New adipate esters from Cunninghamella echinulata: isolation, identification, biosynthesis and in silico prediction of potential opioid/anti-opioid and antidiabetic activities

Abstract Metabolites of the fungus Cunninghamella echinulata NRRL 1382 were investigated under the effect of fusidic acid (1) feeding. In addition to ergosterol (2) which is a fungal sterol, two novel adipate esters (3, 4) were isolated, and their structures were fully investigated using various spectroscopic analyses, including 1 D, 2 D NMR and HRESIMS. In silico biological target prediction and molecular docking investigation revealed a potential agonist/antagonist activity for compound 3 by binding to µ opioid receptor and antidiabetic effect by aldose reductase inhibitory activity for compound 4. Graphical Abstract


Introduction
Adipic acid has an industrial importance as plasticizer and for nylon production (Kruyer et al. 2020).There are no studies reporting the possible use of adipic acid as a drug.It was not isolated previously from fungi.Cunninghamella echinulata is a fungus widely used in transforming compounds to investigate their fate in mammals or for producing analogues with different chemical and/or biological profiles (Carrizo et al. 1998, Chokchaisiri et al. 2019, Dong et al. 2010, Siddiqui et al. 2017).Few studies reported the constituents produced naturally by C. echinulata as ubiquinone q9 and fatty acids such as c-linolenic (Shiosaki et al. 2001).Herein, two novel adipate esters were isolated alongside ergosterol from C. echinulata in the course of fusidic acid feeding.In silico investigation of the potential biological targets was carried out.

Identification of compounds
The metabolites were isolated using column chromatography and characterised using NMR and HR-ESI-MS analyses.By comparison to the literature, 2 was identified as ergosterol (Figures 1, S1-S7) (Carvalho et al. 2016 Also, the signal at d H 3.15 showed HMBC correlation with the aromatic carbons at d C 126.6, 128.0, 129.6 and 135.9 and with carbonyl carbons at d C 165.5 and 169.5 suggesting that the naphthalene ring is connected through a methylene group to 2-hydroxy propionic acid, which is esterified at its hydroxyl to the rest of the structure.The methylene at d H 1.80 showed correlation with two methylene groups at d H 1.25 & 2.10 and d H 3.40 & 3.52.Correlation of the methylene at d H 1.25 & 2.10 with a methine at d H 4.05 and d C 58.6 proposed oxygenation at the latter site.In the HMBC spectrum, the methylene at d H 1.25 & 2.10 and the methine at d H 4.05 showed correlation with a carbonyl at d C 171.8.Thus, the methine at d H 4.05 is substituted with hydroxyl and carboxyl groups.These data proposed 2-hydroxy adipic acid esterified with a 2-hydroxy-1-naphthyl propionic acid (Table S1, Figures 1, S8-S14).
Compound 4 exhibited a pseudo molecular ion at m/z 318.16542 for [C 10 H 18 O 6 þisopropanolþHþNa] þ (calculated 318.16544). 13CNMR spectra indicated 2 CH 3 , 3 CH 2 , 3 CH and 2 quaternary carbons. 1 HNMR data showed two doublets at d H 0.95 and 1.10 each integrating for 3 protons of two diastereomeric methyl groups which showed a COSY correlation with a methine at d H 2.45 and d C 28.1, thus, proposing an isopropyl group.The latter methine was also correlated with another methine at d H 4.05 and d C 60.1, implying hydroxylation at this site.The latter proton, in the HMBC spectrum, was correlated to a carbonyl at d C 166.1 for an ester.Hence, the methine at d C 60.1 is substituted with one free and another esterified hydroxyl groups. 1 HNMR data also showed a methylene at d H 2.01, which in the COSY spectrum was correlated with two methylene groups at d H 3.55 and d H 1.95, 2.33.The latter methylene at d C 28.1 was correlated with a methine at d H 4.20 and d C 58.01 proposing oxygenation at the latter site.In HMBC, the methylene signals at d H 1.95 and 2.33 were correlated with a carbonyl at d C 171.1.Therefore, the methine at d C 58.01 is hydroxylated and connected to a carboxyl group proposing 2-hydoxy adipic acid esterified with a dihydroxy isobutyl alcohol (Table S1, Figures 1, S15-S21).
To our knowledge, 2-hydroxy adipic acid is not a natural compound.Thus, the isolated esters are therefore new.2-Aminoadipic acid is biosynthesized through a-aminoadipic pathway for lysine biosynthesis (Fazius et al. 2012).The biosynthesis of 2hydroxy adipate could be related to this pathway (Figure S22).Two pathways could be investigated, one is by the reduction of 2-ketoadipate to 2-hydroxy adipate, and the other pathway is via the transamination from 2-aminoadipate to generate 2-ketoadipate followed by the reduction.There is little data regarding the biosynthesis of naphthyl structures.Gossypol, a bisnaphthyl-based compound, originates from farnesyl pyrophosphate (Heinstein et al. 1970).Therefore, the origin of the naphthyl part of 3 was proposed to be farnesyl pyrophosphate.In 4, the alcohol part of the ester could be a reduction product of isobutyric acid.
The exposure of microorganisms to chemicals has an impact on their biochemistry (Li et al. 2018, Sobo n et al. 2018).The exposure of C. echinulata to tributyltin led to the accumulation of glycolysis intermediates and tricarboxylic acid cycle disruption (Sobo n et al. 2018).Trehalose increased c-linolenic acid accumulation (Li et al. 2018).Thus, the production of adipate esters could be affected by the growth medium, which will be investigated in future studies.
Swiss TagetPrediction tool was used for checking the potential biological targets for 3 and 4. The top 5 hits for each compound had the same probability (Figure S23).m Opioid receptor (MOP) and aldose reductase (AR) were selected for docking studies of 3 and 4, respectively.Compound 3 exhibited a docking score of 5.2 kcal/mol, while BU72 showed a score of 3.1 kcal/mol.Compound 4 had a docking score of 6.5 compared to that of sulindac sulfone, which was 5.4 kcal/mol.MOR is the target for agonist analgesics and its antagonists are of the therapeutic value for the treatment of drug and alcohol abuse (Pasternak 2014).Both agonist (morphinan BU72) and antagonist (naltrexone) bind to Asp147 and Tyr148 residues (Kaserer et al. 2016).Additionally, naltrexone showed hydrogen bonding with Lys 233.Kaserer et al. claimed that binding to Asp147 and Tyr148 is common for agonists and antagonists, while additional binding to other residues specifies the antagonists.Compound 3 formed hydrogen bonding with Asp147, Tyr148 and also with Tyr326 (Figure S24A).However, we cannot claim whether it is an agonist or antagonist.
AR is a target for type 2 diabetic complications.It catalyses the rate limiting step in the polyol pathway (Schemmel et al. 2010).Analysis of AR complex with sulindac sulfone, a potent inhibitor of AR with a strong evidence on its benefits in type 2 diabetes, revealed hydrogen bonding to Tyr48, His110 and Trp111 residues (Zheng et al. 2012).The same bonding was exhibited by 4 with a slightly better score (Figure S24B).These results encouraged us for future in vitro investigation for this virtual activity.

Conclusion
Novel adipate esters were isolated and identified for the first time from C. echinulata.Biological target prediction revealed MOR and AR as probable targets for 3 and 4, respectively.Future in vitro investigations for these targets are required.

Figure 1
Figure 1 Structures of the isolated metabolites.