immune response [54,55].Thus, we subjected the compounds to in vitro enzymatic assay with Eno1.Surprisingly, compounds A4, B4, B5, C5, and C7 exhibited weaker Eno1 enzyme inhibition activity compared to BE, despite their enhanced antifungal activity when compared to BE.
Therefore, we further used the computer-aided techniques IFD and MD to analyze the possible mechanisms of this class of compounds [56][57][58][59].Firstly, we found that the changes on the B-ring may lead to a decrease in the number of hydrogen bonds to the complex with Eno1 and compound B5, which reduced the inhibitory ability on Eno1.Meanwhile, the amino acid residues around the key residues ASP263, SER269, and LYS273 were more flexible, which was not conducive to the stability of the complex.These may contribute to the fact that B5 possessed lower enzyme-inhibiting ability than BE.These results imply that there may be other targets for this class of compounds, other than just Eno1.Therefore, we further investigated the potential mechanisms of this class of compounds.Our findings revealed that these compounds exhibited greater selectivity for the 1,3-β-D-glucan synthase catalytic subunit, 1,3-β-D-glucan-UDP glucosyltransferase, and glycosyl-phosphatidylinositol protein, as evidenced by IFD and MD analysis.In addition, the results of the IFD and MD analysis also indicated weak binding with sterol biosynthesis pathway related proteins δ-sterol 5-desaturase and 14-α demethylase, which aligned with the results from the in vitro sterol composition assay.
In addition, Lv's work shows that BE enhances the oxidative stress effect of C. albicans by upregulating the expression of CPD2, thereby triggering oxidative damage and apoptosis in fungus [60].These results also suggest that there could be multiple targets for BE.Related studies are still in progress.

Chemistry
Reagents were purchased from common commercial suppliers and were used without further purification.Analytical TLC was carried out on silica gel F254 precoated (0.2 mm thickness) plastic TLC sheets.The TLC plates were spotted with samples using a fine glass capillary tube and developed in a chromatographic tank saturated with solvent vapor at room temperature. 1H NMR and 13 C NMR spectra were recorded on a Bruker Ac 300 or 600 MHz spectrometer in DMSO-d 6 or CDCl 3 solution.Chemical shifts were recorded in δ ppm relative to internal TMS.Mass spectra were obtained by electron spray ionization (ESI) in positive or negative mode using an Aglilent LC/MS-6210 spectrometer.Melting points were determined with an electrothermal melting point apparatus (SGW ® X-4, Shanghai Shenguang Optical Instrument Factory, Shanghai, China) and were uncorrected.

6-hydroxy-2-phenyl-4H-chromen-4-one (A2)
To a solution of MeOH (20 mL), 2 -hydroxy-4 -methoxyacetophenone (185 mg, 1.11 mmol) and benzaldehyde (141.2 mg, 1.33 mmol) were added.Then, 40% NaOH aqueous solution was added dropwise.The mixture was kept at room temperature along with stirring for 24 h.Upon completion of the reaction (monitored by TLC), 50 mL of water were added into the reaction mixture.Then, 2 mol/L diluted hydrochloric acid were added to the mixture to adjust the pH value of the solution to pH 5-6, and the yellow solid precipitate was collected by filtration.After recrystallization with ethyl alcohol, 193 mg of pure white products of 2 -hydroxy-4 -methoxychalcone were obtained with a yield of 60%.To a solution of DMSO (20 mL), 2 -hydroxy-4 -methoxychalcone (127 mg, 0.5 mmol) and I 2 (253 mg, 0.1 mmol) were added, then the mixture was heated to 120-140 • C and kept for 4-8 h.Upon completion of the reaction (monitored by TLC), the reaction mixture was cooled.After 100 mL of water and 100 mg Na 2 S 2 O 3 were added into the reaction mixture, the yellow solid precipitate was collected by filtration.After recrystallization with ethyl alcohol, 73 mg of pure yellow products of 6-methoxyflavone (A23) were obtained with a yield of 59%.To a solution of anhydrous DCM (10 mL), A23 (71 mg, 0.28 mmol) and 1 M of BBr 3 /DCM solution (2.8 mL, 1.4 mmol) were added at a temperature of −15 • C under argon atmosphere condition.Then the mixture was kept, along with stirring at room temperature, for another 12 h.After quenching the reaction by adding 10 mL of water at the temperature of −15 • C, the mixture was poured into a separatory funnel and separated.The aqueous layer was extracted with DCM (30 mL) three times.The combined organic layers were washed with saturated NaCl solution and dried over anhydrous Na 2 SO 4 and evaporated to dryness to give crude products.After recrystallization with ethyl alcohol, 50 mg of pure yellow products of 6-hydroxyflavone (A2) were obtained with a yield of 42%. 1 H NMR (300 MHz, DMSO-d 6 ) δ 10.04 (s, 1H), 8.12-8.03(m, 2H), 7.66 (d, J = 9.0 Hz, 1H), 7.66-7.51(m, 3H), 7.33 (d, J = 2.9 Hz, 1H), 7.26 (dd, J = 9.0, 3.0 Hz, 1H), 6.96 (s, 1H). 13

