A Comprehensive in vitro and in silico Assessment of Eugenol Glycoconjugates against Azole and Amphotericin B Resistant Rhizopus delemar var arrhizus

A Comprehensive in vitro and in silico Assessment of Eugenol Glycoconjugates against Azole and Amphotericin B Resistant Rhizopus delemar var arrhizus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Comprehensive in vitro and in silico Assessment of Eugenol Glycoconjugates against Azole and Amphotericin B Resistant Rhizopus delemar var arrhizus Pooja Sen, Lovely Gupta, Aastha Chauhan, Lakshmi Goswami, Asish K Bhattacharya, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6360712/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Rhizopus delemar var arrhizus is a major cause of mucormycosis, a severe infectious disease with high morbidity and mortality. Treatment is challenging due to rising antifungal resistance. Glycosylation is a crucial technique for enhancing the properties of phenolic compounds like eugenol. The present study tries to examine the antifungal efficacy of eugenol glycoconjugates against azole and amphotericin B-resistant R. delemar. Methods and Results: Out of 50 soil samples, 12 Mucor isolates were isolated with 7 identified as R. delemar via 18S ITS sequencing. Antifungal susceptibility testing (AST) revealed that all R. delemar isolates were resistant to amphotericin B (MIC >1 μg/mL). Most isolates also showed resistance to posaconazole (MIC >1 μg/mL) and itraconazole (MIC >2 μg/mL). AST of eugenol glycoconjugate (coded 6g) showed efficacy against resistant R. delemar isolates, with MIC values ranging from 6.25μg/mL to 25μg/mL. Flow cytometry confirmed its fungicidal activity, correlating with MIC data. Compound 6g significantly reduced conidial germination within 24h and exhibited no cytotoxicity on A549 lung cancer cells. In-silico analysis revealed a negative binding affinity of compound 6g for the spore coat protein CotH3, which could a potential antifungal target. Conclusions: Compound 6g could be an potential antifungal molecule against resistant R. delemar isolates, which requires further studies. Resistance Antifungals Mucormycosis eugenol glycoconjugates virulence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Mucormycosis, also known as black fungus infection, poses a grave threat, particularly to individuals with weakened immune systems such as those with uncontrolled diabetes, COVID-19, or hematological cancers followed by Aspergillus fumigatus (Petrikkos et al. 2012; Cornely et al. 2019; Galicia-Garcia et al. 2020). It is a difficult-to-diagnose disease, with 40%-80% mortality rate. The WHO recently listed the Mucorales order as a high priority due to rising infections, resistance issues, and treatability challenges (WHO report 2022). Within the order of Mucorales, Rhizopus arrhizus (also known as R. delemar ) is the most prevalent species causing mucormycosis worldwide. It is common fungi found in soil and decaying organic matter (Hoffmann et al. 2013; Richardson and Rautemaa-Richardson 2019) and is often associated with food spoilage, especially in fruits and vegetables (Hoffmann et al. 2013; Walther et al. 2013). It is known for its ability to produce spores rapidly, making it a concern in food safety and healthcare settings, and predominantly spread through inhalation of spores, traumatic inoculation and ingestion are possible routes (Prakash and Chakrabarti 2019; Khanna et al. 2021; Chauhan et al. 2024). The class of drugs crucial in treating mucormycosis are amphotericin B (Amp B) and posaconazole (POS) (Cornely et al. 2019). These can be administered orally or through injection. Other commonly used antifungals like echinocandins, fluconazole (FLC), and voriconazole (VOR) are not effective against the fungi responsible for mucormycosis due to innate resistance against these drugs (Pfaller et al. 2022) which further restricts treatment options for mucormycosis. The combination of drug and surgical therapy has significantly improved survival rates compared to drug treatment alone. However, treating mucormycosis is financially challenging, especially in low-income countries and even in the US, due to the high costs of drugs, surgery, and hospitalization (Jian et al. 2022; Sigera and Denning 2024). With the rising number of mucormycosis infections in low- and middle-income countries and the increased occurrence of antifungal resistance, new and affordable therapeutic approaches are urgently needed. Bioactive compounds possess a diverse array of biological activities with therapeutic potential including potent antifungal activity (da Silva et al. 2018; Fisher et al. 2018; Gupta et al. 2024; Song 2024). Eugenol is a natural bioactive compound that was discovered in 1929 as a volatile compound from Eugenia caryophyllata . It was commercially produced in the US in 1940 and is now utilized as a parent molecule for the synthesis of new analogues (da Silva et al. 2018). It has been extensively studied for its various biological and therapeutic applications, but its use is limited due to low solubility, tendency to sublimate and pungent odor. To overcome these limitations, glycosylation has been widely suggested as an important method for structural modification to improve the physico-chemical and biological properties of phenolic compounds like eugenol (Zhang et al. 2013; Gupta et al. 2022; Goswami et al. 2022). The objective of the present study is to investigate the antifungal activity glycoconjugates of eugenol against azole and Amp B resistant R. delemar/ R. arrhizus . MATERIALS AND METHODS Collection and processing of soil samples A total number of 50 soil samples were obtained from diverse locations of Delhi and Delhi-NCR regions including agricultural farms, plant nurseries, hospital compound areas, and open food stalls. The sampling process was meticulous and soil samples were collected from these defined locations after cleaning surface debris such as pebbles and leaves, following digging to a depth of 5–10 cm. Each soil sample (2g by weight) was mixed with sterile 0.9% saline solution and vigorously vortexed for 5 min and left to settle for a period of 4–5 h. Isolation, identification and molecular characterisation of Rhizopus spp For the isolation of Rhizopus spp , 0.1 mL supernatant of the prepared soil solution was spread on potato dextrose agar (PDA) plate containing kanamycin (50µg/mL). Plates were incubated at 28 ± 2ºC for 2–4 days. Daily monitoring of fungal colony growth was done as per established protocols (Hoog 2000; Sen et al. 2023). The typical morphological characteristics of Rhizopus spp were analysed which included the colony shape, colour, and texture of the colony. The microscopic features of Rhizopus spp were viewed under the light microscope (40x magnification) using lactophenol cotton blue staining. Conidia were harvested from Rhizopus spp isolates using sterile phosphate-buffered saline (1x PBS) supplemented with 0.05% Tween 20 (anionic detergent). Approximately 10mL of this solution was carefully poured onto the culture plate, facilitating the dispersion of conidia within the PBS solution. The addition of Tween 20, known for its hydrophobic properties, aided in the efficient separation and collection of the conidia from the culture plate. Conidial suspension was set and adjusted with the concentration of 1x10 6 conidia/mL using UV-vis spectrophotometer at a wavelength of 530nm at an optical density of 0.08–0.1 (Clinical and Laboratory Standards Institute 2008) (Alexander and Clinical and Laboratory Standards Institute, 2017). Further, a final concentration of the conidia was set to 1x10 4 conidia/mL for further experiments. Furthermore, isolates Rhizopus spp. were identified by 18S internal transcribed spacer (ITS) region sequencing using the Sanger sequencing method. Genomic DNA of isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) (Zhang et al. 2010; Sen et al. 2024). The 18S ITS region was amplified using polymerase chain reaction (PCR) with the universal ITS primers ITS1 (5' TCC GTA GGT GAA CCT GCGG 3') and ITS4 (5' TCC TCC GCT TAT TGA TATGC 3') and sequenced. The resulting sequences were analysed using Sequence Scanner Software 2 v2.0 (Applied Biosystems) and identified using basic local alignment search tool (BLAST) from National Centre for Biotechnology Information (NCBI) ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and identification was confirmed when 99–100% sequence identity was observed. Rhizopus spp. identification was also performed by establishing the phylogenetic tree using MEGA X software. Screening for azole and AmpB resistant Rhizopus spp To assess azole and AmpB resistance among Rhizopus spp (n = 7), of which three R. delemar and four R. arrhizus isolates, were grown at epidemiological cutoff values (ECVs) for antifungals amphotericin B (AmpB), posaconazole (POS), itraconazole (ITR)), based on CLSI guidelines (comprising ≥ 95% of the statistical value) (Espinel-Ingroff et al. 2015). Each isolate was subjected to testing at the reported ECVs for Mucorales isolates: (a) Amp B (4µg/mL), (b) POS (4µg/mL), and (c) ITR (2µg/mL) (Espinel-Ingroff et al. 2015; Dannaoui 2017). Isolates were grown at and above ECVs for the respective antifungal agent and were further classified as resistant to the corresponding drug. Further, all seven Rhizopus isolates were evaluated for their resistance using E-strip method, following the protocol outlined by Pfaller et al. (2003) with minor modifications (Pfaller et al. 2003). Briefly, the final conidial suspension (50µl) was inoculated and evenly spread on the PDA plate. E-strips containing AmpB (ranging from 0.002 to 32µg/mL), ITR (ranging from 0.002 to 32µg/mL) and POS (ranging from 0.002 to 32µg/mL) were placed on the plate and incubated at 28°C, and observations were made over a period of 3 days post-inoculation. The ellipse of growth inhibition at the intersection point with the strip was carefully observed, and the upper value was selected for the determination of the minimum inhibitory concentration (MIC). The R. delemar and R. arrhizus isolates showing no ellipse were resistant isolates. Antifungal activity of synthesized glycoconjugate of eugenol The synthesis, and characterisation of ten glycoconjugates of eugenol, which were encoded as compound 6a, 6b, 6c, 6d, 4f, 4e, 6g, 6h, 6i, 6j, has been reported in our previously published research work (Goswami et al. 2022). Their antifungal activity has been analysed against susceptible A. fumigatus (ATCC 46645) isolates. In the present work, antifungal activity of these glycoconjugates were tested against three R. delemar and four R. arrhizus isolates in a 96-well flat-bottom polystyrene plate (Tarsons, India) using the CLSI M38-A2 broth microdilution method (Clinical and Laboratory Standards Institute 2008) (Alexander and Clinical and Laboratory Standards Institute, 2017). The experiment was carried out in biological triplicate to ensure the reliability and consistency of the results. The two-fold serial dilution of all 10 compounds in a 96-well microplate to obtain the concentration range from 200 µg/mL to 0.39 µg/mL. The final conidial suspension was adjusted to 10 4 conidia/mL in RPMI 1640 media. Each well of the microplate was then inoculated with 100µL of conidial suspension except the negative control. The microplate was kept at 28 ± 2˚C for 48–72 h, and the growth in each well was observed and compared with that of the positive control (untreated control). MIC value was determined by the lowest concentration at which no visible growth was observed relative to the drug-free control. Germination assay In the conidial germination assay, Rhizopus isolates ( R. delemar and R. arrhizus ), which showed high resistance towards POS, were analysed. For this, conidial suspensions of isolates containing 1 × 10 5 cells/mL were incubated at MIC of compound 6g supplemented with RPMI 1640 medium, and then incubated for up to 24 h at 28 ± 2°C. At predetermined time points (4, 8, 12, 16, 20 and 24 h), the number of conidia and germinated conidia were observed from treated and untreated cultures using light microscope. For imaging, 1 ml of the sample at each studied time point was spun down at 5,000 rpm for 