Research paperIn silico/in vitro screening and hit evaluation identified new phenothiazine anti-prion derivatives
Graphical abstract
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 (PrPSc), the abnormal, misfolded isoform of the PrPC [11]. PrPC 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: PrPC is rich in α-helices, is soluble in nonionic detergents and sensitive to protease K (PK) digestion while PrPSc is mostly rich in β-sheets, is insoluble in nonionic detergents and partially resistant to PK [13].
Although the conversion of PrPC to PrPSc 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 PrPC or blocking PrPSc 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 PrPSc 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 PrPC 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.
Section snippets
Computational details
Two hundred compounds with known anti-prion activities (IC50) were identified by a literature search (Table S1) and included into 5 datasets, which are distinguished by inhibiting prion replication in cell lines and showing different incubation times in animal models of TSEs (Table S2). Our QSAR model uses Multiple Linear Regression (MLR) to obtain a linear relationship between the pIC50 values against the molecular descriptors of these compounds. For this, we used ordinary least squares (OLS)
QSAR model
We first constructed a list of ca. 200 compounds with known anti-prion activities (IC50) by collecting data from 14 publications (Table S1). The compounds were divided into 5 datasets depending on the type of cell line used in the assays and their incubation time (see Table S2). For each dataset, we generated a QSAR model. Here we present the best model (according to criteria specified in the Materials and Methods section and below) across the five datasets: this is the QSAR model based on the
Conclusions
Prion diseases are triggered by the accumulation of aberrant misfolded isoforms of PrPC in the central nervous system (CNS). The conformational change and replication of the PrPC into its pathological isoform PrPSc are followed by aggregation and cell spreading in the CNS, which lead to fatal neurodegeneration. So far, no therapies against TSEs have been identified. A number of molecules have been developed and many have been tested in human clinical trials with no positive results [18,22,24,26,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by Scuola Internazionale di Studi Superiori Avanzati, University of Bologna and Forschungszentrum Jülich (CDP6 “Drug design” project). We would like to thank Paolo Neviani (Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna) for performing high resolution mass spectroscopy.
References (57)
- et al.
The cerebral proteopathies
Neurobiol. Aging
(2000) In vivo spreading of tau pathology
Neuron
(2012)- et al.
Chapter fourteen - neuropathology of human prion diseases
- et al.
Prion protein gene variation among primates
J. Mol. Biol.
(1995) - et al.
Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein
Biochim. Biophys. Acta BBA - Proteins Proteomics
(2012) - et al.
In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrPC to PrPSc
Mol. Dyn. New Adv. Drug Discov.
(2015) - et al.
Repeated suppression OF creutzfeldt-jakob disease with VIDARABINE
Orig. Publ.
(1982) - et al.
Safety and efficacy of quinacrine in human prion disease (PRION-1 study): a patient-preference trial
Lancet Neurol.
(2009) - et al.
Creutzfeldt-Jakob disease acquired via a dural graft: failure of therapy with quinacrine and chlorpromazine
Surg. Neurol.
(2005) - et al.
Cerebroventricular infusion of pentosan polysulphate in human variant Creutzfeldt–Jakob disease
J. Infect.
(2005)
Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial
Lancet Neurol.
Identification of novel fluorescent probes preventing PrPSc replication in prion diseases
Eur. J. Med. Chem.
Chapter twenty - therapeutic approaches to prion diseases
Development of a high throughput equilibrium solubility assay using miniaturized shake-flask method in early drug discovery
J. Pharmacol. Sci.
Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings1PII of original article: S0169-409X(96)00423-1. The article was originally published in Advanced Drug Delivery Reviews 23 (1997) 3–25.1, Spec. Issue Dedic. Dr Eric Tomlinson Adv. Drug Deliv
Rev. Sel. Most Highly Cited Artic
A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets
J. Biol. Chem.
Microwave-assisted synthesis of 4-quinolylhydrazines followed by nickel boride reduction: a convenient approach to 4-aminoquinolines and derivatives
Tetrahedron Lett.
Differential effects of a new amphotericin B derivative, MS-8209, on mouse bse and scrapie: implications for the mechanism of action of polyene antibiotics
Res. Virol.
Comparison of protease-resistant prion protein inhibitors in cell cultures infected with two strains of mouse and sheep scrapie
Neurosci. Lett.
Structural basis of prion inhibition by phenothiazine compounds
Structure
Protein misfolding diseases
Annu. Rev. Biochem.
Cell biology. A unifying role for prions in neurodegenerative diseases
Science
Approaches for discovering anti-prion compounds: lessons learned and challenges ahead
Expet Opin. Drug Discov.
Defined α-synuclein prion-like molecular assemblies spreading in cell culture
BMC Neurosci.
Transmission and spreading of tauopathy in transgenic mouse brain
Nat. Cell Biol.
Neuron-to-neuron α-synuclein propagation in vivo is independent of neuronal injury
Acta Neuropathol. Commun.
Prions and the potential transmissibility of protein misfolding diseases
Annu. Rev. Microbiol.
Proc. Natl. Acad. Sci. U. S. A.
Cited by (8)
Ellagic acid and pentagalloylglucose are potential inhibitors of prion protein fibrillization
2021, International Journal of Biological MacromoleculesCitation Excerpt :To date, there are no approved effective therapeutic drugs for prion diseases. In recent years, structure-based drug discovery using computer simulations has generated a series of new anti-prion candidates such as trimethoxychalcone and other chalcone derivatives, which need to be further studied [16–19]. One of the advantages of natural molecules, including polyphenols originating from daily diets, is their better toxicity profiles.
Therapeutic strategies for identifying small molecules against prion diseases
2023, Cell and Tissue ResearchResearch Progress in the Synthesis and Biological Activity of Phenothiazines
2022, Chinese Pharmaceutical JournalInnovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance
2022, Journal of Medicinal Chemistry
- 1
Current affiliation: Dementia Research Institute, University College London, Cruciform Building Gower street, London, United Kingdom.