In silico/in vitro screening and hit evaluation identified new phenothiazine anti-prion derivatives

Highlights

• Prions.

• In silico drug screening.

• Phenothiazine anti-prion derivatives.

• In vitro drug screening validation.

• Cellular models of prion disease.

Abstract

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare neurodegenerative disorders. TSEs are characterized by the accumulation of prions (PrP^Sc) that represent pathological isoforms of the physiological cellular prion protein PrP^C. Although the conversion of PrP^C to PrP^Sc is still not completely understood, blocking this process may lead to develop new therapies. Here, we have generated a pharmacophore model, based on anti-prion molecules reported in literature to be effective in phenotypic assay. The model was used to conduct a virtual screen of commercial compound databases that selected a small library of ten compounds. These molecules were then screened in mouse neuroblastoma cell line chronically infected with prions (ScN2a) after excluding neurotoxicity. 1 has been identified as the therapeutic hit on the basis of the following evidence: chronic treatments of ScN2a cells using 1 eliminate PrP^Sc loaded in both Western blotting analysis and Real-Time Quaking-Induced Conversion (RT-QuIC) assay. We also proposed the mechanism of action of 1 by which it has the ability to bind PrP^C and consequentially blocks prion conversion. Herein we describe the results of these efforts.

Introduction

In recent years, our understanding of the pathogenic mechanisms of neurodegenerative diseases has grown steadily. However, no effective therapies have yet been developed. These diseases are recognized as neurological protein misfolding disorders (PMDs) since they are associated with conformational changes of native proteins into disease-associated conformers [1]. PMDs include Alzheimer’s disease, involving accumulation of misfolded amyloid β (Aβ) and tau, Parkinson’s disease in which α-synuclein aggregates, and prion diseases where the physiological form of the prion protein is converted into its pathological isoforms [2].

In the latter case, prion diseases or TSEs present as sporadic, inherited and infectious disorders [3]. Prions were long thought to be unique diseases. However, accumulating evidence suggests that in other PMDs other proteins might follow a similar mechanism of seeding, self-propagation and cell-to-cell spreading [[4], [5], [6], [7], [8]].

Neuropathological changes in TSEs are mainly gliosis, vacuolation and neuronal loss paralleled by cognitive and motor impairments [9,10]. TSEs include kuru, Creutzfeldt-Jacob disease, Gerstmann Sträussler-Scheinker syndrome and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, and chronic wasting disease in elk, deer and other cervids [9]. The etiological agent is the scrapie prion protein (PrP^Sc), the abnormal, misfolded isoform of the PrP^C [11]. PrP^C is anchored to the cell surface through a C-terminal moiety of glycophosphatidyl-inositol or GPI [12]. Even though the two isoforms share the same primary sequence, they have several different biochemical and biophysical properties: PrP^C is rich in α-helices, is soluble in nonionic detergents and sensitive to protease K (PK) digestion while PrP^Sc is mostly rich in β-sheets, is insoluble in nonionic detergents and partially resistant to PK [13].

Although the conversion of PrP^C to PrP^Sc is still not completely understood, blocking this process may lead to the development of effective new therapies. Several studies have focused on the ability of small molecules to interfere with the conversion process, by either binding and stabilizing PrP^C or blocking PrP^Sc aggregation and accumulation [[14], [15], [16]]. A widely employed strategy has been the repositioning of compounds registered as antivirals [17], antimalarials [18,19], antifungals [20] and antidepressants [21]. Drug repositioning is the application of available drugs for treating conditions different from the original treatment purposes. By using this approach quinacrine (antimalarial) [18,19], pentosan polysulfate (heparin mimetic) [22,23], doxycycline (antibiotic) [24,25] and flupirtine (analgesic) [26] were tested in human clinical trials, but with no encouraging results. Other approaches to develop anti-prion therapies rely on rational medicinal chemistry [[26], [27], [28]], multi-target approaches [29] and in silico methods [16,30]. However, most of the approaches attempted so far have not resulted in molecules to progress into clinical investigations and all the identified drugs have inevitably failed [31]. Therefore, development of effective anti-prion small molecules that have drug-likeness and therapeutic potential remains a major challenge.

Toward this aim, our experimental program began with the generation of a pharmacophore model, based on anti-prion ligands reported to be effective in phenotypic assays, which was used to conduct a virtual screen of commercial compounds databases. This approach led to a small target-biased library, which was then screened in cellular model of the disease. Immortalized neuroblastoma (N2a) and hypothalamic (GT1) mouse cell lines chronically infected with different prion strains (RML and 22L) were used to measure anti-prion efficacy, after excluding neurotoxicity. 1 emerged as a therapeutic hit; it is able to eliminate PrP^Sc after chronic treatments of N2a-RML cells as shown in both Western blotting analysis and Real-Time Quaking-Induced Conversion (RT-QuIC) assay. A mechanism by which prion conversion is blocked upon binding of 1 to PrP^C is proposed. However, 1 suffered from poor solubility. We therefore carried out preliminary SAR studies on this compound, particularly focused on improving this physicochemical property.