5 mins and conidia were fixed with 2% formaldehyde solution to prevent further growth. Further, images were captured at 40× magnification with a digital camera (Magcam DC 5) and 20× objective on a confocal microscope using Zeiss Axio Observer after 24 h to assess germination characteristics in response to compounds. Prior to imaging, live conidia were incubated with calcofluor white (CFW) and propidium iodide dye (PI). Flow cytometry-based susceptibility testing of compound 6g Flow cytometry (FC) for antifungal susceptibility assessment yields rapid and reproducible results comparable to the CLSI method (Ramani and Chaturvedi 2000). FC parameters are established based on the analysis of live and dead cells stained with propidium iodide (PI), which selectively permeates cells with severe membrane lesions, resulting in enhanced fluorescence signal detected by a fluorescence-activated cell detector (Khan et al. 2011). The conidial suspension of R. delemar and R. arrhizus (S40, MS and AG4B isolates) was adjusted to 1 x 10 5 conidia/mL spectrophotometrically and inoculated in a glass test tube containing MIC concentration of compound 6g. The tubes were then incubated in a shaker incubator for 24 h at 28 ± 2°C. The positive growth control tube contained fungal conidial suspension and RPMI 1640 without any drug was also incubated at the same condition. POS was taken as an antifungal drug control. All samples were prepared in triplicate. After the incubation period, 1 mL of the suspension of compound and R. delemar and R. arrhizus isolate conidia was taken and centrifuged at 10,000 rpm for 10 min to pellet the cells. The pellet was washed and resuspended in 1× PBS. To this suspension, 5 µl of PI (stock: 1 mg/mL) was added to set a final concentration of 1 µg/mL PI, and the tubes were incubated for 2 min in the dark at 35°C. Unstained cells were included as auto-fluorescence controls. Cell-associated fluorescence was measured using a flow cytometer (BD Accuri C6) equipped with a blue argon laser of 488 nm and 15mW power, and the results were analyzed using Accuri C6 software. The instrument was calibrated, and DNA beads were aligned daily following the manufacturer's instructions. Assessment of cytotoxicity of eugenol glycoconjugate (compound 6g) in human lung cancer cell line A549. Cell cytotoxicity analysis of compound 6g, was performed with A549 cell line using MTT assay. The human lung cancer cell line A549 was obtained from the American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/ streptomycin in a humidified atmosphere of 5% CO 2 at 37 o C. Briefly, A549 cells were seeded at density 1 x 10 4 cells per well in 100 µl culture medium containing 10% FBS in 96 multi-well culture plates and incubated overnight for adherence. After that, the medium was removed, and cells were incubated with various serially diluted concentrations of glycoconjugate 6g and AmpB (antifungal drug) in individual wells having concentrations 1000, 333.3, 111.1, 37.03, 12.34, 4.1, 1.3, 0.45 µg/mL in FBS free medium for another 24 h. The wells containing media with A549 cells are considered as positive control and only media as negative control. After 24 h, the reaction medium was removed, and the adhering cells were washed with 1× PBS. 100 µl of MTT solution (0.5g/L in medium) was added to each culture well and incubated for 4 h at 37 o C. After removal of the medium, formazan blue was solubilized in 100 µl of DMSO and the absorbance was measured at 570 nm using micro-plate reader (Cloud-Clone smart microplate reader, Model no. SMR-16.1). All experiments were performed in technical triplicates and biological duplicates. The percentage relative cell viability was calculated as (A 570 of treated samples/A 570 of untreated samples) * 100 (Vajrabhaya and Korsuwannawong 2018). In-silico screening of compound 6g for therapeutic activity In-silico study of compound 6g was conducted for the prediction of physiochemical and quantitative parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) using Swiss ADME program ( http://www.swissadme.ch/index.php ). The major ADMET properties include molecular weight, number of hydrogen donors, number of hydrogen acceptor atoms, number of rotatable bonds, LogP value, LogS value, and total polar surface area (TPSA) were calculated (Lipinski et al. 2001). The parameters deployed to predict the physicochemical properties of the compound are summerised in Table. Molecular docking studies Eugenol glycoconjugate compound 6g was tested for its interaction with virulence spore coat protein homolog CotH3. The 3D structure of invasin CotH3 protein of R. delemar (Alphafold ID: I1CFE1; Figure) was obtained from Uniprot database ( https://www.uniprot.org/ ). Initial preparation of the proteins involved in various tasks such as charge assignment, determination of solvation parameters, and calculation of fragmental volumes using SPDVB-4.10 version software. Further optimization of the protein molecules was performed using the AutoDock4 Tool for molecular docking (Sharma et al. 2020; Kamboj et al. 2022). The two-dimensional (2D) structures of ligands (compound 6g) were generated using ACD/Chemsketch and saved in mol file format, further mol file format was converted to the PDB file format using Open Babel tool. To predict potential binding pockets within the proteins, the Computed Atlas of Surface Topography of proteins (CASTp), was employed. CASTp 3.0 facilitated the identification and characterization of binding sites, surface structural pockets, as well as the area, shape, and volume of each pocket and internal cavities within the proteins. The docking analysis was conducted using the molecular docking program AutoDock4.2.3 Tool, to explore the binding poses of potential inhibitors within the active site of the targets. For protein-ligand interactions, the Lamarckian genetic algorithm was employed with preset parameters. A total of 50 poses were generated, which were subsequently clustered using an all-atom RMSD cutoff of 0.3 Å to eliminate redundancy, resulting in an average of 20 cluster representatives being retained. All other parameters for docking and scoring were set to default values. The protein structure remained rigid throughout the docking process. The docking poses and interaction analysis was performed using Biovia Discovery Studio Visualizer programs. Statistics Statistical analysis was performed to calculate analog cytotoxicity applying Nonlinear regression dose-inhibition curve fit with 95% confidence interval (CI) using GraphPad Prism software version 8.0.2.263. All tests were performed in biological and technical duplicates. RESULTS Morphological and molecular characterisation of Rhizopus isolates Typical morphological characteristics of Rhizopus spp were observed on the PDA culture plates including fluffy, dense colonies, with intertwined aerial mycelium of whitish-grey cottony appearance and later became heavily speckled with fruiting structures containing sporangiophores (Supplementary Fig. 1). Upon microscopic examination, aseptate or sparsely septate hyphae, along with the presence of rhizoids, sporangium shape, sporangiophore length, columella morphology, as well as organization and branching of stolons were observed (Supplementary Fig. 1). Based on the macroscopic and microscopic characteristics, there are 12 identified species of Rhizopus from 50 collected soil samples, of which 7 were identified as Rhizopus spp and 5 were Mucor spp. Molecular characterization of Rhizopus isolates using ITS1 and ITS4 nucleotide sequences confirmed the species identification of the fungal isolates. The sequences of 12 isolates were 99%-100% identical to the respective genera, of which three were identified as R. delemar , four R. arrhizus , four M. circinelloides and one M. indicus. These 18S ITS sequences of Mucorales isolates were submitted to the NCBI database as listed in Supplementary Table 1. A neighbor-joining phylogenetic tree, constructed using nearly complete 18S ITS sequences, illustrates the phylogenetic positioning of Mucorale isolates within the Mucoraceae family using MEGA X software (Fig. 1 ). Screening of azole and AmpB-resistant isolates of Rhizopus spp Among seven isolates of Rhizopus spp. evaluated in this study, 3 were R. delemar and 4 R. arrhizus . Screening of azole and ampB-resistant isolates of R. delemar and R. arrhizus revealed significant resistance profiles (Fig. 2 ). The MIC of AmpB exceeded its epidemiological cutoff value (ECV) of 1 µg/mL, indicating resistance in the R. delemar and R. arrhizus isolates. Of two azole antifungals tested, POS demonstrated efficacy against only two isolates (S4 and YV), while the remaining isolates exhibited MICs above the ECV (> 1 µg/mL). ITR displayed effectiveness against only one isolate (S4 isolate), with the majority exhibiting MICs surpassing the ECV (> 2 µg/mL). The results are shown in Supplementary Table 2, indicating emergence of resistance to both AmpB and azole antifungals within the R. delemar and R. arrhizus isolates studied. Further, the resistance pattern was verified using E-strips of antifungals (ITR, POS and AmpB) as shown in Fig. 3 , and similar results were observed for 96-well MIC susceptibility. Antifungal activity of eugenol glycoconjugates Antifungal susceptibility testing was conducted on 10 eugenol glycoconjugates—specifically compounds 6a, 6b, 6c, 6d, 4f, 4e, 6g, 6h, 6i, and 6j against three R. delemar and four R. arrhizus isolates that are resistant to azole and AmpB. The MIC values revealed that most compounds (6a, 6b, 6c, 6d, 4f, 4e, 6h, 6i, and 6j) effectively inhibited the growth of resistant isolates at concentrations of ≥ 25 µg/ml (Table 1 ). A compound 6g exhibited significant antifungal activity with MIC values of ≤ 25 µg/ml against all seven resistant isolates, specifically showing MICs of 6.25 µg/ml for S4, MS, and AG4B isolates, 12.5 µg/ml for GM and S40 isolates, and 25 µg/ml for YV and VA2 isolates. Additionally, compound 4f demonstrated an MIC of 6.25 µg/ml against the AG4B isolate. The compound 6g is distinguished by its unique structural features, including a rhamnose sugar moiety, a triazole ring, and a eugenol-based framework. These characteristics likely contribute to its enhanced antifungal efficacy compared to the other synthesised compounds. Table 1 In vitro antifungal activity of 10 glycoconjugates of eugenol against seven azole and AmpB resistant R. delemar and R.arrhzius isolates. Isolates Compounds MIC (µg/mL) 6a 6b 6c 6d 6g 6h 6i 6j 4f 4e S4 25 25 25 50 6.25 50 25 25 25 25 MS 25 25 25 25 6.25 25 25 25 12.5 50 GM 12.5 12.5 25 - 12.5 25 25 25 25 - YV 12.5 12.5 25 25 25 25 25 25 25 25 S40 25 25 25 25 12.5 - - 50 25 25 VA2 - - - 25 25 25 25 - 25 25 AG4B 25 25 25 - 6.25 25 50 25 6.25 - Germination assay The germination of conidia in untreated control cultures of R. delemar and R. arrhizus isolates was initiated after 4 h of incubation. The conidia shift from the dormant stage to the swelling stage in 4–6 h after providing the minimal media and optimum conditions for conidia germination. However, at the MIC of compound 6g, there was a significant reduction in germlings formation at 4, 8, 12, and 16 h of incubation in all isolates. The experiment was designed to observe conidial germination at the MIC of compound 6g for R. delemar and R. arrhizus isolates having high POS MIC values (S40, MS and AG4B) under light microscope. At different time points, compound 6g inhibited the formation of germ tubes by conidia, as shown in the Fig. 4. The antifungal drug POS was used as a drug control. In the untreated control culture, conidia exhibited swelling after 4 h of incubation along with the formation of buds. In contrast, compound 6g halted growth and germination by 8 h of incubation in resistant R. delemar and R. arrhizus isolates. At varying MIC concentrations for each isolate (as mentioned in Table 1 ), POS also showed a reduction in conidia germination compared to the untreated control at 16, 20, and 24 h of incubation under a light microscope. The effect of compound 6g on fungal conidial germination was observed after 24 h of incubation through confocal microscopy using CFW and PI dyes. In the treated cultures, there was