Using Virtual AChE Homology Screening to Identify Small Molecules With the Ability to Inhibit Marine Biofouling

Abstract

The search for effective yet environmentally friendly strategies to prevent marine biofouling is hampered by the large taxonomic diversity amongst fouling organisms and a lack of well-defined conserved molecular targets. The acetylcholinesterase enzyme catalyses the breakdown of the neurotransmitter acetylcholine, and several natural antifouling allelochemicals have been reported to display acetylcholinesterase inhibitory activity. Our study is focussed on establishing if acetylcholinesterase can be used as a well-defined molecular target to accelerate discovery and development of novel antifoulants via sequential high-throughput in silico screening, in vitro enzymatic studies of identified compound libraries, and in vivo assessment of the most promising lead compounds. Using this approach, we identified potent cholinesterase inhibitors with inhibitory concentrations down to 3 μM from a 10,000 compound library. The most potent inhibitors were screened against five microfouling marine bacteria and marine microalgae and the macrofouling tunicate Ciona savignyi. No activity was seen against the microfoulers but a potent novel inhibitor of tunicate settlement and metamorphosis was discovered. Although only one of the identified active cholinesterase inhibitors displayed antifouling activity suggesting the link between cholinesterase inhibition and antifouling is limited to certain compound classes, the study highlights how in silico screening employed regularly for drug discovery can also facilitate discovery of antifouling leads.

Predicting Antifouling Activity and Acetylcholinesterase Inhibition of Marine-Derived Compounds Using a Computer-Aided Drug Design Approach

“…The binding of donepezil to the PAS of AChE is in accordance with its proposed peculiar inhibitory mechanism, which involves a reversible double-binding site interaction with the catalytic anionic site and PAS of the enzyme [54]. Unlike our approach and in other reported studies [46,54], Arabshahi et al [50] performed a screening docking of AChE at the catalytic anionic site and not at the PAS. Although none of the 11 reported compounds [50] passed the QSAR model threshold to be subjected to molecular docking, we still performed the molecular docking and the docking scores are presented in Table S5 (Supplementary Data).…”

“…Unlike our approach and in other reported studies [46,54], Arabshahi et al [50] performed a virtual screening by molecular docking of AChE at the catalytic anionic site and not at the PAS. Although none of the 11 reported compounds [50] passed the QSAR model threshold to be subjected to molecular docking, we still performed the molecular docking and the docking scores are presented in Table S5 (Supplementary Data). It was verified that none of these compounds exceeded the established threshold in the molecular docking experiments, ΔGB ≤ −7 kcal/mol.…”

“…The list of eleven lead-like AChE inhibitors against antifouling activity generated from the AChE homology virtual screening, which were experimentally screened in in vitro and micro-and macrofouling assays reported by Arabshahi et al [50], was used in this study as a second virtual screening library (Supplementary Data, Table S5). Only one out of the eleven lead-like AChE inhibitors was predicted to have antifouling activity with a Prob_active higher than 0.59 (Table S5), the morpholine derivative (Figure 2), in which experimental antifouling activity IC 50 = 16 µg/mL was reported (51.7 µM) [50]. However, none of the eleven compounds passed the established threshold, which was more demanding (Prob_active ≥ 0.68), to be selected for the molecular docking experiments.…”

“…Acetylcholinesterase (AChE) inhibitors are a class of drugs used for the treatment of Alzheimer's disease, glaucoma and autoimmune disorders [47][48][49]. The enzymes AChE [28] and tyrosinase (Tyr) were associated with the adhesive processes in the settlement of different biofouling species [28,46,50]. Almeida et al reported a molecular docking study conducted by modulation of Electrophorus electric (fish) AChE of the two most promising (thio)xanthone antifouling agents [46].…”

“…Almeida et al reported a molecular docking study conducted by modulation of Electrophorus electric (fish) AChE of the two most promising (thio)xanthone antifouling agents [46]. Recently, Arabshahi et al [50] reported an extensive virtual Tetronarce californica (fish) AChE homology screening campaign for 10,000 small organic molecules from the Chembridge library. The authors also reported the experimental screening of the most promising AChE inhibitors proposed by the in silico model, against five microfouling marine bacteria and marine microalgae macrofouling tunicate Ciona savignyi, discovering a potent novel inhibitor of tunicate settlement [50].…”