Nephthea sp. inhibits biofilm, DNA gyrase, HSP90, and DHFR: in vitro, in silico, and pharmacokinetics studies

Abstract This study attempts to identify and assess a novel marine-derived antibiofilm agent. The antibacterial activity of n-hexane, dichloromethane, ethyl acetate, and butanol fractions from the crude extract of soft coral Nephthea sp. was evaluated against six microorganisms.Ethyl acetate fraction considered the most effective one against Bacillus subtilis, Escherichia coli, and Candida, investigated potential biofilm inhibition against the tested strains. Seventeen secondary metabolites were identified using (UPLC-Q/TOF-MS) responsible for these biological activities of the active fraction. Additionally, a molecular docking study showed free binding energy of −7.5 kcal/mol; Azamial A had the highest binding affinity for the DNA gyrase enzyme, while Sinularectin had −8.3 and −7.6 kcal/mol for the DHFR and HSP90 enzymes, respectively. Moreover, pharmacokinetics and (ADME) studies for Azamial A and Sinularectin were performed. Finally, results were confirmed by the in vitro enzymatic inhibitory effect of ethyl acetate fraction suggested in the in-silico study. Graphical Abstract


Introduction
The most distinguishing feature of biofilms is the survival of bacteria in the microenvironment as long as conditions are favorable (Alam et al. 2020).Biofilms are complex microbial communities consisting of proteins, extracellular nucleic acids (eDNA), exo-polysaccharides, and lipids.They are absorbed into biotic surfaces to provide the initial binding to the bacterial cell for success in the infection process (Okba et al. 2021).Increasing biofilm resistance to conventional treatments such as antibiotics or chemical preservatives and associated adverse side effects are leading to great interest in searching for safer alternatives (Nasr-Eldin et al. 2018).
DNA gyrase is classified as topoisomerase II, an ATP-dependent enzyme that is vital in gram-positive bacteria growth and biofilm formation (Khan et al. 2018).Also, the Heat shock protein 90 (HSP 90) enzyme is involved in the formation of the fungi C. Albicans biofilm (O'Meara et al. 2017).On the other hand, (Zhang et al. 2015)indicate that inhibition of Streptococcus mutans biofilms is related to reduced Dihydrofolate reductase (DHFR) enzyme activity as plays a critical role in regulating the metabolism of folate by the conversion of folic acid to its reduced form, tetrahydro-folic acid (THF).
A Survey of natural medicine, obtained from marine soft coral that has antibiofilm and antimicrobial activity represents an important step in finding an alternative treatment for the infectious disease (Elkin et al. 2011).Increasing the episodes of resistance among pathogenic micro zooplankton bacteria or in the biofilm have encouraged the treatment with natural molecules and marine products (Noumi et al. 2017).Therefore, steps are taken to develop natural medicine that ensures no side effects and a reliable cure for harmful microorganisms (Yuan et al. 2016).
In this work, we examined in this work the antimicrobial potency of four different fractions (n-hexane, dichloromethane, ethyl acetate, and butanol fractions) extracted from the soft coral Nephthea sp.assembled from the Red Sea region.The antibiofilm activity of the most active antimicrobial fraction (ethyl acetate) was evaluated.In addition, Metabolomics characteristics based on the UPLC-Q/TOF-MS data were recorded for this active fraction of soft coral followed by pharmacokinetics and ADME profiling.Finally, it is the first record of the in-vitro inhibitory activity of an ethyl acetate fraction, and the in-silico study of the identified major components against DNA gyrase, DHFR, and HSP90 enzyme was scored (Scheme 1).

Antimicrobial activity
The potential assessment of microbial inhibition using the agar well diffusion method was evaluated.The results revealed that the Nephthea sp.fractions showed good antimicrobial activity against A. niger and S. aureus while low antimicrobial activity was obtained against P. vulgais and E. cloacae (Table S1).The most active fractions of Nephthea sp. were ethyl acetate and butanol compared to the control drug while the tested bacterial strains were resistant to n-hexane and dichloromethane fractions.Thus, MIC values of the active fractions against the tested strains confirmed the results of antimicrobial activity as recorded in Table S2.
The ethyl acetate fraction of Nephthea sp.exhibited potential activity against B. subtilis, C. Albicans, and E. coli where the areas of inhibition were as much as the reference drug Gentamicin (Figure S1).

Biofilm inhibition activity
The antibiofilm activity of ethyl acetate fraction which is the most powerful antimicrobial fraction of Nephthea sp. was assessed against bacteria with great biofilm-forming capacity as E. coli (Ramalingam et al. 2019), B. subtilis (Verma et al. 2020) and fungi as C. Albicans (Meenambiga and Rajagopal 2018).
Figures S2 and S3, illustrated that the highest biofilm inhibition percentage was recorded by ethyl acetate fraction against B. subtills which was as much as 83% inhibition followed by C. albicans at 81% then Ecoli 68.71%, respectively.

