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 Ab and Tau proteins, Parkinson’s disease with a-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 (PrP), the abnormal, misfolded form of the cellular prion protein (PrP). 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: PrP is rich in a-helices, is soluble and protease K (PK) sensitive; while the PrP has a high level of b-sheets, is insoluble and partially PK resistant. The molecular mechanism underlying the conversion of PrP to PrP is still not completely understood. Several studies in literature focus on the ability of small molecules to inhibit the conversion by either binding the PrP or blocking the PrP 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 antiprion therapies are medicinal chemistry, multi-target approaches and in silico methods. __________________________________________________________________________ 6 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 (RTQuIC) assay, compound 1 emerged as the most promising molecule, since it was able to completely cure N2a-RML cells from PrP, 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. __________________________________________________________________________ 7 __________________________________________________________________________ 8 LIST OF PUBLICATIONS: All the work reported here arises from my own experiments and data analysis performed in SISSA or as a result of joint collaborations with other groups. -Zaccagnini L. , Brogi S. , Brindisi M. , Gemma S. , Chemi G. , Legname G. , Campiani G. and Butini S. Identification of novel fluorescent probes preventing PrPSc replication in prion diseases. Eur J Med Chem. 2017 Feb 15;127:859-873. -Zaccagnini L. , Rossetti G. , Tran TH. , Gandini A. , Salzano G. , Bolognesi ML. , Carloni P., Legname G. In silico and in vitro screening of acridine and phenothiazine derivatives as antiprion agents. Eur J Med Chem. 2018 submitted __________________________________________________________________________ 9 __________________________________________________________________________ 10 LIST OF ABBREVIATIONS 3D: three-dimensional a-syn: a-synuclein protein Ab: amyloid-b protein AD: Alzheimer’s disease ALS: amyotrophic lateral sclerosis ASA: amyloid seeding assay BBB: blood brain barrier CC1: charged aminoacid cluster 1(residue 23-27) in the HuPrP sequence CC2: charged aminoacid cluster 2 (residue 95-110) in the HuPrP sequence CNS: central nervous system CJD: Creutzfeldt-Jakob disease CSF: cerebral spinal fluid CWD: chronic wasting disease Dpl: Doppel DWI: Diffusion-weighted magnetic resonance imaging Edbg: Edimburg, a PrP-null mice strain EEG: Electroencephalography ER: endoplasmic reticulum f: familial FL: full length FLAIR: fluid attenuated inversion recovery FFI: Fatal familial insomnia FSE: Feline spongiform encephalopathy FT: Fourier Transform FTIR: Fourier transform infrared spectroscopy GAGs: glycosaminoglycans GPI: glycosylphosphatidylinositol GSS: Gerstmann-Sträussler–Scheinker GT1: mouse hypothalamic cells line HD: hydrophobic domain HD: Hungtington’s diseases Hu: human i: iatrogenic IC50: half maximal inhibitory concentration Ig: immunoglobulin LRP1: low density lipoprotein receptor-related protein LTP: long term potentiation MD: molecular dynamic MM: minimal medium Mo: mouse MRI: Magnetic resonance imaging N2a: mouse neuroblastoma cells line NFTs: neurofibrillary tangles Ngsk: Nagasaki, a PrP-null mice strain NMDA: N-methyl-D-aspartate glutamate receptors NMR: nuclear magnetic resonance OR: octapeptide-repeat region ORF: open reading frame __________________________________________________________________________ 11 PCD: protein conformational disorder PD: Parkinson’s disease pIC50: predicted IC50 PMCA: Protein Misfolding Cyclic Amplification PNS: peripheral nervous system PrP: prion protein PrP: physiological cellular form of PrP PrP: misfolded, pathogenic form of PrP, also denoted as prion. PrP: C-terminal transmembrane PrP form PrP: N-terminal transmembrane PrP form PrP: PK-resistant PrP PK: protease K PRNP: human prion protein gene Prnp: prion protein gene in non human species Prnd: Doppel gene in non human species PSS: RTQuIC: Real-time quaking-induced conversion Sh: Syrian Hamster SOD-1: Superoxide dismutase rec: recombinant RT: room temperature s: sporadic ScGT1: chronically PrP infected mouse hypothalamic cells line ScN2a: chronically PrP infected mouse neuroblastoma cells line SPR: surface plasma resonance STED: stimulated emission depletion nanoscopy STI1: stress-inducible protein I TDP-43: TAR DNA-binding protein 43 Tg: transgenic ThT: thioflavin T TME: Transmissible Mink Encephalopathy TSEs: transmissible spongiform encephalopathies, also denoted as prion diseases v: variant VGCC: voltage-gated calcium channel WHO: world health organization WT: wild-type ZrchI: Zurich I or Prnp, a PrP-null mice strain __________________________________________________________________________ 12 __________________________________________________________________________ 13