Antifungal Activity (Broth Microdilution Method)
The clinical isolates of FLC-resistant C. albicans 103, 938 and 939 were used in this study, and C. albicans ATCC 90028 was used as a quality control.The antifungal properties of the title compounds were evaluated by the broth microdilution method according to the NCCLS reference document M27-A3 [22].BE was used as positive control.The MIC 80 of FLC against the FLC-resistant C. albicans (clinical isolate 103, 938 and 939) was determined to be 128 µg/mL.The MIC 80 of each title compound when used alone and when combined with FLC (8.0 µg/mL) are described in Tables 1-3.Each compound was tested in triplicate.Furthermore, the fractional inhibitory concentration index (FICI) of each agent was calculated by adding the ratios of the MIC 80 (with FLC)/MIC 80 (used alone).The interaction modes-synergistic or indifferent-were defined according to FICI values of ≤0.5 or >0.5, respectively.

In Vitro Hyphal Formation Assay
The clinical isolates of FLC-resistant C. albicans 901,904 underwent exponential growth in YEPD medium.The cells were then diluted with 1.5 mL of spider medium to obtain a concentration of 1 × 10 5 CFU/mL.The fungal cell suspension was subsequently added to a 12-well plate.Compound A4, B4, B5 at a concentration of 8 µg/mL, along with FLC as the positive drug, were added.After incubating the fungal cells at 37 • C for 7 h, the morphological differences between the drug-treated group and the group without drugs were recorded using a live cell imaging inverted microscope [61].

Analysis of Fungal Sterol Composition Assay
After treatment with compound A4 with a concentration of 8 µg/mL in YEPD medium, the C. albicans SC5314 cells were collected (wet weight about 1.0 g).Subsequently, the mixtures were combined with 10 mL of saponifier (a solution of 90% ethanol and 15% NaOH) and heated to 80 • C for 1 h.Following this, 10 mL of petroleum ether (with a boiling range of 30-60 • C) was added three times to extract the sterols from each group.The extracted solutions were then evaporated under reduced pressure, and the residue was dissolved in 1 mL of cyclohexane.Finally, the composition of sterols in each compound group was analyzed using GC-MS.To identify the sterols, we matched the molecular fragments from each peak in the GC-MS chromatograms with the corresponding sterol compounds in the NIST (the National Institute of Standards and Technology) reference database [61].

Enolase Activity Analysis
Enolase activity was determined by direct spectrophotometric assay via measuring the increase of absorbance at 240 nm of phosphoenolpyruvate (PEP) as described previously with some modifications [62].Briefly, the reaction buffer (pH 7.0) containing 10 mM imidazole, 200 mM KCl, and 0.5 mM MgAc in a final reaction volume of 100 mL was mixed with Eno1 (a final concentration of 30 nM), followed by the mixture of 2-phosphoglycerate (2-PG) (Yuanye Biotech Shanghai, China) with a final concentration of 1 mM.The enolase activity of Eno1 was evaluated by measuring the increase of absorbance (OD240) at room temperature for 10 min.For the inhibition study of enolase by compounds, compounds at a concentration of 200 µM were used to mix well with Eno1 and were incubated at room temperature for 5 min, and subsequent operations were described above.