clear indication of conidial damage sans conidial germination, which indicates the efficacy of compound 6g in inhibiting the fungal growth at initial stage. In contrast, the untreated cultures exhibited hyphal growth of fungal pathogen as visualized under confocal microscopy (Fig. 5 ). The flouroscence of the PI dye within the treated conidia indicated cellular damage as depicted in the Fig. 5 B. The absorbtion of PI fluorescence inside the treated conidia indicates that compound 6g induced significant membrane damage, leading to cellular disintegration. Flow cytometry-based susceptibility testing of compound 6g Flow cytometry was used to investigate the effect of compounds on the membrane integrity of the fungal cells by calculating the mean fluorescence of PI dye. A significant increase in MCF percentage was observed at 24h of incubation with compound 6g and standard drug POS (Fig. 6 ). In Rhizopus isolates at 24 h, compound 6g demonstrated MCF values of 60.2%, 57.5% and 53.2% in S40, MS, and AG4B isolates, respectively. MCF values of POS antifungal drug were also analysed at MIC which showed efficacy at higher concentrations than reported ECV. The MIC with respect to FC was defined as the lowest concentration of analogs that showed an increase of 50% in MCF compared to that of the control growth. Assessment of cytotoxicity of compound 6g in human lung cancer cell line A549 Compound 6g was tested against human lung cancer cell line A549 for assessing the cytotoxic potential of the bioactive compound. The results reveled that compound 6g showed no cytotoxic activity against the A549 cell line, with an IC 50 of 39.29 µg/mL (95% CI) compared to AmpB as the standard drug (IC 50 of 19.59 µg/mL) (Supplementary Fig. 2). The experiments were set in triplicates and analysis was conducted over a period of 24 h. In-silico screening of analogues for therapeutic activity The analysis of the properties of compound 6g against various drug-likeness criteria reveals some challenges but also its potential as an orally available drug (Supplementary Table 3). Compound 6g has a molecular weight of 505.52 g/mol, exceeding the preferred limit of 500 g/mol. While it meets hydrogen donor criterion, it greatly exceeds the acceptable number of hydrogen acceptors with 11. It follows the rotational bond criterion with 9 bonds and has a topological polar surface area (TPSA) of 117.59 Ų, which is within the acceptable range of less than 140 Ų. The compound 6g displays favourable lipophilicity with a LogP value of 0.83 but has low solubility with a LogS value of -4.55. Despite having a bioavailability score of 0.17, it violates multiple drug-likeness filters. Specifically, it fails Lipinski's rule due to its high molecular weight and number of hydrogen acceptors, and it does not comply with the Ghose rule due to its molecular weight and molar refractivity. However, it meets Veber and Egan criteria, suggesting better potential for oral bioavailability. Despite these issues, overall profile of compound 6g indicates fewer significant barriers to drug development, suggesting it may be a more promising candidate for further optimization. Molecular docking studies CotH3 is reported as a significant virulence factor in R. delemar . It encodes for spore coat proteins that mediate attachment to GRP78 during host cell invasion. In the present study, utilizing the CASTp server, the active sites of CotH3 for both species were identified, revealing substantial differences in area and volume. For R. delemar , the best pocket had an area of 5640.334 (SA) and a volume of 18821.824 (SA). This suggests that CotH3 plays a crucial role in the pathogenicity of these organisms, possibly through interactions at these active sites. The 3D structure of compound 6g and virulence CotH3 protein has been shown in the Fig. 7 A and 7 B. Compounds 6g was docked against CotH3 active site domain of R. delemar (Fig. 7 C). The docking results demonstrated that compound 6g demonstrated a binding affinity score of -7.23 kcal/mol at the active site. It formed hydrogen bonds with ALA397 and GLY418 (Fig. 7 D). Van der Waals interactions were observed with PRO417, GLN366, GLY285, SER284, ASP360, THR367, LEU396, GLY401, ASN395, PRO399, ALA398, and ARG415. Additionally, it forms hydrophobic interactions with one alkyl interaction (ALA397), three pi-sigma interactions (TRP359, ALA397), and two pi-alkyl interactions (TYR416, MET364) at the active site. These interactions highlight the detailed binding mechanisms of compound 6g with the CotH3 active sites, illustrating their potential as inhibitors to reduce the virulence of these pathogenic fungi. Discussion Fungal infections have become more common over the past few decades, significantly contributing to high mortality and morbidity, especially among immunocompromised individuals. Recently, COVID-19 pandemic has facilitated the emergence of secondary infections post SARS-CoV2. The primary fungal pathogen responsible for these infections is Rhizopus spp (agent of mucormycosis). The Mucorales order encompasses a diverse range of genera, with 38 different species reported to cause mucormycosis (Walther et al. 2013). According to WHO report, mucormycosis is rare worldwide, but in India, it is much more common, and significantly higher than in developed countries ( https://www.who.int/ ). Diabetes has been recognized as the most frequent underlying condition in 60% of mucor cases, with only 2.1% showing no underlying co-morbidity (Sigera and Denning 2024). In the present study, Rhizopus spps were isolated from environmental sources (soil samples from Delhi- NCR region). Similar to previous studies, our findings indicated that Rhizopus spp . and Mucor spp . were the most commonly isolated Mucorales from collected soil samples (Ziaee et al. 2016; Mousavi et al. 2018; Prakash and Chakrabarti 2021). Further, 18S ITS sequencing confirmed 3 isolates to be R. delemar , 4 isolates as R. arrhizus , 4 isolates as M. circinelloides and 1 isolate of M. indicus using Sanger’s sequencing. The present study mainly focuses on R. delemar and R. arrhizus environmental isolates for their antifungal susceptibility pattern. Studying R. delemar and R. arrhizus for antifungal development is crucial because these species are among the most common and virulent causes of mucormycosis. Their prevalence in clinical settings, particularly in immunocompromised patients, underscores their significant impact on public health. Moreover, the increasing resistance to existing antifungal treatments in these species makes them prime targets for research, aiming to develop more effective therapeutic strategies. The antifungal susceptibility of Rhizopus isolates was assessed using the CLSI protocol. There is a lack of data correlating MIC values of antifungals with clinical outcomes for Mucorales infections. Studies on the establishment of breakpoints or ECVs for antifungals against Rhizopus spp and Mucor spp are very limited (Espinel-Ingroff et al. 2015). In this study, all Rhizopus isolates exhibited resistance to AmpB. Two isolates (S4 and YV) were susceptible to POS, while the remaining five isolates were resistant, with their MIC values exceeding the ECV. Furthermore, six out of seven isolates were resistant to ITZ, except for the S4 isolate. For AmpB, the CLSI- ECV is 2 µg/ml for both R. arrhizus and R. microsporus . The ECV for POS is 1 µg/ml for both R. arrhizus and R. microsporus (covering 95% of the modeled populations). For ITZ, the proposed ECV for R. arrhizus is 2 µg/ml, applicable to both 95% and 97.5% of the modeled populations. These ECVs are based on data from 8 to 14 laboratories, incorporating 100 MICs for each species and antifungal agent evaluated (Espinel-Ingroff et al. 2015). The prolonged and excessive use of azoles as antifungal medications has resulted in increased drug resistance among certain fungal pathogens, including Mucorales . This issue underscores the urgent need for alternative therapeutic compounds. Natural compounds, especially phytochemicals, have emerged as promising candidates due to their significant antifungal properties. Eugenol, a natural derivative of guaiacol, is known for its significant antifungal, antibacterial, and antiviral properties. This broad spectrum of biological activity makes eugenol a potential molecule for structural modifications in order to enhance its therapeutic properties. Minor structural changes can substantially improve its efficacy; for instance, the IC 50 value of eugenol as antifungal can be reduced from 149 to 109 ppm by conjugating the side chain double bond with the aromatic system, resulting in increased growth inhibition (Olea et al. 2019). Similarly in the present study, eugenol was structurally modified to synthesize glycoconjugates, which are anticipated to have enhanced antifungal efficacy and bioavailability. The synthesis and characterization of these glycoconjugates were published in our previous paper (Goswami et al. 2022). These glycoconjugates have significantly improved the efficacy of eugenol against resistant isolates of R. delemar and R. arrhizus . Among the 10 synthesized glycoconjugates, compound 6g demonstrated antifungal efficacy against all resistant isolates of R. delemar and R. arrhizus , with effective concentrations ranging from 6.25 µg/mL to 25 µg/mL. This compound has rhamnose as an attached sugar moiety. Compounds containing rhamnose are particularly intriguing due to their potential applications, such as antibacterial vaccines and tumor eradication (Karmakar et al. 2016; Chen et al. 2018). Germination, a fundamental process in the lifecycle of filamentous fungi, marks the transition from a dormant spore to an actively growing hyphal cell. This intricate process encompasses several distinct transitions, each crucial for the initiation and progression of fungal growth. Among filamentous fungi, including Rhizopus spp , germination unfolds through a series of well-defined stages, although the precise timing and regulatory mechanisms can vary across species. In the present study, compound 6g has completely inhibited the germination within 24 h of incubation. Therefore, the effect of compound 6g on the germination pattern of conidia was observed at various time points. Both light microscopy and confocal microscopy images confirmed that conidial germination was completely inhibited within 24 h of incubation with compound 6g in resistant isolates of R. delemar and R. arrhizus . This was further validated by the intercalation of PI dye into the conidia treated with compound 6g at MIC levels, indicating effective inhibition under confocal microscope. In comparison, conidia treated with POS showed delayed germination, whereas the control group displayed normal germination patterns. Similarly, minimum fungicidal concentration values for fungal cells within 24 h of treatment via FC effectively indicated a drug's antifungal activity against R. delemar and R. arrhizus isolates, showing a strong correlation with the MICs obtained by the CLSI broth microdilution method (M38-A2). Our findings also revealed that compound 6g exhibited better antifungal activity within 24 h of incubation than POS against R. delemar and R. arrhizus isolates. According to literature, FC studies have been employed to measure antifungal susceptibility in pathogenic yeasts, including Candida spp , Cryptococcus neoformans , and Aspergillus fumigatus (Ramani et al. 2003; Canturk 2018). Additionally, cytotoxicity analysis on the human lung cancer cell line A549 revealed an IC 50 of 39.29 µg/mL for compound 6g. Although the ADME-Tox study indicated that compound 6g violates Lipinski’s Rule of Five and Ghose rule, its overall profile suggests that the barriers to oral bioavailability are manageable. Consequently, compound 6g could emerge as a promising candidate for further optimization to enhance its oral bioavailability and therapeutic potential. According to the literature, structural modifications of naturally active lead moieties can enhance drug efficacy and reduce side effects (Olea et al. 2019; Goswami et al. 2022; Mishra et al. 2024). One effective approach is the synthesis of glyconjugates of bioactive molecule, yielding derivatives