Papers by Giuseppe Legname
European journal of medicinal chemistry, 2023
Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (P... more Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrP Sc) of the cellular prion protein (PrP C). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrP C conformation and prevents its pathogenic conversion. A focused library was synthesized via a fourstep route, and a representative member was confirmed to have native fluorescence, including a band in the nearinfrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrP Sc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrP C levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrP Sc aggregates by 2b was confirmed in
Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance
Journal of Medicinal Chemistry, Jun 30, 2022
Approaches for discovering anti-prion compounds: lessons learned and challenges ahead
Expert Opinion on Drug Discovery, Feb 15, 2015
Introduction: Recent years have witnessed major advances in our understanding of the molecular ba... more Introduction: Recent years have witnessed major advances in our understanding of the molecular bases of prion diseases. These studies not only highlight the protein misfolding as a potential initiator of a neurodegenerative process, they also provide a foundation for considering whether such a process can be common to many neurodegenerative diseases, including Alzheimer's disease. This makes prion diseases a sort of prototype of neurodegenerative disease, endowed with some intrinsic positive features in terms of drug development. Thanks to the fact that disappearance of the scrapie protein can serve as a clear readout of drug efficiency, phenotypic approaches have high potential for prion disease drug discovery. Areas covered: In this review, the authors discuss phenotypic screening and how it lends itself to drug repositioning. Furthermore, they discuss the advantages of working with a molecule with proven safety, tolerability and drug-like properties in combination with a reliable phenotypic screening and how it could improve the success rate for prion drug development. They also provide examples of several interesting candidates that have been identified using this approach, including quinacrine, astemizole, guanabenz and doxycycline. Expert opinion: The availability of persistently scrapie-infected murine neuroblastoma cells has greatly helped to identify compounds that inhibit prion formation. However, a human neuronal model infected with the human isoform would ultimately serve as the ideal disease model toward the discovery of effective drugs.
PLOS ONE, Feb 16, 2010
The action of dopamine on the aggregation of the unstructured alpha-synuclein (a-syn) protein may... more The action of dopamine on the aggregation of the unstructured alpha-synuclein (a-syn) protein may be linked to the pathogenesis of Parkinson's disease. Dopamine and its oxidation derivatives may inhibit a-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of a-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to a-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.
Proceedings of the National Academy of Sciences of the United States of America, Dec 12, 2011
Journal of Biological Chemistry, Jun 1, 2002
In vivo under pathological conditions, the normal cellular form of the prion protein, PrP C (resi... more In vivo under pathological conditions, the normal cellular form of the prion protein, PrP C (residues 23-231), misfolds to the pathogenic isoform PrP Sc , a -rich aggregated pathogenic multimer. Proteinase K digestion of PrP Sc leads to a proteolytically resistant core, PrP 27-30 (residues 90-231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP Sc and found that it can adopt two non-native abnormal isoforms, a -oligomer and an amyloid fibril. Several lines of kinetic data suggest that the -oligomer is not on the pathway to amyloid formation. The preferences for forming either a -oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the -oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high -sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct -sheet-rich abnormal isoforms may explain the difficulties in refolding PrP Sc in vitro, the need for a PrP Sc template, and the significant variation in disease presentation and neuropathology.
bioRxiv (Cold Spring Harbor Laboratory), Dec 28, 2018
Prions are pathological isoforms of the cellular prion protein (PrP C) responsible for transmissi... more Prions are pathological isoforms of the cellular prion protein (PrP C) responsible for transmissible spongiform encephalopathies (TSE). PrP C interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrP C suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrP C (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrP C carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrP C models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.
Alzheimers & Dementia, Jul 1, 2006
Rational approach to an antiprion compound with a multiple mechanism of action
Future Medicinal Chemistry, Oct 1, 2015
Background: The main pathogenic event of prion disorders has been identified in the deposition of... more Background: The main pathogenic event of prion disorders has been identified in the deposition of the disease-associated prion protein (PrPSc), which is accompanied by metal dyshomeostasis. Results: The multitarget-directed ligand 1, designed by combining a heteroaromatic prion recognition motif to an 8-hydroxyquinoline metal chelator, has been developed as a potential antiprion disease-modifying agent. Importantly, 1 was found to effectively clear PrPSc from scrapie-infected cells, and, at the same time, inhibit metal-induced prion aggregation and reactive oxygen species generation. 1 was also characterized in terms of pharmacokinetic properties in a preliminary in vitro investigation. Conclusion: Compound 1 has emerged as a suitable lead candidate against prion diseases and as a good starting point for a further optimization process.
