Monomethyl Suberate Screening for Antifungal Activity , Molecular Docking and Drug-Like Properties

Antifungal activity of suberic acid monomethyl ester (monomethyl suberate) was investigated in a growth inhibition assay comprising of 11 different fungi and 3 Phytophthora oomycetes strains relevant in agriculture. In comparison to standard antifungal hymexazol, monomethyl suberate showed moderate antifungal effects at a concentration range of 100–300 μg/mL. Alternaria alternata, Fusarium equiseti, Fusarium fujikuroi and Phytophtora infestans GL-1 were the most sensitive fungi showing inhibition rates up to 100%. Physico-chemical descriptors of monomethyl suberate revealed its low toxicity profile. Molecular docking analysis comprising several known antifungal targets points to the N-myristoyltransferase as the most probable site of interaction.

To our knowledge, reports on physiological role(s) and potential biotechnological application(s) of suberic acid or its derivatives are scarce.Staphylococcus aureus and Candida albicans were shown to be sensitive to a chloroform extract of Polysiphonia denudate f. fragilis (Rhodophyceae), which contained suberic acid dimethyl ester (0.2%) among other biologically active substances. 7The latter was also detected by GC-MS in the larval and pupal internal lipids of medically important flies Calliphora vicina (0.15-0.20 µg/g) and Sarcophaga carnaria (0.14-0.21 µg/g). 8In the same study it was shown, that the substance itself slightly inhibited the growth of entomopathogenic fungi of Beauveria bassiana (Dv-1/07), B. bassiana (Tve-N39), Lecanicillium lecanii, Metarhizium anisopliae, Paecilomyces fumosoroseus and Paecilomyces lilacinus with a minimal inhibitory concentration (MIC) of 512 µg/mL.Antimicrobial tests carried out with diverse gram-positive and gram-negative bacterial strains, as well as with the fungi Candida albicans and Candida tropicalis, were without any positive results. 8ecently, Iornumbe et al. 9 investigated the antifungal activity of suberic acid organotin derivatives against Microsporum gypseum, M. audounii, M. distortum, M. gallinae and Trichoephyton: mentagrophytes and T. equinum.They found a decreasing average activity according to diverse octandioate rests: potassium triphenyltin(IV) oc- Suberic acid is known to be obtained along with azelaic acid through oxidation of ricinoleic acid 2 or as a component isolated from oil extracted from Vernonia galamensis. 3Antonova et al. described its synthesis by oxidation of cyclooctane-1,2-diol. 4 Suberic acid (4.13%) and its monomethyl ester (2.38%) were also detected by gas chromatography-mass spectrometry (GC-MS) in methanolic extracts of Hibiscus micranthus stem along with 56 other compounds. 5Furthermore, monomethyl suberate was found by GC-MS in an ethylacetate/hexane extract of Pestalotiopsis JCM2A4, an endophyte growing on Chinese mangrove plant Rhizophora mucronata. 6anedioate > potassium tributyltin(IV) octanedioate > potassium dibutyltin(IV) octanedioate > potassium diphenyltin(IV) octanedioate.The activities were comparable to standard antifungals fulcin and fluconazole.The leading compounds MICs were found to be 25 μg/mL.The free suberic acid or its monopotassium salt exhibited a weaker antifungal activity than synthesized organotin compounds, inhibiting growth of only M. distortum and T. equinum.This fits to the observation that many biologically compounds enhance their activity upon complexation.10 Only few suberic acid derivatives were reported to have non-pharmacological applications: octacalcium phosphate carboxylates as bone reconstructors for biomedical applications; 11 suberate as thermotropic liquid crystalline polymers; 12 poly(propylene suberate)s 13 and poly(butylene suberate)s 14,15 as biodegradable polyesters for sutures, implant materials for tissue engineering, and biologically active controlled drug-release devices; anhydrous copper suberates as polymers with extended bridged structures, which are interesting materials to study spin exchange and charge transfer between metal ions.16 Interestingly, in a very different context suberic bishydroxamate was found to be a potent agent in overcoming resistance of melanoma to "TNF-related apoptosis-inducing ligand", which induces apoptosis by acting as a histone deacetylase inhibitor.17 Here we present an investigation of antifungal activity of monomethyl suberate.

1. Antifungal Activity
We decided to work on monomethyl suberate, because dimethyl ester was already described having only a moderate antifungal activity (512 μg/mL MIC). 8Furthermore, the monoethyl ester of azelaic acid, which bears a molecular scaffold structure very similar to suberate, showed a pronounced antifungal activity against Pyricularia oryzae with an MIC 50 of 50 µg/mL (free acid: MIC 50 at 95 µg/mL). 18,19This observation is probably due to higher lipophilicity of the monoethyl ester form. 20Thus, antifungal studies of shorter in one carbon dicarboxylic acid, namely, suberic, seemed promising with enhancing its lipophilicity in monomethyl ester form.

