In silico and in vitro screening of acridine and phenothiazine derivatives as anti-prion agents

Although in the last decades there has been a growing interest in neurodegenerative diseases, there are still no effective therapies. Some of these diseases are considered protein misfolding disorders (PCDs), since they are mainly caused by a conformational change of a native protein into a disease-associated one, that can aggregate and form an infective seed; Alzheimer\u2019s disease with A\u3b2 and Tau proteins, Parkinson\u2019s disease with \u3b1 -synuclein, and prion diseases with prion protein. Recently, transmissible spongiform encephalopathies (TSEs), also known as prion diseases, have been considered as a prototype of neurodegenerative diseases for their ability to be sporadic, inherited and also infectious. Indeed, the infectivity was long thought to belong exclusively to prion diseases, but in the last years accumulating evidence suggest that other proteins follow a similar mechanism of seed self-propagation and cell-to-cell spreading in vitro and in vivo. TSEs are mainly characterized by vacuolation, neuronal loss, cognitive and motor impairments. They include kuru, Creutzfeldt-Jacob disease, Gerstmann Str\ue4ussler-Scheinker syndrome and fatal familial insomnia in human, bovine spongiform encephalopathy in cattle, scrapie in sheep and chronic wasting disease in elk and deer. The etiology agent is the scrapie prion protein (PrPSc), the abnormal, misfolded form of the cellular prion protein (PrPC). The normal protein is anchored to the cell surface through a C-terminal moiety of glycophosphatidyl-inositol (GPI). Even though the two isoforms share the same primary sequence, they have several different aspects: PrPC is rich in \u3b1 -helices, is soluble and protease K (PK) sensitive; while the PrPSc has a high level of \u3b2 -sheets, is insoluble and partially PK resistant. The molecular mechanism underlying the conversion of PrPC to PrPSc is still not completely understood. Several studies in literature focus on the ability of small molecules to inhibit the conversion by either binding the PrPC or blocking the PrPSc aggregation. The majority of compounds screened for this action were already used as antivirals, antimalarials, antifungals and antidepressants. Drug repositioning is a strategy that uses known compounds to treat new diseases. By using this approach quinacrine (antimalarial), Pentosan Polysulfate (heparin mimetic), Doxycycline (antibiotic) and Flupirtine (analgesic) were tested in human clinical trials, unfortunately without the expected results. Other approaches applied to develop anti-prion therapies are medicinal chemistry, multi-target approaches and in silico methods. In drug screening, the in silico method is useful to increase the discovery speed of new drugs, thus reducing costs and lab work. In particular, we developed a quantitative structure-activity relationship model (QSAR) and by using it performed a virtual screening of some purchasable compounds. With the help of the QSAR model, we obtained a library of 10 molecules with a predicted IC50 in the nanomolar range. Immortalized neuroblastoma (N2a) and hypothalamic (GT1) mouse cell lines chronically infected with prions were used to assess the cell viabilty of the library and to measure the anti-prion infectivity. After performing western blot analysis and Real-Time Quaking-Induced Conversion (RT-QuIC) assay, compound 1 emerged as the most promising molecule, since it was able to completely cure N2a-RML cells from PrPSc, either after acute or chronic treatments. Additionally, we designed a competitional assay, which shows that compound 1 blocks prion conversion by binding to the cellular prion protein. These results make this molecule an interesting and promising therapeutic tool for prion diseases

Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other 'synucleinopathies'. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity

Brain region-specific susceptibility of Lewy body pathology in synucleinopathies is governed by α-synuclein conformations

The protein α-synuclein, a key player in Parkinson's disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible "prion-like" propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a "prion-like" aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and "prion-like" aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein

Identification of novel fluorescent probes preventing PrPSc replication in prion diseases

Prion diseases are serious, not curable neurodegenerative disorders caused by the accumulation of the misfolded protein PrPScthat represents the pathological variant of the normally folded cellular protein PrPC. Molecules that bind the cellular isoform PrPCpreventing its misfolding, could arrest the progression of pathological conditions related to the abnormal PrP protein. In this context, by combining 3D-QSAR model, derived from pharmacophore-based alignment, with molecular docking procedures and physico-chemical properties prediction we have developed a virtual screening protocol to find novel chemicals able to prevent PrPCmisfolding. We identified different hits characterized by low toxicity and able to inhibit PrPScaccumulation in\uc2\ua0vitro in prion-infected neuroblastoma cell lines (ScN2a). In this assay, the pyrroloquinoxaline hydrazone 96 showed the higest potency with an IC50value of 1.6\uc2\ua0\uce\ubcM. Pyrroloquinoxaline 96 was demonstrated also to bind PrPScaggregates in infected ScN2a cells with a fluorescence pattern comparable to that found for Thioflavin-T. In consideration of its satisfactory physico-chemical properties, including predicted blood brain barrier permeability, 96 could represent an interesting prototypic hit for the development of diagnostic and therapeutic probes for prion diseases

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models. Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation. These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate