Evaluation of bioactive natural products from traditional Chinese medicines against a-synuclein-targeted drug screening

Abstract

Parkinson’s Disease belongs to a group of overlapping neurodegenerative disorders called α-Synucleinopathies. Patients are diagnosed by degeneration of dopaminergic neurons and the presence of Lewy bodies within surviving neurons of the Substantia Nigra Pars Compacta. These Lewy bodies are proteinaceous deposits of α-Synuclein. This intrinsically disordered protein pathologically aggregates into toxic oligomers or β-sheet fibril species. Currently there are no known drugs available to treat or reverse the pathogenesis of this disease. Therefore there is an unmet clinical need to find small molecules that can inhibit αS aggregation to mediate Lewy Body pathology. The proposed mechanism to stop Lewy Body pathology is to identify small molecule inhibitors that can preferentially bind to monomeric α-Synuclein to inhibit aggregation by encouraging it to fold into a non-toxic species. Although there are no drugs available to stop or reverse Parkinson’s Disease, 67 small molecule inhibitors were identified from literature to stop the aggregation of α-Synuclein. These inhibitors belonged to ten structurally diverse classes, phenolic or terpenoids were most common, and were predominantly isolated from plants used as traditional Chinese medicines. This review indicated that traditional Chinese medicine is a promising avenue to discover new small molecule binders and inhibitors of α-Synuclein aggregation. A TCM pure compound library was collated from 139 bioactive molecules that had not been previously evaluated for their binding potential against α-Synuclein. Compounds were previously isolated and reported from Phlegmariurus carinatus, Asarum sieboldii var. Seoulense, Macleaya cordata, Gastrodia elata Bl. and Ligusticum chuanxiong Hort as they are commonly used for their antioxidant properties and ability to treat symptoms of brain related disorders, common amongst Parkinson’s Disease patients. The compounds were screened using laboratory synthesised monomeric α-Synuclein (>95% purity). α-Synuclein was expressed in house by selectively synthesising the protein in pET-22b (+) vector and grown in Escherichia coli BL21 (DE3) cells. Non-chromatographic purification expelled α-Synuclein protein from the periplasm and separated it from other proteins and nucleic acids. Further purification was performed by anion exchange fast protein liquid chromatography. Mass spectrometry is an emerging high-throughput technique that uses small concentrations of protein and compounds to sensitively detect protein-ligand complexes. It has many benefits and has since been favoured as a drug screening tool for Parkinson’s Disease. α-Synuclein samples were buffer exchanged and Quadrupole-time-of-flight Mass Spectrometry was used to assess purity of protein and screen pure compounds at a 7.5:1 concentration. Three TCM compounds bound to monomeric α-Synuclein at 7.5:1 ratio. Terpenoid compounds 3.12, 3.15 bound non-covalently whereas glucoside compound 3.124 bound covalently to α-Synuclein protein. All other TCM compounds did not bind at a 7.5:1 ratio and are not plausible drug candidates despite being bioactive against other neurodegenerative disorders. All TCM compounds and known inhibitors were evaluated for their oral bioavailability and blood brain barrier permeability by an in silico study. Lipinski’s Rule of 5 and Veber’s Rule were used to assess the drug-likeness of molecules to be orally active. Whereas a BOILED-Egg plot was used to predict gastrointestinal tract and blood brain barrier permeation. Drug candidates must satisfy these criteria in order to be a successful orally administered Parkinson’s Disease drug. All three TCM binders were calculated to be orally bioavailable and were predicted to permeate the gastrointestinal tract however not the blood brain barrier. TCM compounds were predicted to permeate the blood brain barrier at a higher rate than the known inhibitors and are a good source of drug candidates. Comparatively, 13 of the 67 known α-Synuclein aggregation inhibitors were predicted to have good oral bioavailability and cross the blood brain barrier. These 13 inhibitors belonged to either phenolic or terpenoid compound classes, the same structural class of the three α-Synuclein binders. Thus, these phenolic and terpenoid compounds are promising agents and should be prioritised for development into a neurotherapeutic Parkinson’s Disease drug.