Drug-Likeness Physico-Chemical Parameters and Promiscuity Score
Considering the found moderate antifungal activity of monomethyl suberate at 300 μg/mL, its physico-chemical parameters were calculated by Molinspiration engine 21 (Table 1) in order to predict the level of drug-likeness, toxicity [22][23][24] and substance promiscuity. 25These properties may be of value if monomethyl suberate will be considered as a compound in human medical care, food processing or as an antifungal in agriculture.For comparison, corresponding data for the standard antifungal hymexazol are also shown (CHEMBL244877). 26s it is seen from the Table 1, monomethyl suberate complies to all presented criteria for molecular properties, that influence the oral bioavailability of drug candidates, [22][23][24] except of molecular polar surface area (TPSA).Its surface is larger (63.30Å 2 ) than that of hymexazol (42.26 Å 2 ), which implies its penetration of the blood brain barrier is less likely.Calculating the promiscuity of biological activity of monomethyl suberate with "bioactivity data associative promiscuity pattern learning engine" (Badapple), 25 no data were found in the database, which means a neutral result with respect to toxicity prediction.At least the predicted promiscuity was not found to be high.For hymexazol the pScore was shown with a moderate true value (238) based on reported biological activity data of drugs with isoxazole scaffold.So, the reference antifungal hymexazol demonstrated higher level of potentially binding to a variety of bimolecular targets, and thereby may have higher level of toxicity than tested natural monomethyl suberate. 27

3. Molecular Docking
A literature survey did not give us any indication with respect to a biological target, to which monomethyl suberate may bind and thereby reveal a mode of action of growth inhibition.Only PubChem BioAssays (CHEM-BL1162491) 26 reported suberic acid to be an antagonist of the retinoid-related orphan receptor gamma, farnesoid-X-receptor, thyroid hormone receptor beta and NFkB signaling pathways.All these reports are related to human health studies.So, investigations to elucidate antifungal mechanism(s) of suberate's with respect to fungi of agricultural importance are worth to study.
Analysis of in silico molecular docking predicted affinity scores 28 to six common fungal targets (enzymes) 29 and showed, that monomethyl suberate may interact with them with higher probability, than hymexazol (Table 2).
The highest affinity score (-6.0) of monomethyl suberate was calculated to N-myristoyltransferase (NMT).In Figure 3 it is shown how it fits into the active site of this enzyme.
Two conventional hydrogen bonds are formed with HIS B:227 (3.05 Å) and ASN B:392 (3.04 Å) due to carbonyl oxygen in methyl ester residue of suberate.A further hydrophobic Pi-sigma bond build up between PHE B:240 (3.76 Å) and MeC(O)CH 2 fragment.Thus, N-myristoyltransferase (NMT) should be among priority antifungal targets for further in vitro enzymatic studies.

1. Antifungal Studies
The mycelial growth rate assay was used for antifungal studies. 30Strains of filamentous fungi were obtained from the following sources: Asperillus niger DSM 246, Altenaria alternata DSM 1102, Fusarium equiseti DSM 21725,   A clear stock solution of 5 mg/mL was made of 0.050 g of reference substance hymexazol in 10 mL of deionized sterile water as solvent. 1 ml of each stock solution was mixed in situ into 99 ml of PDA prior to solidification to obtain a final concentration of 50 µg/mL.In the same way mixtures of PDA with monomethyl suberate were prepared with final concentrations of 50, 100 and 300 µg/mL.9 mL of each mixture were poured into 6 cm diameter petri dishes.After solidification central hole (diameter: 2.5 mm) was cut out and inoculated with 6.5 µL spore suspension.Plates were incubated at 25 °C (+/-1 °C) for 6 d.Control plates containing only PDA and water were prepared in the same way.Inhibitory effects (I %) were determined by analyzing growth zone diameters and calculated as described by Tang et al.: where C (mm) represents the growth zone of control PDA, and T (mm) the average growth zone in presence of methyl suberate. 30All growth experiments were carried out in triplicate.Means and standard deviations were calculated with software "Exel 2016" (Microsoft, USA).

Conclusion
The antifungal spectrum of monomethyl suberate was investigated against 11 different fungi and 3 Phytophthora oomycetes strains of agricultural importance.The monomethyl ester derivative revealed a significantly higher activity than the dimethyl ester 8 , but an average lower activity than reference antifungal hymexazol.Nevertheless, an extraordinary activity was observed against strain GL-1 of the devastating oomycete P. infestans.Furthermore, monomethyl suberate as a naturally occurring substance has a more environmentally friendly structure with less promiscuity score than conventional antifungals with heterocyclic ring systems.Therefore, we expect to have at hand an antifungal drug with an attractive profile with respect to potential toxicity and mutagenicity.

Figure 3 .
Figure 3. Visual representation (3D and 2D) of the monomethyl suberate showing bonds formation and position in the active site of N-myristoyltransferase (NMT) of Candida albicans.29

Table 2 .
Calculated affinities of monomethyl suberate and reference hymexazol to common antifungal enzymatic targets, Kcal/Mol