with promising antifungal activity. The conjugation of glycones with aglycone molecules, such as eugenol, results in glycohybrids that possess unique characteristics, including multifunctionality, enhanced stability, and targeted action. This method often leads to improved pharmacological properties, such as better solubility, bioavailability, efficacy, and reduced toxicity. In the search for novel antifungal agents, targeting spore coat protein homologs (CotH) appears to be a promising strategy for treating mucormycosis. CotH proteins, a family of kinase proteins, play a crucial role in morphogenesis (such as conidia formation and cell wall structure), stress adaptation, and virulence (Szebenyi et al. 2023). In this study, compound 6g was found to inhibit conidia germination and the growth of R. delemar . Additionally, compound 6g demonstrated a negative binding affinity with the active site of the CotH3 protein in R. delemar , indicating that CotH3 is a potential target of compound 6g. According to the literature, CotH3, a surface protein of the Mucorales, interacts with the glucose-regulated protein 78 (GRP78) receptor on the surface of human nasal epithelial cells and endothelial cells, facilitating tissue invasion and angio-invasion (Liu et al. 2010; Gebremariam et al. 2014; Alqarihi et al. 2020). Elevated levels of iron, hyperglycemia, and ketone bodies increase the expression of the GRP78 receptor. In contrast, R. oryzae mutant with reduced CotH3 expression showed a diminished ability to invade endothelial cells and reduced virulence in diabetic ketoacidosis (DKA) murine model of invasive mucormycosis (IM) (Gebremariam et al. 2014). Compound 6g could potentially replicate this effect by targeting CotH3, offering protection against mucormycosis in both DKA and neutropenic mice, suggesting a promising direction for future research. In conclusion, this study highlights the potential of compound 6g as a promising antifungal agent against mucormycosis caused by R. delemar and R. arrhizus . The compound demonstrated significant inhibition of conidia germination and fungal growth, along with a strong antifungal efficacy within 24 h of incubation. Its interaction with the CotH3 protein suggests a targeted mechanism, offering protection in diabetic and neutropenic murine models. Despite challenges in oral bioavailability, compound 6g's structural modifications and glycoconjugate formulation show enhanced antifungal activity and reduced cytotoxicity. These findings underscore the importance of further optimization and development of compound 6g, aiming to address the increasing resistance and improve therapeutic strategies for mucormycosis. Declarations Funding The authors would also like to thank Council of Scientific & Industrial Research (CSIR) for providing financial support in the form of Senior Research Fellowship [09/915(0013)/2018-EMR-I] to PS. Competing Interests No potential conflict of interest was reported by the authors. The authors are responsible for the content and paper writing. Author Contributions PS and LG conducted literature search, performed experiments, results analysis and drafted the manuscript; AC also performed experiments and helped in manuscript editing; LG synthesized compounds; and AKB critically reviewed and corrected the manuscript; AS helped in In-silico analysis; PV conceptualised the idea and critically analysed the results and manuscript. Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article or supplementary information file . 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Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 14 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 25 Apr, 2025 Reviewers agreed at journal 22 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Editor assigned by journal 04 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 02 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6360712","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446504893,"identity":"681c0cd2-5d37-492b-93e8-8e346d0bf235","order_by":0,"name":"Pooja Sen","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Pooja","middleName":"","lastName":"Sen","suffix":""},{"id":446504894,"identity":"43ae9149-acfb-48ba-ba12-7726f7edddfc","order_by":1,"name":"Lovely Gupta","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Lovely","middleName":"","lastName":"Gupta","suffix":""},{"id":446504895,"identity":"af652db0-5654-4634-aa48-eb5d823a6853","order_by":2,"name":"Aastha Chauhan","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Aastha","middleName":"","lastName":"Chauhan","suffix":""},{"id":446504896,"identity":"e73c5dd4-5aaa-45b9-8181-376e46dce54e","order_by":3,"name":"Lakshmi Goswami","email":"","orcid":"","institution":"National Chemical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Lakshmi","middleName":"","lastName":"Goswami","suffix":""},{"id":446504897,"identity":"03516799-00de-4a9c-9746-7600c265c801","order_by":4,"name":"Asish K Bhattacharya","email":"","orcid":"","institution":"National Chemical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Asish","middleName":"K","lastName":"Bhattacharya","suffix":""},{"id":446504898,"identity":"3b872f04-209e-4021-8421-31aba5db5329","order_by":5,"name":"Abhishek Sengupta","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Sengupta","suffix":""},{"id":446504899,"identity":"4ee95d8b-829f-4d26-92ad-2dd0ad3f98f8","order_by":6,"name":"Pooja Vijayaraghavan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYNACHgY5BgbGBhATQhKjxRiouLGBBC0MDIkNUNWEtRjcPnvwwQcZu/T+aYfbH/Mw2MhuOEBIy7m8ZMMZPMm5M24nNjbzMKQZE9Qi2cNjJs3Dw5zbANFyOJFYLfXp8hAt/wlr4ecBazmcYADRcoAYLXwgvxw33AjUMnOOQbLxTEJa2Hh4Dz742FMtL3c7/cGHNxV2sn2EtICikYGxB8Jk4jEgqByqheEHhMn4gygdo2AUjIJRMNIAACpyQWyzWrI9AAAAAElFTkSuQmCC","orcid":"","institution":"Amity University","correspondingAuthor":true,"prefix":"","firstName":"Pooja","middleName":"","lastName":"Vijayaraghavan","suffix":""}],"badges":[],"createdAt":"2025-04-02 11:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6360712/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6360712/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-10673-2","type":"published","date":"2025-06-12T15:56:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81353982,"identity":"2a679d20-d401-4b0c-a9d5-d453c069c424","added_by":"auto","created_at":"2025-04-25 07:08:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138342,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic tree based on nearly complete 18S ITS sequences showing the phylogenetic position of \u003cem\u003eMucorale\u003c/em\u003e isolates within the Mucorasea species. Numbers at the nodes indicate the percentage of 1000 bootstrap resampling procedures and only values over 50% are given. Bar: 0.01 substitutions per nucleotide position\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/041c36c0e9bae7c3e6d9e3c4.png"},{"id":81354566,"identity":"60e25323-084f-4216-aa9b-584deeda60d2","added_by":"auto","created_at":"2025-04-25 07:17:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26111,"visible":true,"origin":"","legend":"\u003cp\u003eBar graph indicating Minimum Inhibitory Concentration (MIC) (µg/ml) for antifungal drugs (Itraconazole (ITR), Posaconazole (POS), and Amphotericin B (AmpB)) against \u003cem\u003eR. delemar \u003c/em\u003eand \u003cem\u003eR.\u003c/em\u003e \u003cem\u003earrhizus\u003c/em\u003e isolates\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/b2b0a71c3ecb1eb6e16b0c17.png"},{"id":81353978,"identity":"e4143ecd-18ad-4c47-b60d-79908ad5e80f","added_by":"auto","created_at":"2025-04-25 07:08:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":851572,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of Minimum Inhibitory Concentration (MIC) of azole antifungals, including itraconazole (ITR), posaconazole (POS), and amphotericin B (AmpB) against\u003cem\u003e R. delemar \u003c/em\u003eand \u003cem\u003eR. arrhizus \u003c/em\u003eisolates using E-test strips\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/5afdb454ca972a0a6bd1f98d.png"},{"id":81353995,"identity":"e8065b1a-8e85-4b61-8eab-e986a64a67ac","added_by":"auto","created_at":"2025-04-25 07:08:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1584808,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic characterisation of germination pattern of conidia under light microscope. Conidial germination in effect of compound 6g was imaged at different time points (4h, 8h, 12h, 16h, 20h and 24h) against S40, MS and AG4B isolates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/e61cd7dd00e294384948371e.png"},{"id":81353996,"identity":"ef4650c3-3414-48ca-80cb-828d30a42714","added_by":"auto","created_at":"2025-04-25 07:08:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":219892,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of compound 6g on \u003cem\u003eR. delemar \u003c/em\u003eand \u003cem\u003eR.\u003c/em\u003e \u003cem\u003earrhizus \u003c/em\u003eisolates\u003cem\u003e \u003c/em\u003econidial germination and damage at 24 h incubation under confocal laser scanning microscope. Visualization of \u003cem\u003eR. delemar \u003c/em\u003eand \u003cem\u003eR.\u003c/em\u003e \u003cem\u003earrhizus \u003c/em\u003econidial germination (A) untreated control; (B) compound 6g treated; and (C) POS treated, stained with calcofluor white (CFW) and propidium iodide (PI) dye at 20X Magnification. (Scale Bar: 10 µm.)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/6312624d332d85edb5f6cf21.png"},{"id":81354011,"identity":"1ec36fa0-1ddf-4b81-96e0-793306d9dbd1","added_by":"auto","created_at":"2025-04-25 07:09:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":933146,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis of \u003cem\u003eR. delemar \u003c/em\u003evar \u003cem\u003earrhius \u003c/em\u003eisolate following exposure to compound 6g using propidium iodide (PI) dye. The 2D plots illustrate percent mean channel fluorescence (MCF) results determined for control (untreated) and compounds treated (A) S40 isolate, (B) MS isolate and (C) AG4B\u003cem\u003e \u003c/em\u003eisolate at MIC value. Percent MCF indicated the dead cell counts in the effect of compound 6g treatment in comparison with control (untreated). There was a significant increase in MCF, with a 50% or more increase observed at 24 h incubation with compound 6g\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/2cfd4066930280096fefe776.png"},{"id":81353985,"identity":"240b8903-521f-4957-951f-f4b475048e76","added_by":"auto","created_at":"2025-04-25 07:08:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":443423,"visible":true,"origin":"","legend":"\u003cp\u003e3D structure of (A) Compound 6g and (B) virulence spore coat protein CotH3 of \u003cem\u003eR. delemar.\u003c/em\u003e Molecular docking of compound 6g with CotH3 virulence protein (C) and (D) 3D and 2D interaction profile of compound 6g with CotH3, respectively\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/8c3a0cdf79f309aa0483583a.png"},{"id":84726443,"identity":"a2c9a1b4-d899-4f92-9222-080373acf75c","added_by":"auto","created_at":"2025-06-16 16:03:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5225840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/f18d2781-1e83-4168-934d-548188607ada.pdf"},{"id":81354563,"identity":"4758db29-fdcf-4d0c-820c-e9925019ac93","added_by":"auto","created_at":"2025-04-25 07:16:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":192096,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6360712/v1/2b5a714dfa1c267238b4a7e3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Comprehensive in vitro and in silico Assessment of Eugenol Glycoconjugates against Azole and Amphotericin B Resistant Rhizopus delemar var arrhizus ","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMucormycosis, also known as black fungus infection, poses a grave threat, particularly to individuals with weakened immune systems such as those with uncontrolled diabetes, COVID-19, or hematological cancers followed by \u003cem\u003eAspergillus fumigatus\u003c/em\u003e (Petrikkos et al. 2012; Cornely et al. 2019; Galicia-Garcia et al. 2020). It is a difficult-to-diagnose disease, with 40%-80% mortality rate. The WHO recently listed the Mucorales order as a high priority due to rising infections, resistance issues, and treatability challenges (WHO report 2022). Within the order of Mucorales, \u003cem\u003eRhizopus arrhizus\u003c/em\u003e (also known as \u003cem\u003eR. delemar\u003c/em\u003e) is the most prevalent species causing mucormycosis worldwide. It is common fungi found in soil and decaying organic matter (Hoffmann et al. 2013; Richardson and Rautemaa-Richardson 2019) and is often associated with food spoilage, especially in fruits and vegetables (Hoffmann et al. 2013; Walther et al. 2013). It is known for its ability to produce spores rapidly, making it a concern in food safety and healthcare settings, and predominantly spread through inhalation of spores, traumatic inoculation and ingestion are possible routes (Prakash and Chakrabarti 2019; Khanna et al. 2021; Chauhan et al. 2024).