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’s disease with Aβ and Tau proteins, Parkinson’s disease with α -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 Sträussler-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 α -helices, is soluble and protease K (PK) sensitive; while the PrPSc has a high level of β -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.