The EMBO Journal, Apr 2, 2001
It is hypothesized that infectious prions are generated as the cellular form of the prion protein... more It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP C) undergoes pronounced conformational change under the direction of an infectious PrP Sc template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90±120), which has an extended¯exible structure in the PrP C isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of speci®c unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.
Journal of Medicinal Chemistry, Aug 4, 2018
Several findings propose altered tau protein network as an important target for Alzheimer's disea... more Several findings propose altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β, and tau aggregation process. In light of this and based on our interest in multi-target paradigms in AD, we report on the discovery of 2,4-thiazolidinedionederivatives endowed with such profile. 28 and 30 displayed micromolar IC50 values towards GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 µM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, non-toxic, dual-acting compounds hitting tau cascade at two different hubs.
In silico/in vitro screening and hit evaluation identified new phenothiazine anti-prion derivatives
European journal of medicinal chemistry, Jun 1, 2020
Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare neurodegen... more Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare neurodegenerative disorders. TSEs are characterized by the accumulation of prions (PrPSc) that represent pathological isoforms of the physiological cellular prion protein PrPC. Although the conversion of PrPC to PrPSc 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 PrPSc 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 PrPC and consequentially blocks prion conversion. Herein we describe the results of these efforts.
PLOS Pathogens, Feb 18, 2021
Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian spec... more Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian species, including humans. During the course of the disease, the abnormally folded scrapie prion protein (PrP Sc) accumulates in the central nervous system where it causes neurodegeneration. In prion disorders, the diverse spectrum of illnesses exists because of the presence of different isoforms of PrP Sc where they occupy distinct conformational states called strains. Strains are biochemically distinguished by a characteristic three-band immunoblot pattern, defined by differences in the occupancy of two glycosylation sites on the prion protein (PrP). Characterization of the exact N-glycan structures attached on either PrP C or PrP Sc is lacking. Here we report the characterization and comparison of N-glycans from two different sheep prion strains. PrP Sc from both strains was isolated from brain tissue and enzymatically digested with trypsin. By using liquid chromatography coupled to electrospray mass spectrometry, a site-specific analysis was performed. A total of 100 structures were detected on both glycosylation sites. The N-glycan profile was shown to be similar to the one on mouse PrP, however, with additional 40 structures reported. The results presented here show no major differences in glycan composition, suggesting that glycans may not be responsible for the differences in the two analyzed prion strains.
ChemMedChem, Mar 15, 2011
Prion diseases or transmissible spongiform encephalopathies are a group of invariably fatal disor... more Prion diseases or transmissible spongiform encephalopathies are a group of invariably fatal disorders, for which there is neither early diagnosis nor a cure. [1, 2] These maladies are characterized by spongiform brain neurodegeneration caused by a misfolded protein with unique infective properties: prion protein scrapie (PrP Sc). According to the protein-only hypothesis, [1] in the central nervous system of the infected host the cellular prion protein (PrP), PrP C , is converted into an abnormal insoluble amyloidogenic isoform, that is, PrP Sc or prion. The latter acts as a template for PrP C leading to nascent PrP Sc molecules. The process of conversion is associated with conformational changes of secondary structure from a-helices to b-sheets. [1, 2] While this hypothesis is supported by in vitro conversion of PrP C to PrP Sc , the mechanism underlying in vivo conversion, although not yet fully elucidated, seems to be more complex, and possibly involves some molecular chaperones. In recent years, it has been gradually accepted that prion disease pathogenesis involves a complex array of processes that operate simultaneously and synergistically. [3] These include: 1) protein aggregation; [4, 5] 2) oxidative stress (OS) accompanied by lipid and protein oxidation; [6-10] 3) decreased levels of potent freeradical scavengers such as polyunsaturated fatty acids, a-tocopherol, and glutathione; [6, 11] 4) an imbalance of metal ions; [12] and 5) brain inflammation with activation of astrocytes and microglia. [13]
Bioorganic & Medicinal Chemistry Letters, Mar 1, 2010
A small library combining two different benzoquinone cores with seven (L) amino acid methyl ester... more A small library combining two different benzoquinone cores with seven (L) amino acid methyl esters (alanine, Nx-nitro-arginine, Ne-BOC-lysine, isoleucine, methionine, phenylalanine and tryptophan) was prepared and tested for prion replication inhibition in ScGT1 cells. The most potent hit, 6a, displayed an EC 50 value of 0.87 lM, which is very close to that of quinacrine (0.4 lM).