\u003c/p\u003e \u003cp\u003eThe class of drugs crucial in treating mucormycosis are amphotericin B (Amp B) and posaconazole (POS) (Cornely et al. 2019). These can be administered orally or through injection. Other commonly used antifungals like echinocandins, fluconazole (FLC), and voriconazole (VOR) are not effective against the fungi responsible for mucormycosis due to innate resistance against these drugs (Pfaller et al. 2022) which further restricts treatment options for mucormycosis. The combination of drug and surgical therapy has significantly improved survival rates compared to drug treatment alone. However, treating mucormycosis is financially challenging, especially in low-income countries and even in the US, due to the high costs of drugs, surgery, and hospitalization (Jian et al. 2022; Sigera and Denning 2024). With the rising number of mucormycosis infections in low- and middle-income countries and the increased occurrence of antifungal resistance, new and affordable therapeutic approaches are urgently needed.\u003c/p\u003e \u003cp\u003eBioactive compounds possess a diverse array of biological activities with therapeutic potential including potent antifungal activity (da Silva et al. 2018; Fisher et al. 2018; Gupta et al. 2024; Song 2024). Eugenol is a natural bioactive compound that was discovered in 1929 as a volatile compound from \u003cem\u003eEugenia caryophyllata\u003c/em\u003e. It was commercially produced in the US in 1940 and is now utilized as a parent molecule for the synthesis of new analogues (da Silva et al. 2018). It has been extensively studied for its various biological and therapeutic applications, but its use is limited due to low solubility, tendency to sublimate and pungent odor. To overcome these limitations, glycosylation has been widely suggested as an important method for structural modification to improve the physico-chemical and biological properties of phenolic compounds like eugenol (Zhang et al. 2013; Gupta et al. 2022; Goswami et al. 2022). The objective of the present study is to investigate the antifungal activity glycoconjugates of eugenol against azole and Amp B resistant \u003cem\u003eR. delemar/ R. arrhizus\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and processing of soil samples\u003c/h2\u003e \u003cp\u003eA total number of 50 soil samples were obtained from diverse locations of Delhi and Delhi-NCR regions including agricultural farms, plant nurseries, hospital compound areas, and open food stalls. The sampling process was meticulous and soil samples were collected from these defined locations after cleaning surface debris such as pebbles and leaves, following digging to a depth of 5\u0026ndash;10 cm. Each soil sample (2g by weight) was mixed with sterile 0.9% saline solution and vigorously vortexed for 5 min and left to settle for a period of 4\u0026ndash;5 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation, identification and molecular characterisation of\u003c/b\u003e \u003cb\u003eRhizopus spp\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the isolation of \u003cem\u003eRhizopus spp\u003c/em\u003e, 0.1 mL supernatant of the prepared soil solution was spread on potato dextrose agar (PDA) plate containing kanamycin (50\u0026micro;g/mL). Plates were incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C for 2\u0026ndash;4 days. Daily monitoring of fungal colony growth was done as per established protocols (Hoog 2000; Sen et al. 2023). The typical morphological characteristics of \u003cem\u003eRhizopus spp\u003c/em\u003e were analysed which included the colony shape, colour, and texture of the colony. The microscopic features of \u003cem\u003eRhizopus spp\u003c/em\u003e were viewed under the light microscope (40x magnification) using lactophenol cotton blue staining.\u003c/p\u003e \u003cp\u003eConidia were harvested from \u003cem\u003eRhizopus spp\u003c/em\u003e isolates using sterile phosphate-buffered saline (1x PBS) supplemented with 0.05% Tween 20 (anionic detergent). Approximately 10mL of this solution was carefully poured onto the culture plate, facilitating the dispersion of conidia within the PBS solution. The addition of Tween 20, known for its hydrophobic properties, aided in the efficient separation and collection of the conidia from the culture plate. Conidial suspension was set and adjusted with the concentration of 1x10\u003csup\u003e6\u003c/sup\u003e conidia/mL using UV-vis spectrophotometer at a wavelength of 530nm at an optical density of 0.08\u0026ndash;0.1 (Clinical and Laboratory Standards Institute 2008) (Alexander and Clinical and Laboratory Standards Institute, 2017). Further, a final concentration of the conidia was set to 1x10\u003csup\u003e4\u003c/sup\u003e conidia/mL for further experiments.\u003c/p\u003e \u003cp\u003eFurthermore, isolates \u003cem\u003eRhizopus spp.\u003c/em\u003e were identified by 18S internal transcribed spacer (ITS) region sequencing using the Sanger sequencing method. Genomic DNA of isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) (Zhang et al. 2010; Sen et al. 2024). The 18S ITS region was amplified using polymerase chain reaction (PCR) with the universal ITS primers ITS1 (5' TCC GTA GGT GAA CCT GCGG 3') and ITS4 (5' TCC TCC GCT TAT TGA TATGC 3') and sequenced. The resulting sequences were analysed using Sequence Scanner Software 2 v2.0 (Applied Biosystems) and identified using basic local alignment search tool (BLAST) from National Centre for Biotechnology Information (NCBI) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and identification was confirmed when 99\u0026ndash;100% sequence identity was observed. \u003cem\u003eRhizopus spp.\u003c/em\u003e identification was also performed by establishing the phylogenetic tree using MEGA X software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScreening for azole and AmpB resistant\u003c/b\u003e \u003cb\u003eRhizopus spp\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess azole and AmpB resistance among \u003cem\u003eRhizopus spp\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7), of which three \u003cem\u003eR. delemar\u003c/em\u003e and four \u003cem\u003eR. arrhizus\u003c/em\u003e isolates, were grown at epidemiological cutoff values (ECVs) for antifungals amphotericin B (AmpB), posaconazole (POS), itraconazole (ITR)), based on CLSI guidelines (comprising\u0026thinsp;\u0026ge;\u0026thinsp;95% of the statistical value) (Espinel-Ingroff et al. 2015). Each isolate was subjected to testing at the reported ECVs for Mucorales isolates: (a) Amp B (4\u0026micro;g/mL), (b) POS (4\u0026micro;g/mL), and (c) ITR (2\u0026micro;g/mL) (Espinel-Ingroff et al. 2015; Dannaoui 2017). Isolates were grown at and above ECVs for the respective antifungal agent and were further classified as resistant to the corresponding drug. Further, all seven \u003cem\u003eRhizopus\u003c/em\u003e isolates were evaluated for their resistance using E-strip method, following the protocol outlined by Pfaller et al. (2003) with minor modifications (Pfaller et al. 2003).\u003c/p\u003e \u003cp\u003eBriefly, the final conidial suspension (50\u0026micro;l) was inoculated and evenly spread on the PDA plate. E-strips containing AmpB (ranging from 0.002 to 32\u0026micro;g/mL), ITR (ranging from 0.002 to 32\u0026micro;g/mL) and POS (ranging from 0.002 to 32\u0026micro;g/mL) were placed on the plate and incubated at 28\u0026deg;C, and observations were made over a period of 3 days post-inoculation. The ellipse of growth inhibition at the intersection point with the strip was carefully observed, and the upper value was selected for the determination of the minimum inhibitory concentration (MIC). The \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates showing no ellipse were resistant isolates.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntifungal activity of synthesized glycoconjugate of eugenol\u003c/h3\u003e\n\u003cp\u003eThe synthesis, and characterisation of ten glycoconjugates of eugenol, which were encoded as compound 6a, 6b, 6c, 6d, 4f, 4e, 6g, 6h, 6i, 6j, has been reported in our previously published research work (Goswami et al. 2022). Their antifungal activity has been analysed against susceptible \u003cem\u003eA. fumigatus\u003c/em\u003e (ATCC 46645) isolates. In the present work, antifungal activity of these glycoconjugates were tested against three \u003cem\u003eR. delemar\u003c/em\u003e and four \u003cem\u003eR. arrhizus\u003c/em\u003e isolates in a 96-well flat-bottom polystyrene plate (Tarsons, India) using the CLSI M38-A2 broth microdilution method (Clinical and Laboratory Standards Institute 2008) (Alexander and Clinical and Laboratory Standards Institute, 2017). The experiment was carried out in biological triplicate to ensure the reliability and consistency of the results. The two-fold serial dilution of all 10 compounds in a 96-well microplate to obtain the concentration range from 200 \u0026micro;g/mL to 0.39 \u0026micro;g/mL. The final conidial suspension was adjusted to 10\u003csup\u003e4\u003c/sup\u003e conidia/mL in RPMI 1640 media. Each well of the microplate was then inoculated with 100\u0026micro;L of conidial suspension except the negative control. The microplate was kept at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2˚C for 48\u0026ndash;72 h, and the growth in each well was observed and compared with that of the positive control (untreated control). MIC value was determined by the lowest concentration at which no visible growth was observed relative to the drug-free control.\u003c/p\u003e\n\u003ch3\u003eGermination assay\u003c/h3\u003e\n\u003cp\u003eIn the conidial germination assay, \u003cem\u003eRhizopus\u003c/em\u003e isolates (\u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e), which showed high resistance towards POS, were analysed. For this, conidial suspensions of isolates containing 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL were incubated at MIC of compound 6g supplemented with RPMI 1640 medium, and then incubated for up to 24 h at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. At predetermined time points (4, 8, 12, 16, 20 and 24 h), the number of conidia and germinated conidia were observed from treated and untreated cultures using light microscope. For imaging, 1 ml of the sample at each studied time point was spun down at 5,000 rpm for 5 mins and conidia were fixed with 2% formaldehyde solution to prevent further growth. Further, images were captured at 40\u0026times; magnification with a digital camera (Magcam DC 5) and 20\u0026times; objective on a confocal microscope using Zeiss Axio Observer after 24 h to assess germination characteristics in response to compounds. Prior to imaging, live conidia were incubated with calcofluor white (CFW) and propidium iodide dye (PI).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry-based susceptibility testing of compound 6g\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlow cytometry (FC) for antifungal susceptibility assessment yields rapid and reproducible results comparable to the CLSI method (Ramani and Chaturvedi 2000). FC parameters are established based on the analysis of live and dead cells stained with propidium iodide (PI), which selectively permeates cells with severe membrane lesions, resulting in enhanced fluorescence signal detected by a fluorescence-activated cell detector (Khan et al. 2011).