Docking Studies
Docking studies of all baicalein derivatives were performed in the Discovery Studio.The protein crystal structure of CaEno1 (PDB ID: 7V67) was obtained by downloading from the PDB (https://www.rcsb.org/,accessed on 12 January 2023).The protein is prepared using Prepare Protein module, including hydrogenation, removal of water-free molecules, repair of protein side chains and modification of C and N termini, and repair of disordered chemical bonds and atoms.The parameters related to this process are default values.After importing the compound set into DS, these compounds as ligands are prepared using Prepare Ligands module for docking, including changing the ionization state, generating reciprocal isomers and stereoisomers, etc.The parameters related to this procedure are default values.The binding sites were set regarding the literature [18], with key residues ASP 263, SER 269, and LYS 273 set as binding pockets.the radius was set to 15 Å.Semi-flexible docking was performed using CDOCKER, and the parameters were all default values.
Molecular dynamic simulation of complexes was generated by docking of compound B5 using the Standard Dynamic Cascade module.The force field was chosen as charmm36 [63] while solventization was performed afterward.All parameters are at default values.A total of 9795 water molecules, 26 sodium ions, and 26 chloride ions were added.Afterward, the equilibrium phase was set to 20 ps and the runtime phase was set to 200 ps to perform molecular dynamic simulation.

Conclusions
The emergence of drug resistance in fungi is outpacing the development of new antifungal treatments.To address this, alternative approaches such as combination therapy, anti-virulence agents, and modulation of host immune responses are being explored [64].Combination therapy has shown promise in overcoming resistance by targeting multiple mechanisms of action and reducing pathogen populations.It can also reverse drug resistance and unveil new treatment options.Additionally, combination therapy increases the efficiency of drugs and makes it more difficult for pathogens to develop resistance [65,66].Our BE-core derivatives exhibited a strong inhibitory effect on hypha formation, which makes these compounds act as ideal synergistic small molecules, since hypha are one of the important virulence factors in C. albicans infections.
SAR study provides o-dihydroxyls, and vic-trihydroxy are an essential pharmacophore, either on the A ring or the B ring of these derivatives.Analyses of MoA showed that these compounds retained the effect on inhibition of hypha formation.However, there was little effect on ergosterol biosynthesis and Eno1.In silico studies explained that these results were due to the hydrogen bond reduction and key residues instability.Further studies indicated that the 1,3-β-D-glucan synthase catalytic subunit, 1,3-β-D-glucan-UDP glucosyltransferase, and glycosyl-phosphatidylinositol protein could be the candidate targets of these compounds.Taken together, this study suggests some potential for synergistic antifungal agents and an effective strategy for treatment of FLC-resistant C. albicans infections.

Figure 1 .
Figure 1.General structures of BE analogues.

2. 7 .
In Silico Studies 2.7.1.Molecular Dynamic Simulation To understand the reason for the limited affinity of potent compounds binding to Eno1, we chose compound B5 to perform Molecular Dynamics Simulations (MDs) in

26 Figure 6 .
Figure 6.2D diagram of the interaction between B5 and Eno1.

Figure 6 .
Figure 6.2D diagram of the interaction between B5 and Eno1.

Figure 6 .
2D diagram of the interaction between B5 and Eno1.

Figure 7 .
Figure 7. Potential diagram of the combined pocket between B5 and Eno1.

Figure 7 .
Figure 7. Potential diagram of the combined pocket between B5 and Eno1.

Figure 9 .
Figure 9. Results of amino acid residue analysis between B5 and Eno1 complexes.Figure 9. Results of amino acid residue analysis between B5 and Eno1 complexes.

Figure 9 . 26 Figure 10 .
Figure 9. Results of amino acid residue analysis between B5 and Eno1 complexes.Figure 9. Results of amino acid residue analysis between B5 and Eno1 complexes.

Figure 10 .
Figure 10.Heat map of hydrogen bond formation in different conformations between B5 and Eno1 complexes.
[a]MIC 80 value of compound in combination with 8.0 µg/mL FLC.

4 Alone with FLC [a] FICI Mode of Interaction
[a]MIC 80 value of compound in combination with 8.0 µg/mL FLC.-: no substitution.
NA * indicates that the complex is unable to bind spontaneously in MDs.