Anti-Amyloid Drug Screening Methods Using Bacterial Inclusion Bodies.

Amyloid aggregation is linked to a number of human disorders that range from non-neurological illnesses such as type 2 diabetes to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The formation of insoluble protein aggregates with amyloid conformation inside bacteria, namely, in bacterial inclusion bodies, offers the possibility to use bacteria as simple models to study amyloid aggregation processes and potential effects of both anti-amyloid drugs and/or pro-aggregative compounds. This chapter describes fast, simple, inexpensive, highly reproducible, and tunable in vitro and in cellulo methods that use bacterial inclusion bodies as preliminary screening tools for anti-amyloid drugs.

From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects.

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aß42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead.

Bacterial Inclusion Bodies for Anti-Amyloid Drug Discovery: Current and Future Screening Methods.

Amyloid aggregation is linked to an increasing number of human disorders from nonneurological pathologies such as type-2 diabetes to neurodegenerative ones such as Alzheimer or Parkinson's diseases. Thirty-six human proteins have shown the capacity to aggregate into pathological amyloid structures. To date, it is widely accepted that amyloid folding/aggregation is a universal process present in eukaryotic and prokaryotic cells. In the last decade, several studies have unequivocally demonstrated that bacterial inclusion bodies - insoluble protein aggregates usually formed during heterologous protein overexpression in bacteria - are mainly composed of overexpressed proteins in amyloid conformation. This fact shows that amyloid-prone proteins display a similar aggregation propensity in humans and bacteria, opening the possibility to use bacteria as simple models to study amyloid aggregation process and the potential effect of both anti-amyloid drugs and pro-aggregative compounds. Under these considerations, several in vitro and in cellulo methods, which exploit the amyloid properties of bacterial inclusion bodies, have been proposed in the last few years. Since these new methods are fast, simple, inexpensive, highly reproducible, and tunable, they have aroused great interest as preliminary screening tools in the search for anti-amyloid (beta-blocker) drugs for conformational diseases. The aim of this mini-review is to compile recently developed methods aimed at tracking amyloid aggregation in bacteria, discussing their advantages and limitations, and the future potential applications of inclusion bodies in anti-amyloid drug discovery.

Natural Xanthones from Garcinia mangostana with Multifunctional Activities for the Therapy of Alzheimer's Disease.

Natural xanthones have diversity pharmacological activities. Here, a series of xanthones isolated from the pericarps of Garcinia mangostana Linn, named α-Mangostin, 8-Deoxygartanin, Gartanin, Garciniafuran, Garcinone C, Garcinone D, and γ-Mangostin were investigated. Biological screening performed in vitro and in Escherichia coli cells indicated that most of the xanthones exhibited significant inhibition of self-induced ß-amyloid (Aß) aggregation and also ß-site amyloid precursor protein-cleaving enzyme 1, acted as potential antioxidants and biometal chelators. Among these compounds, α-Mangostin, Gartanin, Garcinone C and γ-Mangostin showed better antioxidant properties to scavenge Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) free radical than Trolox, and potent neuroprotective effects against glutamate-induced HT22 cell death partly by up-regulating HO-1 protein level and then scavenging reactive oxygen species. Moreover, Gartanin, Garcinone C and γ-Mangostin could be able to penetrate the blood-brain barrier (BBB) in vitro. These findings suggest that the natural xanthones have multifunctional activities against Alzheimer's disease (AD) and could be promising compounds for the therapy of AD.

Ultra rapid in vivo screening for anti-Alzheimer anti-amyloid drugs.

More than 46 million people worldwide suffer from Alzheimer's disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid ß-peptide (Aß), considered one of the main culprits in Alzheimer's disease. Limitations in monitoring the aggregation of Aß in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aß aggregation in vivo in real-time, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aß in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aß aggregation and the effect of potential anti-aggregating agents.