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe conidial suspension of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e (S40, MS and AG4B isolates) was adjusted to 1 x 10\u003csup\u003e5\u003c/sup\u003e conidia/mL spectrophotometrically and inoculated in a glass test tube containing MIC concentration of compound 6g. The tubes were then incubated in a shaker incubator for 24 h at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. The positive growth control tube contained fungal conidial suspension and RPMI 1640 without any drug was also incubated at the same condition. POS was taken as an antifungal drug control. All samples were prepared in triplicate. After the incubation period, 1 mL of the suspension of compound and \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolate conidia was taken and centrifuged at 10,000 rpm for 10 min to pellet the cells. The pellet was washed and resuspended in 1\u0026times; PBS. To this suspension, 5 \u0026micro;l of PI (stock: 1 mg/mL) was added to set a final concentration of 1 \u0026micro;g/mL PI, and the tubes were incubated for 2 min in the dark at 35\u0026deg;C. Unstained cells were included as auto-fluorescence controls. Cell-associated fluorescence was measured using a flow cytometer (BD Accuri C6) equipped with a blue argon laser of 488 nm and 15mW power, and the results were analyzed using Accuri C6 software. The instrument was calibrated, and DNA beads were aligned daily following the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of cytotoxicity of eugenol glycoconjugate (compound 6g) in human lung cancer cell line A549.\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell cytotoxicity analysis of compound 6g, was performed with A549 cell line using MTT assay. The human lung cancer cell line A549 was obtained from the American Type Culture Collection (ATCC). Cells were grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/ streptomycin in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003eBriefly, A549 cells were seeded at density 1 x 10\u003csup\u003e4\u003c/sup\u003e cells per well in 100 \u0026micro;l culture medium containing 10% FBS in 96 multi-well culture plates and incubated overnight for adherence. After that, the medium was removed, and cells were incubated with various serially diluted concentrations of glycoconjugate 6g and AmpB (antifungal drug) in individual wells having concentrations 1000, 333.3, 111.1, 37.03, 12.34, 4.1, 1.3, 0.45 \u0026micro;g/mL in FBS free medium for another 24 h. The wells containing media with A549 cells are considered as positive control and only media as negative control. After 24 h, the reaction medium was removed, and the adhering cells were washed with 1\u0026times; PBS. 100 \u0026micro;l of MTT solution (0.5g/L in medium) was added to each culture well and incubated for 4 h at 37\u003csup\u003eo\u003c/sup\u003eC. After removal of the medium, formazan blue was solubilized in 100 \u0026micro;l of DMSO and the absorbance was measured at 570 nm using micro-plate reader (Cloud-Clone smart microplate reader, Model no. SMR-16.1). All experiments were performed in technical triplicates and biological duplicates.\u003c/p\u003e \u003cp\u003eThe percentage relative cell viability was calculated as (A\u003csub\u003e570\u003c/sub\u003e of treated samples/A\u003csub\u003e570\u003c/sub\u003e of untreated samples) * 100 (Vajrabhaya and Korsuwannawong 2018).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-silico\u003c/b\u003e \u003cb\u003escreening of compound 6g for therapeutic activity\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cem\u003eIn-silico\u003c/em\u003e study of compound 6g was conducted for the prediction of physiochemical and quantitative parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) using Swiss ADME program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/index.php\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The major ADMET properties include molecular weight, number of hydrogen donors, number of hydrogen acceptor atoms, number of rotatable bonds, LogP value, LogS value, and total polar surface area (TPSA) were calculated (Lipinski et al. 2001). The parameters deployed to predict the physicochemical properties of the compound are summerised in Table.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eMolecular docking studies\u003c/h3\u003e\n\u003cp\u003eEugenol glycoconjugate compound 6g was tested for its interaction with virulence spore coat protein homolog CotH3. The 3D structure of invasin CotH3 protein of \u003cem\u003eR. delemar\u003c/em\u003e (Alphafold ID: I1CFE1; Figure) was obtained from Uniprot database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Initial preparation of the proteins involved in various tasks such as charge assignment, determination of solvation parameters, and calculation of fragmental volumes using SPDVB-4.10 version software. Further optimization of the protein molecules was performed using the AutoDock4 Tool for molecular docking (Sharma et al. 2020; Kamboj et al. 2022).\u003c/p\u003e \u003cp\u003eThe two-dimensional (2D) structures of ligands (compound 6g) were generated using ACD/Chemsketch and saved in mol file format, further mol file format was converted to the PDB file format using Open Babel tool. To predict potential binding pockets within the proteins, the Computed Atlas of Surface Topography of proteins (CASTp), was employed. CASTp 3.0 facilitated the identification and characterization of binding sites, surface structural pockets, as well as the area, shape, and volume of each pocket and internal cavities within the proteins.\u003c/p\u003e \u003cp\u003eThe docking analysis was conducted using the molecular docking program AutoDock4.2.3 Tool, to explore the binding poses of potential inhibitors within the active site of the targets. For protein-ligand interactions, the Lamarckian genetic algorithm was employed with preset parameters. A total of 50 poses were generated, which were subsequently clustered using an all-atom RMSD cutoff of 0.3 \u0026Aring; to eliminate redundancy, resulting in an average of 20 cluster representatives being retained. All other parameters for docking and scoring were set to default values. The protein structure remained rigid throughout the docking process. The docking poses and interaction analysis was performed using Biovia Discovery Studio Visualizer programs.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed to calculate analog cytotoxicity applying Nonlinear regression dose-inhibition curve fit with 95% confidence interval (CI) using GraphPad Prism software version 8.0.2.263. All tests were performed in biological and technical duplicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMorphological and molecular characterisation of\u003c/b\u003e \u003cb\u003eRhizopus\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTypical morphological characteristics of \u003cem\u003eRhizopus spp\u003c/em\u003e were observed on the PDA culture plates including fluffy, dense colonies, with intertwined aerial mycelium of whitish-grey cottony appearance and later became heavily speckled with fruiting structures containing sporangiophores (Supplementary Fig.\u0026nbsp;1). Upon microscopic examination, aseptate or sparsely septate hyphae, along with the presence of rhizoids, sporangium shape, sporangiophore length, columella morphology, as well as organization and branching of stolons were observed (Supplementary Fig.\u0026nbsp;1). Based on the macroscopic and microscopic characteristics, there are 12 identified species of \u003cem\u003eRhizopus\u003c/em\u003e from 50 collected soil samples, of which 7 were identified as \u003cem\u003eRhizopus spp\u003c/em\u003e and 5 were \u003cem\u003eMucor spp.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMolecular characterization of \u003cem\u003eRhizopus\u003c/em\u003e isolates using ITS1 and ITS4 nucleotide sequences confirmed the species identification of the fungal isolates. The sequences of 12 isolates were 99%-100% identical to the respective genera, of which three were identified as \u003cem\u003eR. delemar\u003c/em\u003e, four \u003cem\u003eR. arrhizus\u003c/em\u003e, four \u003cem\u003eM. circinelloides\u003c/em\u003e and one \u003cem\u003eM. indicus.\u003c/em\u003e These 18S ITS sequences of \u003cem\u003eMucorales\u003c/em\u003e isolates were submitted to the NCBI database as listed in Supplementary Table\u0026nbsp;1. A neighbor-joining phylogenetic tree, constructed using nearly complete 18S ITS sequences, illustrates the phylogenetic positioning of Mucorale isolates within the Mucoraceae family using MEGA X software (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eScreening of azole and AmpB-resistant isolates of\u003c/b\u003e \u003cb\u003eRhizopus spp\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong seven isolates of \u003cem\u003eRhizopus spp.\u003c/em\u003e evaluated in this study, 3 were \u003cem\u003eR. delemar\u003c/em\u003e and 4 \u003cem\u003eR. arrhizus\u003c/em\u003e. Screening of azole and ampB-resistant isolates of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e revealed significant resistance profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The MIC of AmpB exceeded its epidemiological cutoff value (ECV) of 1 \u0026micro;g/mL, indicating resistance in the \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates. Of two azole antifungals tested, POS demonstrated efficacy against only two isolates (S4 and YV), while the remaining isolates exhibited MICs above the ECV (\u0026gt;\u0026thinsp;1 \u0026micro;g/mL). ITR displayed effectiveness against only one isolate (S4 isolate), with the majority exhibiting MICs surpassing the ECV (\u0026gt;\u0026thinsp;2 \u0026micro;g/mL). The results are shown in Supplementary Table\u0026nbsp;2, indicating emergence of resistance to both AmpB and azole antifungals within the \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates studied. Further, the resistance pattern was verified using E-strips of antifungals (ITR, POS and AmpB) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and similar results were observed for 96-well MIC susceptibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAntifungal activity of eugenol glycoconjugates\u003c/h3\u003e\n\u003cp\u003eAntifungal susceptibility testing was conducted on 10 eugenol glycoconjugates\u0026mdash;specifically compounds 6a, 6b, 6c, 6d, 4f, 4e, 6g, 6h, 6i, and 6j against three \u003cem\u003eR. delemar\u003c/em\u003e and four \u003cem\u003eR. arrhizus\u003c/em\u003e isolates that are resistant to azole and AmpB.\u003c/p\u003e \u003cp\u003eThe MIC values revealed that most compounds (6a, 6b, 6c, 6d, 4f, 4e, 6h, 6i, and 6j) effectively inhibited the growth of resistant isolates at concentrations of \u0026ge;\u0026thinsp;25 \u0026micro;g/ml (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A compound 6g exhibited significant antifungal activity with MIC values of \u0026le;\u0026thinsp;25 \u0026micro;g/ml against all seven resistant isolates, specifically showing MICs of 6.25 \u0026micro;g/ml for S4, MS, and AG4B isolates, 12.5 \u0026micro;g/ml for GM and S40 isolates, and 25 \u0026micro;g/ml for YV and VA2 isolates. Additionally, compound 4f demonstrated an MIC of 6.25 \u0026micro;g/ml against the AG4B isolate.\u003c/p\u003e \u003cp\u003eThe compound 6g is distinguished by its unique structural features, including a rhamnose sugar moiety, a triazole ring, and a eugenol-based framework. These characteristics likely contribute to its enhanced antifungal efficacy compared to the other synthesised compounds.