Inhibition of acetylcholinesterase (AChE) is considered a promising strategy for the treatment of Alzheimer's disease (AD) and dementia. Members of the Amaryllidaceae family are well known for their pharmacologically active alkaloids, including galanthamine, which is used to treat AD. The aim of this study was to evaluate the potential of South African Amaryllidaceae species to inhibit AChE, to isolate the active compounds, and probe their ability to bind the enzyme using molecular docking. The AChE inhibitory activity of extracts of 42 samples, representing 14 genera and 28 species, as well as isolated compounds, were evaluated in vitro using a qualitative thin layer chromatography (TLC) bio-autography assay and Ellman's method in a quantitative 96-well microplate assay. Targeted isolation of compounds was achieved with the aid of preparative-high perfomance liquid chromatography-mass spectrometry. The structures of the isolates were elucidated using nuclear magnetic resonance spectrocopy, and were docked into the active site of AChE to rationalise their biological activities. The most active species were found to be Amaryllis belladonna L (IC50 14.3+/-2.6mug/mL), Nerine huttoniae Schonland (IC50 45.3+/-0.4mug/mL) and Nerine undulata (L.) Herb. (IC50 52.8+/-0.5mug/mL), while TLC bio-autography indicated the presence of several active compounds in the methanol extracts. Four compounds, isolated from A. belladonna, were identified as belladine, undulatine, buphanidrine and acetylcaranine. Acetylcaranine and undulatine were previously isolated from A. belladonna, while belladine and buphanidrine were reported from other South African Amaryllidaceae species. Using Ellman's method, acetylcaranine was found to be the most active of the isolates towards AChE, with an IC50 of 11.7+/-0.7muM, comparable to that of galanthamine (IC50=6.19+/-2.60muM). Molecular docking successfully predicted the binding modes of ligands within receptor binding sites. Acetylcaranine was predicted by the docking workflow to have the highest activity, which corresponds to the in vitro results. Both qualitative and quantitative assays indicate that several South African Amaryllidaceae species are notable AChE inhibitors.
An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7beta, CyC8beta, and CyC17 bind covalently to the catalytic Ser(124) residue in Ag85C; inhibit mycolyltransferase activity (i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8beta disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.
BACKGROUND: Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak 'Guangfen #1' and 10 Cavendish 'Brazilian' plants. Fusaric acid and BEA were detected in all the tissues, including the fruits. CONCLUSIONS/SIGNFICANCE: The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.
Homozygous lipoprotein lipase (LPL) deficiency is an ultra-orphan disease associated with increased rates of pancreatitis. Current treatments based on acute plasmapheresis allied with ultra-low fat diets are inadequate as responses to fibrates or other triglyceride-lowering therapies tend to be poor. Alipogene tiparvovec is an adeno-associated virus type I (AAV1) gene therapy using a hyper-functional LPL serine(447)-stop (S447X) insert administered intramuscularly under general anaesthetic with allied immunosuppression. Treatment results in histological muscle expression of LPL allied with a transient 40% reduction in triglycerides and improvements in postprandial chylomicron triglyceride content. Alipogene tiparvovec is the first possibly curative treatment for LPL deficiency.
