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1.
Commun Biol ; 4(1): 62, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33437023

RESUMO

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Dobramento de Proteína , Animais , Sítios de Ligação , Simulação por Computador , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes
2.
Bioorg Med Chem ; 28(21): 115717, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065443

RESUMO

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.


Assuntos
Benzilaminas/química , Proteínas PrPSc/metabolismo , Animais , Benzilaminas/síntese química , Benzilaminas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Camundongos , Mutagênese , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/genética , Relação Estrutura-Atividade
3.
ACS Appl Bio Mater ; 3(12): 8361-8374, 2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35019608

RESUMO

The in vitro degradation profile and the cytotoxicity of the degradation products of a silk fibroin (SF)-based nerve conduit (SilkBridge), with a complex three-layered wall architecture comprising both native and regenerated (electrospun) fibers, are reported. The bacterial protease type XIV from Streptomyces griseus was used as a hydrolytic agent at three different enzyme/substrate ratios (1:8, 1:80, and 1:800 w/w) to account for the different susceptibility to degradation of the native and regenerated components. The incubation time was extended up to 91 days. At fixed time points, the remaining device, the insoluble debris, and the incubation buffers containing soluble degradation products were separated and analyzed. The electrospun fibers forming the inner and outer layers of the conduit wall were almost completely degraded within 10 days of incubation at an enzyme/substrate ratio of 1:80 w/w. The progression of degradation was highlighted by the emergence of zones of erosion and discontinuity along the electrospun fibers, weakening of the electrospun layers, and decrease in resistance to compressive stress. Native SF microfibers forming the middle layer of the conduit wall displayed a higher resistance to enzymatic degradation. When incubated at an enzyme/substrate ratio of 1:8 w/w, the weight decreased gradually over the incubation time as a consequence of fiber erosion and fragmentation. Analogously, the tensile properties markedly decreased. Both spectroscopic and thermal analyses confirmed the gradual increase in the crystalline character of the fibers. The incubation buffers containing the soluble degradation products were subjected to cytotoxicity testing with human HEK293 cells and mouse neuroblastoma N2a cells. No detrimental effects on cell viability were observed, suggesting that the degradation products do not retain any toxic property. Finally, the mass spectrometry analysis of degradation products showed that the SF polypeptides recovered in the incubation buffers were representative of the aminoacidic sequence of the fibroin light chain and of the highly repetitive fibroin heavy chain, indicating that virtually the entire sequence of the fibroin protein constituent of SilkBridge was degraded.

4.
J Neurochem ; 152(1): 136-150, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264722

RESUMO

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.


Assuntos
Membrana Celular/química , Proteínas PrPC/análise , Príons/antagonistas & inibidores , Animais , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HEK293 , Harmalina/análogos & derivados , Harmalina/farmacologia , Hematoxilina/análogos & derivados , Hematoxilina/farmacologia , Humanos , Camundongos , Neuroblastoma , Proteínas PrPC/genética , Príons/biossíntese , Príons/toxicidade , Quinacrina/farmacologia , Tacrolimo/farmacologia
5.
Pathogens ; 7(1)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518975

RESUMO

Prion diseases are associated with the conversion of the cellular prion protein (PrPC), a glycoprotein expressed at the surface of a wide variety of cell types, into a misfolded conformer (the scrapie form of PrP, or PrPSc) that accumulates in brain tissues of affected individuals. PrPSc is a self-catalytic protein assembly capable of recruiting native conformers of PrPC, and causing their rearrangement into new PrPSc molecules. Several previous attempts to identify therapeutic agents against prion diseases have targeted PrPSc, and a number of compounds have shown potent anti-prion effects in experimental models. Unfortunately, so far, none of these molecules has successfully been translated into effective therapies for prion diseases. Moreover, mounting evidence suggests that PrPSc might be a difficult pharmacological target because of its poorly defined structure, heterogeneous composition, and ability to generate different structural conformers (known as prion strains) that can elude pharmacological intervention. In the last decade, a less intuitive strategy to overcome all these problems has emerged: targeting PrPC, the common substrate of any prion strain replication. This alternative approach possesses several technical and theoretical advantages, including the possibility of providing therapeutic effects also for other neurodegenerative disorders, based on recent observations indicating a role for PrPC in delivering neurotoxic signals of different misfolded proteins. Here, we provide an overview of compounds claimed to exert anti-prion effects by directly binding to PrPC, discussing pharmacological properties and therapeutic potentials of each chemical class.

6.
PLoS One ; 12(8): e0182589, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28787011

RESUMO

Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrPC may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrPC. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrPC at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrPC from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrPC, as well as in the propagation of infectious prions.


Assuntos
Antipsicóticos/farmacologia , Clorpromazina/farmacologia , Proteínas Priônicas/metabolismo , Antipsicóticos/metabolismo , Linhagem Celular , Clorpromazina/metabolismo , Dinaminas/antagonistas & inibidores , Humanos , Ligantes , Mutação , Proteínas Priônicas/genética , Transporte Proteico/efeitos dos fármacos
7.
ChemMedChem ; 12(16): 1286-1292, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28722340

RESUMO

Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self-replicating, misfolded isoform (PrPSc ) of the cellular prion protein (PrPC ). No therapies are available for these pathologies. We capitalized on previously described cell-based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity. Compound 55 may represent the starting point for the development of a completely new class of therapeutics for prion diseases.


Assuntos
Proteínas Priônicas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Mutagênese , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Proteínas Priônicas/antagonistas & inibidores , Proteínas Priônicas/genética , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/toxicidade
8.
Neural Plast ; 2017: 6468356, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28367336

RESUMO

The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity.


Assuntos
Córtex Cerebral/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Terminações Pré-Sinápticas/enzimologia , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas SNARE/metabolismo , Animais , Córtex Cerebral/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Glicina/farmacologia , Masculino , Camundongos , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , N-Metilaspartato/farmacologia , Sinaptossomos/metabolismo
9.
Elife ; 52016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27431613

RESUMO

The RAS pathway is central to epidermal homeostasis, and its activation in tumors or in Rasopathies correlates with hyperproliferation. Downstream of RAS, RAF kinases are actionable targets regulating keratinocyte turnover; however, chemical RAF inhibitors paradoxically activate the pathway, promoting epidermal proliferation. We generated mice with compound epidermis-restricted BRAF/RAF1 ablation. In these animals, transient barrier defects and production of chemokines and Th2-type cytokines by keratinocytes cause a disease akin to human atopic dermatitis, characterized by IgE responses and local and systemic inflammation. Mechanistically, BRAF and RAF1 operate independently to balance MAPK signaling: BRAF promotes ERK activation, while RAF1 dims stress kinase activation. In vivo, JNK inhibition prevents disease onset, while MEK/ERK inhibition in mice lacking epidermal RAF1 phenocopies it. These results support a primary role of keratinocytes in the pathogenesis of atopic dermatitis, and the animals lacking BRAF and RAF1 in the epidermis represent a useful model for this disease.


Assuntos
Dermatite Atópica/patologia , Dermatite Atópica/prevenção & controle , Queratinócitos/fisiologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Camundongos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética
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