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antifungal activity of 10 glycoconjugates of eugenol against seven azole and AmpB resistant \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR.arrhzius\u003c/em\u003e isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eCompounds MIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6a\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6b\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6c\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6i\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6j\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4f\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAG4B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGermination assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe germination of conidia in untreated control cultures of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates was initiated after 4 h of incubation. The conidia shift from the dormant stage to the swelling stage in 4\u0026ndash;6 h after providing the minimal media and optimum conditions for conidia germination. However, at the MIC of compound 6g, there was a significant reduction in germlings formation at 4, 8, 12, and 16 h of incubation in all isolates. The experiment was designed to observe conidial germination at the MIC of compound 6g for \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates having high POS MIC values (S40, MS and AG4B) under light microscope. At different time points, compound 6g inhibited the formation of germ tubes by conidia, as shown in the Fig.\u0026nbsp;4. The antifungal drug POS was used as a drug control. In the untreated control culture, conidia exhibited swelling after 4 h of incubation along with the formation of buds. In contrast, compound 6g halted growth and germination by 8 h of incubation in resistant \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates. At varying MIC concentrations for each isolate (as mentioned in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), POS also showed a reduction in conidia germination compared to the untreated control at 16, 20, and 24 h of incubation under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe effect of compound 6g on fungal conidial germination was observed after 24 h of incubation through confocal microscopy using CFW and PI dyes. In the treated cultures, there was clear indication of conidial damage sans conidial germination, which indicates the efficacy of compound 6g in inhibiting the fungal growth at initial stage. In contrast, the untreated cultures exhibited hyphal growth of fungal pathogen as visualized under confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The flouroscence of the PI dye within the treated conidia indicated cellular damage as depicted in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. The absorbtion of PI fluorescence inside the treated conidia indicates that compound 6g induced significant membrane damage, leading to cellular disintegration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry-based susceptibility testing of compound 6g\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlow cytometry was used to investigate the effect of compounds on the membrane integrity of the fungal cells by calculating the mean fluorescence of PI dye. A significant increase in MCF percentage was observed at 24h of incubation with compound 6g and standard drug POS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In \u003cem\u003eRhizopus\u003c/em\u003e isolates at 24 h, compound 6g demonstrated MCF values of 60.2%, 57.5% and 53.2% in S40, MS, and AG4B isolates, respectively. MCF values of POS antifungal drug were also analysed at MIC which showed efficacy at higher concentrations than reported ECV. The MIC with respect to FC was defined as the lowest concentration of analogs that showed an increase of 50% in MCF compared to that of the control growth.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of cytotoxicity of compound 6g in human lung cancer cell line A549\u003c/h2\u003e \u003cp\u003eCompound 6g was tested against human lung cancer cell line A549 for assessing the cytotoxic potential of the bioactive compound. The results reveled that compound 6g showed no cytotoxic activity against the A549 cell line, with an IC\u003csub\u003e50\u003c/sub\u003e of 39.29 \u0026micro;g/mL (95% CI) compared to AmpB as the standard drug (IC\u003csub\u003e50\u003c/sub\u003e of 19.59 \u0026micro;g/mL) (Supplementary Fig.\u0026nbsp;2). The experiments were set in triplicates and analysis was conducted over a period of 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-silico\u003c/b\u003e \u003cb\u003escreening of analogues for therapeutic activity\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe analysis of the properties of compound 6g against various drug-likeness criteria reveals some challenges but also its potential as an orally available drug (Supplementary Table\u0026nbsp;3). Compound 6g has a molecular weight of 505.52 g/mol, exceeding the preferred limit of 500 g/mol. While it meets hydrogen donor criterion, it greatly exceeds the acceptable number of hydrogen acceptors with 11. It follows the rotational bond criterion with 9 bonds and has a topological polar surface area (TPSA) of 117.59 \u0026Aring;\u0026sup2;, which is within the acceptable range of less than 140 \u0026Aring;\u0026sup2;. The compound 6g displays favourable lipophilicity with a LogP value of 0.83 but has low solubility with a LogS value of -4.55. Despite having a bioavailability score of 0.17, it violates multiple drug-likeness filters. Specifically, it fails Lipinski's rule due to its high molecular weight and number of hydrogen acceptors, and it does not comply with the Ghose rule due to its molecular weight and molar refractivity. However, it meets Veber and Egan criteria, suggesting better potential for oral bioavailability. Despite these issues, overall profile of compound 6g indicates fewer significant barriers to drug development, suggesting it may be a more promising candidate for further optimization.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking studies\u003c/h2\u003e \u003cp\u003eCotH3 is reported as a significant virulence factor in \u003cem\u003eR. delemar\u003c/em\u003e. It encodes for spore coat proteins that mediate attachment to GRP78 during host cell invasion. In the present study, utilizing the CASTp server, the active sites of CotH3 for both species were identified, revealing substantial differences in area and volume. For \u003cem\u003eR. delemar\u003c/em\u003e, the best pocket had an area of 5640.334 (SA) and a volume of 18821.824 (SA). This suggests that CotH3 plays a crucial role in the pathogenicity of these organisms, possibly through interactions at these active sites.\u003c/p\u003e \u003cp\u003eThe 3D structure of compound 6g and virulence CotH3 protein has been shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. Compounds 6g was docked against CotH3 active site domain of \u003cem\u003eR. delemar\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The docking results demonstrated that compound 6g demonstrated a binding affinity score of -7.23 kcal/mol at the active site. It formed hydrogen bonds with ALA397 and GLY418 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Van der Waals interactions were observed with PRO417, GLN366, GLY285, SER284, ASP360, THR367, LEU396, GLY401, ASN395, PRO399, ALA398, and ARG415. Additionally, it forms hydrophobic interactions with one alkyl interaction (ALA397), three pi-sigma interactions (TRP359, ALA397), and two pi-alkyl interactions (TYR416, MET364) at the active site. These interactions highlight the detailed binding mechanisms of compound 6g with the CotH3 active sites, illustrating their potential as inhibitors to reduce the virulence of these pathogenic fungi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFungal infections have become more common over the past few decades, significantly contributing to high mortality and morbidity, especially among immunocompromised individuals. Recently, COVID-19 pandemic has facilitated the emergence of secondary infections post SARS-CoV2. The primary fungal pathogen responsible for these infections is \u003cem\u003eRhizopus spp\u003c/em\u003e (agent of mucormycosis). The Mucorales order encompasses a diverse range of genera, with 38 different species reported to cause mucormycosis (Walther et al. 2013).\u003c/p\u003e \u003cp\u003eAccording to WHO report, mucormycosis is rare worldwide, but in India, it is much more common, and significantly higher than in developed countries (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/\u003c/span\u003e\u003cspan address=\"https://www.who.int/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Diabetes has been recognized as the most frequent underlying condition in 60% of mucor cases, with only 2.1% showing no underlying co-morbidity (Sigera and Denning 2024).\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eRhizopus\u003c/em\u003e spps were isolated from environmental sources (soil samples from Delhi- NCR region). Similar to previous studies, our findings indicated that \u003cem\u003eRhizopus spp\u003c/em\u003e. and \u003cem\u003eMucor spp\u003c/em\u003e. were the most commonly isolated \u003cem\u003eMucorales\u003c/em\u003e from collected soil samples (Ziaee et al. 2016; Mousavi et al. 2018; Prakash and Chakrabarti 2021). Further, 18S ITS sequencing confirmed 3 isolates to be \u003cem\u003eR. delemar\u003c/em\u003e, 4 isolates as \u003cem\u003eR. arrhizus\u003c/em\u003e, 4 isolates as \u003cem\u003eM. circinelloides\u003c/em\u003e and 1 isolate of \u003cem\u003eM. indicus\u003c/em\u003e using Sanger\u0026rsquo;s sequencing.\u003c/p\u003e \u003cp\u003eThe present study mainly focuses on \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e environmental isolates for their antifungal susceptibility pattern. Studying \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e for antifungal development is crucial because these species are among the most common and virulent causes of mucormycosis. Their prevalence in clinical settings, particularly in immunocompromised patients, underscores their significant impact on public health. Moreover, the increasing resistance to existing antifungal treatments in these species makes them prime targets for research, aiming to develop more effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eThe antifungal susceptibility of \u003cem\u003eRhizopus\u003c/em\u003e isolates was assessed using the CLSI protocol. There is a lack of data correlating MIC values of antifungals with clinical outcomes for Mucorales infections. Studies on the establishment of breakpoints or ECVs for antifungals against \u003cem\u003eRhizopus spp\u003c/em\u003e and \u003cem\u003eMucor spp\u003c/em\u003e are very limited (Espinel-Ingroff et al. 2015). In this study, all \u003cem\u003eRhizopus\u003c/em\u003e isolates exhibited resistance to AmpB. Two isolates (S4 and YV) were susceptible to POS, while the remaining five isolates were resistant, with their MIC values exceeding the ECV. Furthermore, six out of seven isolates were resistant to ITZ, except for the S4 isolate. For AmpB, the CLSI- ECV is 2 \u0026micro;g/ml for both \u003cem\u003eR. arrhizus\u003c/em\u003e and \u003cem\u003eR. microsporus\u003c/em\u003e. The ECV for POS is 1 \u0026micro;g/ml for both \u003cem\u003eR. arrhizus\u003c/em\u003e and \u003cem\u003eR. microsporus\u003c/em\u003e (covering 95% of the modeled populations). For ITZ, the proposed ECV for \u003cem\u003eR. arrhizus\u003c/em\u003e is 2 \u0026micro;g/ml, applicable to both 95% and 97.5% of the modeled populations. These ECVs are based on data from 8 to 14 laboratories, incorporating 100 MICs for each species and antifungal agent evaluated (Espinel-Ingroff et al. 2015).