Metabolomics profiling
UPLC-Q/TOF-MS analysis of the ethyl acetate fraction of Nephthea sp.revealed the presence of 17 tentative dereplicated metabolites from various classes as (2) Alkaloids, (2) Amino acids, (2) diterpenoids, (2) Fatty acids, (6) Steroid, (1) oxygenated ergostanoid, (1) meroditerpenoid and one polyketide.The majority of the observed metabolites are found in the positive mode.The compounds were identified and tentatively annotated based on their retention times, and molecular weight, and by searching DNP, as well as comparison with reported data found in literature, the majority of these metabolites, were previously reported from the Nephtheidae family (Abdelhafez et al. 2019;Nautiyal 2013; Figures S4-S6; Table S3, Supplementary material).

Molecular docking
To take an additional step to determine the mode of action of the major identified compounds as potential antibiofilm agents, an in-silico study was employed to determine the binding modes against three important proteins implicated in tested microorganisms growth and biofilm formation including DNA gyrase, DHFR, and HSP 90.The major ten isolates were used to be investigated.The cocrystal ligands for the three proteins were re-docked to the corresponding proteins to assure the validity of the docking parameters and methods to represent the position and orientation of the detected ligand in the crystal structure.The difference in RMSD value between the cocrystalized ligands to the original co-crystalized ligand was <2Å which agreed with the accuracy of the docking protocols and parameters.To elucidate the mode of action for the most promising antimicrobial compounds, precisely compound Azamial A docking study conducted against DNA gyrase protein showed a potential binding affinity on binding the active site of this protein, this was evident by low free binding of energy À7.5 kcal/mol, compared to Gentamicin which has À5.6 kcal/mol free binding of energy, formation of one hydrogen bond with amino acid (VAL29:2.89Å), and hydrophobic interaction.On the other hand, Sinularectin showed the highest binding affinity with low free binding of energy score towards DHFR & HSP 90 at À8.3 and À7.6 kcal/mol, compared to Gentamicin (-7.7, and À7.3 Kcal/mol), respectively (Figures S7-S12; Table S4, Supplementary material).

Pharmacokinetics 'ADME' activity
Because the majority of drug candidates fail during clinical development due to inappropriate ADME features, pharmacokinetic parameters should also be included with the significant filters to further enhance selected lead molecules into drug candidates, and reduce failure rates during clinical trials.Prediction of physicochemical and druglikeness properties based on bioinformatics study of the most potential antibiofilm compounds Azamial A and Sinularectin were carried out (Tables S5 and S6).Both compounds generally show excellent drug-like characteristics, high oral absorption, high bioavailability, and low toxicity (Tables S7-S9).Bioavailability Radar was created to check the suitable physicochemical properties of oral bioavailability, based on 6 characteristics including (lipophilicity: XLOGP3 between À 0.7 and þ 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å, solubility: log S not higher than 6, saturation: fraction of carbons in the 3 hybridizations not less than 0.25, and flexibility: no more than 9 rotatable bonds) as shown in Figure S13, Supplementary material).
2.6.In-vitro inhibition assay of DNA gyrase, DHFR, and HSP 90 enzymes DNA gyrase, DHFR, and HSP 90 enzymes are important targets of antibiofilm drugs as they play a critical role in the synthesis of biofilm for microorganisms.Gentamicin drug was used as a reference drug with IC 50 against DNA gyrase, DHFR, and HSP 90 (26.82 ± 1.51 mg/ml, 0.841 ± 0.277 mg/ml, and 136.2 ± 8.29 mg/ml, respectively.In addition, the ethyl acetate fraction of Nephthea sp.showed inhibitory activity towards DNA gyrase, DHFR, and HSP 90 with IC 50 ¼ 17.97 ± 1.01 mg/ml, 2.151 ± 0.044 mg/ml and 506.3 ± 30.83 mg ml as shown in (Figure S14).

Conclusion
This study highlights the promising role of octocoral Nephthea sp. as an antibiofilm agent against Gram-positive and Gram-negative bacteria and yeast.The UPLC-Q/TOF-MS fingerprint of the ethyl acetate fraction represents the secondary metabolites profile of the soft coral.The efficacy of ethyl acetate fraction against B. subtilis, C. albicans, and E. coli was proved after further in-vitro and in-silico studies.In-silico and ADME studies of the identified compounds assumed their mechanism of action as antibiofilm potential.Our results indicate that Nephthea sp. is a rich source of effective metabolites, making it a promising candidate for bacterial infection control.Finally, we suggest preparing the nano formula of ethyl acetate fraction which could be used as an effective therapy against antibiofilm-resistant pathogens and a lack of eagerness for various artificial drugs.