Non-anticoagulant glycosaminoglycan extracts isolated from aquatic species inhibit BACE-1: a key drug target in Alzheimer’s Disease

Abstract

Currently, only palliative drugs are available for the treatment of Alzheimer’s disease, therefore there is an urgent need to develop therapeutics targeting the underlying aetiology of the disease. The clinical anticoagulant heparin has previously been reported to inhibit the primary neuronal !-secretase, BACE-1, which is responsible for the production of the widely regarded causative agent of Alzheimer’s disease, amyloid peptides. Despite this, the utilisation of pharmaceutical heparin for the treatment of Alzheimer’s disease has largely been precluded due to the potent anticoagulant activity displayed by this polysaccharide. This is primarily as a result of uncontrolled hemostasis being considered as a highly undesirable side-effect for Alzheimer’s disease therapeutics. Furthermore, concerns regarding the use of heparin sources from mammalian species, primarily due to transmissible encephalopathies and religious beliefs, have hindered the deployment of heparin for alternative therapeutic applications. Glycosaminoglycans obtained from aquatic species have previously been shown to possess significantly attenuated anticoagulant activities, in comparison to mammalian heparin, while retaining a diverse array of additional biological activities. This is principally a result of the increased structural diversity displayed by glycosaminoglycans obtained from aquatic species in comparison to mammalian counterparts. Furthermore, the considerable amount of waste produced from the fishery and aquaculture industries offers a sustainable, financially viable alternative to mammalian derived glycosaminoglycan sources for potential therapeutic exploitation. Here several glycosaminoglycan extracts obtained from the tissue of aquatic species, via proteolytic digestion, were observed to potently inhibit BACE-1, while being shown to displaying highly attenuated anticoagulant activities. The BACE-1 inhibitory activities of glycosaminoglycan extracts obtained from Portunus pelagicus, Litopenaeus vannamei and Sardinia pilchardus, were evaluated utilising a Förster resonance transfer assay and then subsequently compared to their respective anticoagulant activities (determined utilising the activated partial thromboplastin time assay). In comparison to mammalian heparin the glycosaminoglycan extracts obtained from Portunus pelagicus, Litopenaeus vannamei and Sardinia pilchardus exhibited increased therapeutic ratios of, 0.7, 22.7, 2.5-6.1 and 84.3, respectively. The extracts were evaluated for structurefunction analysis utilising agarose gel electrophoresis, attenuated total reflectance Fourier transform infrared spectroscopy, circular dichroism spectroscopy, high pressure liquid chromatography disaccharide compositional analysis and nuclear magnetic resonance spectroscopy. Structural-function analysis indicated that the extent of 2- sulphated uronic acid residues, present within the extracts predominantly composed of heparan sulphate (Portunus pelagicus, Litopenaeus vannamei), may be indicative of increased BACE-1 inhibitory activity, with extracts containing higher levels of 2- sulphated uronic acid residues displaying increased BACE-1 inhibitory activity. Furthermore, extracts composed principally of chondroitin sulphate were also observed to inhibit BACE-1, although the potency of this action is likely dependent upon the fine structure of the polysaccharide. The interaction of glycosaminoglycans with BACE-1 was evaluated further through the use of circular dichroism spectroscopy and differential scanning fluorometry. This indicated that glycosaminoglycans inhibit BACE-1 via a mechanism that involves a conformational change, which likely renders the enzyme in an inactive conformation.