\u003c/p\u003e \u003cp\u003eThe prolonged and excessive use of azoles as antifungal medications has resulted in increased drug resistance among certain fungal pathogens, including \u003cem\u003eMucorales\u003c/em\u003e. This issue underscores the urgent need for alternative therapeutic compounds. Natural compounds, especially phytochemicals, have emerged as promising candidates due to their significant antifungal properties. Eugenol, a natural derivative of guaiacol, is known for its significant antifungal, antibacterial, and antiviral properties. This broad spectrum of biological activity makes eugenol a potential molecule for structural modifications in order to enhance its therapeutic properties. Minor structural changes can substantially improve its efficacy; for instance, the IC\u003csub\u003e50\u003c/sub\u003e value of eugenol as antifungal can be reduced from 149 to 109 ppm by conjugating the side chain double bond with the aromatic system, resulting in increased growth inhibition (Olea et al. 2019). Similarly in the present study, eugenol was structurally modified to synthesize glycoconjugates, which are anticipated to have enhanced antifungal efficacy and bioavailability. The synthesis and characterization of these glycoconjugates were published in our previous paper (Goswami et al. 2022). These glycoconjugates have significantly improved the efficacy of eugenol against resistant isolates of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e. Among the 10 synthesized glycoconjugates, compound 6g demonstrated antifungal efficacy against all resistant isolates of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e, with effective concentrations ranging from 6.25 \u0026micro;g/mL to 25 \u0026micro;g/mL. This compound has rhamnose as an attached sugar moiety. Compounds containing rhamnose are particularly intriguing due to their potential applications, such as antibacterial vaccines and tumor eradication (Karmakar et al. 2016; Chen et al. 2018).\u003c/p\u003e \u003cp\u003eGermination, a fundamental process in the lifecycle of filamentous fungi, marks the transition from a dormant spore to an actively growing hyphal cell. This intricate process encompasses several distinct transitions, each crucial for the initiation and progression of fungal growth. Among filamentous fungi, including \u003cem\u003eRhizopus spp\u003c/em\u003e, germination unfolds through a series of well-defined stages, although the precise timing and regulatory mechanisms can vary across species. In the present study, compound 6g has completely inhibited the germination within 24 h of incubation. Therefore, the effect of compound 6g on the germination pattern of conidia was observed at various time points. Both light microscopy and confocal microscopy images confirmed that conidial germination was completely inhibited within 24 h of incubation with compound 6g in resistant isolates of \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e. This was further validated by the intercalation of PI dye into the conidia treated with compound 6g at MIC levels, indicating effective inhibition under confocal microscope. In comparison, conidia treated with POS showed delayed germination, whereas the control group displayed normal germination patterns.\u003c/p\u003e \u003cp\u003eSimilarly, minimum fungicidal concentration values for fungal cells within 24 h of treatment \u003cem\u003evia\u003c/em\u003e FC effectively indicated a drug's antifungal activity against \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates, showing a strong correlation with the MICs obtained by the CLSI broth microdilution method (M38-A2). Our findings also revealed that compound 6g exhibited better antifungal activity within 24 h of incubation than POS against \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e isolates. According to literature, FC studies have been employed to measure antifungal susceptibility in pathogenic yeasts, including \u003cem\u003eCandida spp\u003c/em\u003e, \u003cem\u003eCryptococcus neoformans\u003c/em\u003e, and \u003cem\u003eAspergillus fumigatus\u003c/em\u003e (Ramani et al. 2003; Canturk 2018).\u003c/p\u003e \u003cp\u003eAdditionally, cytotoxicity analysis on the human lung cancer cell line A549 revealed an IC\u003csub\u003e50\u003c/sub\u003e of 39.29 \u0026micro;g/mL for compound 6g. Although the ADME-Tox study indicated that compound 6g violates Lipinski\u0026rsquo;s Rule of Five and Ghose rule, its overall profile suggests that the barriers to oral bioavailability are manageable. Consequently, compound 6g could emerge as a promising candidate for further optimization to enhance its oral bioavailability and therapeutic potential. According to the literature, structural modifications of naturally active lead moieties can enhance drug efficacy and reduce side effects (Olea et al. 2019; Goswami et al. 2022; Mishra et al. 2024). One effective approach is the synthesis of glyconjugates of bioactive molecule, yielding derivatives with promising antifungal activity. The conjugation of glycones with aglycone molecules, such as eugenol, results in glycohybrids that possess unique characteristics, including multifunctionality, enhanced stability, and targeted action. This method often leads to improved pharmacological properties, such as better solubility, bioavailability, efficacy, and reduced toxicity.\u003c/p\u003e \u003cp\u003eIn the search for novel antifungal agents, targeting spore coat protein homologs (CotH) appears to be a promising strategy for treating mucormycosis. CotH proteins, a family of kinase proteins, play a crucial role in morphogenesis (such as conidia formation and cell wall structure), stress adaptation, and virulence (Szebenyi et al. 2023). In this study, compound 6g was found to inhibit conidia germination and the growth of \u003cem\u003eR. delemar\u003c/em\u003e. Additionally, compound 6g demonstrated a negative binding affinity with the active site of the CotH3 protein in \u003cem\u003eR. delemar\u003c/em\u003e, indicating that CotH3 is a potential target of compound 6g.\u003c/p\u003e \u003cp\u003eAccording to the literature, CotH3, a surface protein of the Mucorales, interacts with the glucose-regulated protein 78 (GRP78) receptor on the surface of human nasal epithelial cells and endothelial cells, facilitating tissue invasion and angio-invasion (Liu et al. 2010; Gebremariam et al. 2014; Alqarihi et al. 2020). Elevated levels of iron, hyperglycemia, and ketone bodies increase the expression of the GRP78 receptor. In contrast, \u003cem\u003eR. oryzae\u003c/em\u003e mutant with reduced CotH3 expression showed a diminished ability to invade endothelial cells and reduced virulence in diabetic ketoacidosis (DKA) murine model of invasive mucormycosis (IM) (Gebremariam et al. 2014). Compound 6g could potentially replicate this effect by targeting CotH3, offering protection against mucormycosis in both DKA and neutropenic mice, suggesting a promising direction for future research.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the potential of compound 6g as a promising antifungal agent against mucormycosis caused by \u003cem\u003eR. delemar\u003c/em\u003e and \u003cem\u003eR. arrhizus\u003c/em\u003e. The compound demonstrated significant inhibition of conidia germination and fungal growth, along with a strong antifungal efficacy within 24 h of incubation. Its interaction with the CotH3 protein suggests a targeted mechanism, offering protection in diabetic and neutropenic murine models. Despite challenges in oral bioavailability, compound 6g's structural modifications and glycoconjugate formulation show enhanced antifungal activity and reduced cytotoxicity. These findings underscore the importance of further optimization and development of compound 6g, aiming to address the increasing resistance and improve therapeutic strategies for mucormycosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would also like to thank Council of Scientific \u0026amp; Industrial Research\u0026nbsp;(CSIR) for providing financial support in the form of Senior Research Fellowship [09/915(0013)/2018-EMR-I] to PS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the authors. The authors are responsible for the content and paper writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePS\u003c/strong\u003e and \u003cstrong\u003eLG\u003c/strong\u003e conducted literature search, performed experiments, results analysis and drafted the manuscript; \u003cstrong\u003eAC\u003c/strong\u003e also performed experiments and helped in manuscript editing; \u003cstrong\u003eLG \u003c/strong\u003esynthesized compounds; and \u003cstrong\u003eAKB\u003c/strong\u003e critically reviewed and corrected the manuscript; \u003cstrong\u003eAS \u003c/strong\u003ehelped in \u003cem\u003eIn-silico\u003c/em\u003e analysis; \u003cstrong\u003ePV\u003c/strong\u003e conceptualised the idea and critically analysed the results and manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article or supplementary information file .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlqarihi A, Gebremariam T, Gu Y, Swidergall M, Alkhazraji S, Soliman SSM, Bruno VM, Edwards JE, Filler SG, Uppuluri P, Ibrahim AS (2020) GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis. mBio 11:e01087-20. https://doi.org/10.1128/mBio.01087-20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanturk Z (2018) Evaluation of synergistic anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans. 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Curr Med Mycol 2:13\u0026ndash;19. https://doi.org/10.18869/acadpub.cmm.2.1.13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Resistance, Antifungals, Mucormycosis, eugenol, glycoconjugates, virulence","lastPublishedDoi":"10.21203/rs.3.rs-6360712/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6360712/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003cem\u003e Rhizopus delemar\u003c/em\u003e var arrhizus is a major cause of mucormycosis, a severe infectious disease with high morbidity and mortality. Treatment is challenging due to rising antifungal resistance. Glycosylation is a crucial technique for enhancing the properties of phenolic compounds like eugenol. The present study tries to examine the antifungal efficacy of eugenol glycoconjugates against azole and amphotericin B-resistant \u003cem\u003eR. delemar.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e Out of 50 soil samples, 12 Mucor isolates were isolated with 7 identified as \u003cem\u003eR. delemar \u003c/em\u003evia 18S ITS sequencing. Antifungal susceptibility testing (AST) revealed that all \u003cem\u003eR. delemar\u003c/em\u003eisolates were resistant to amphotericin B (MIC \u0026gt;1 μg/mL). Most isolates also showed resistance to posaconazole (MIC \u0026gt;1 μg/mL) and itraconazole (MIC \u0026gt;2 μg/mL). AST of eugenol glycoconjugate (coded 6g) showed efficacy against resistant \u003cem\u003eR. delemar\u003c/em\u003e isolates, with MIC values ranging from 6.25μg/mL to 25μg/mL. Flow cytometry confirmed its fungicidal activity, correlating with MIC data. Compound 6g significantly reduced conidial germination within 24h and exhibited no cytotoxicity on A549 lung cancer cells. In-silico analysis revealed a negative binding affinity of compound 6g for the spore coat protein CotH3, which could a potential antifungal target.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Compound 6g could be an potential antifungal molecule against resistant \u003cem\u003eR. delemar\u003c/em\u003e isolates, which requires further studies.\u003c/p\u003e","manuscriptTitle":"A Comprehensive in vitro and in silico Assessment of Eugenol Glycoconjugates against Azole and Amphotericin B Resistant Rhizopus delemar var arrhizus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 07:08:41","doi":"10.21203/rs.3.rs-6360712/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-14T16:24:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T11:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188823022520399739969605155690516784831","date":"2025-04-25T06:54:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44903180173762493698346752944992000632","date":"2025-04-22T16:16:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T10:37:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T08:56:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-04T08:55:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-04-02T11:14:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"960621c9-e3b8-4118-9cbb-71ec12aea262","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T15:58:44+00:00","versionOfRecord":{"articleIdentity":"rs-6360712","link":"https://doi.org/10.1007/s11033-025-10673-2","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-06-12 15:56:53","publishedOnDateReadable":"June 12th, 2025"},"versionCreatedAt":"2025-04-25 07:08:41","video":"","vorDoi":"10.1007/s11033-025-10673-2","vorDoiUrl":"https://doi.org/10.1007/s11033-025-10673-2","workflowStages":[]},"version":"v1","identity":"rs-6360712","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6360712","identity":"rs-6360712","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}