Nanoscale, 2010
Gold nanoparticles coated with oppositely charged polyelectrolytes, such as polyallylamine hydroc... more Gold nanoparticles coated with oppositely charged polyelectrolytes, such as polyallylamine hydrochloride and polystyrenesulfonate, were examined for potential inhibition of prion protein aggregation and prion (PrP Sc) conversion and replication. Different coatings, finishing with a positive or negative layer, were tested, and different numbers of layers were investigated for their ability to interact and reduce the accumulation of PrP Sc in scrapie prion infected ScGT1 and ScN2a cells. The particles efficiently hampered the accumulation of PrP Sc in ScN2a cells and showed curing effects on ScGT1 cells with a nanoparticle concentration in the picomolar range. Finally, incubation periods of prion-infected mice treated with nanomolar concentrations of gold nanoparticles were significantly longer compared to untreated controls.
Prion, Jul 1, 2010
Introduction. Seven rapid tests were approved according to the Commission Regulation (EC) 162/200... more Introduction. Seven rapid tests were approved according to the Commission Regulation (EC) 162/2009 for active surveillance of TSE in sheep and goat, however EFSA Scientific Opinion on Analytical Sensitivity of Approved TSE rapid tests-2009 recommends only three of those tests: Bio-Rad TeSeE SAP (A), Bio-Rad TeSeE Sheep/Goat (B) and Idexx HerdChek Antigen Test Kit EIA (C). Aim of the study was to provide a direct comparison of the performances of the three rapid tests when applied to goat brainstem. Results. Differences among the three systems were observed in terms of diagnostic sensitivity, test C being the most sensitive (94,7%, 95%CI:82,3-99,4%) while all three rapid tests displayed 100% specificity. The agreement in reproducibility and repeatability was very good. Patients. A sample size of 73 confirmed positive and negative samples, including both classical and atypical field cases, experimentally inoculated goats and regularly slaughtered animals, were tested. Conclusion. Whenever all three tests displayed good performance, according to results achieved on diagnostic sensitivity a difference in the efficacy of TSE active surveillance system in goat populations, based on the test adopted, can be expected.
PLOS ONE, Aug 18, 2009
Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrP C), together sha... more Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrP C), together sharing common structural and biochemical properties. Unlike PrP C , which is abundantly expressed throughout the central nervous system (CNS), Dpl protein expression is not detectable in the CNS. Interestingly, its ectopic expression in the brain elicits neurodegeneration in transgenic mice. Here, by combining native isoelectric focusing plus non-denaturing polyacrylamide gel electrophoresis and mass spectrometry analysis, we identified two Dpl binding partners: rat alpha-1-inhibitor-3 (a 1 I 3) and, by sequence homology, alpha-2-macroglobulin (a 2 M), two known plasma metalloproteinase inhibitors. Biochemical investigations excluded the direct interaction of PrP C with either a 1 I 3 or a 2 M. Nevertheless, enzyme-linked immunosorbent assays and surface plasmon resonance experiments revealed a high affinity binding occurring between PrP C and Dpl. In light of these findings, we suggest a mechanism for Dpl-induced neurodegeneration in mice expressing Dpl ectopically in the brain, linked to a withdrawal of natural inhibitors of metalloproteinase such as a 2 M. Interestingly, a 2 M has been proven to be a susceptibility factor in Alzheimer's disease, and as our findings imply, it may also play a relevant role in other neurodegenerative disorders, including prion diseases.
Induction of Protease-Resistant Prion Protein and alpha-synuclein Aggregation by Human alpha-synuclein Fibrils in Cultured Cells
Recent Advances of Biology and Medicinal Chemistry of Prion Protein and Prions: On the Road to Therapeutics
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Papers by Giuseppe Legname