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1.
ACS Chem Neurosci ; 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32559370

RESUMO

Oligomers of amyloid ß-protein (Aß) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aß has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aß in vitro and both promote and inhibit the neurotoxicity of Aß, depending on the experimental conditions. Thus, successful inhibitors of Aß self-assembly may have to inhibit the toxicity not only of Aß oligomers themselves, but also of Aß-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aß self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aß42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aß oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aß42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, ß-sheet, and fibril formation by Aß42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aß42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aß42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aß42 oligomerization and Aß42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aß42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aß42 alone. In agreement with these results, CLR01 inhibited ß-sheet and fibril formation in Aß42-Zn2+ complexes. Our data suggest that for development of efficient therapeutic agents, inhibitors of Aß self-assembly and toxicity should be examined in the presence of relevant metal ions, and that molecular tweezers may be particularly attractive candidates for therapy development.

2.
Mol Ther ; 28(4): 1167-1176, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32087148

RESUMO

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ß progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.

3.
Front Chem ; 7: 657, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632951

RESUMO

Molecular tweezers (MTs) are supramolecular host molecules equipped with two aromatic pincers linked together by a spacer (Gakh, 2018). They are endowed with fascinating properties originating from their ability to hold guests between their aromatic pincers (Chen and Whitlock, 1978; Zimmerman, 1991; Harmata, 2004). MTs are finding an increasing number of medicinal applications, e.g., as bis-intercalators for DNA such as the anticancer drug Ditercalinium (Gao et al., 1991), drug activity reverters such as the bisglycoluril tweezers Calabadion 1 (Ma et al., 2012) as well as radioimmuno detectors such as Venus flytrap clusters (Paxton et al., 1991). We recently embarked on a program to create water-soluble tweezers which selectively bind the side chains of lysine and arginine inside their cavity. This unique recognition mode is enabled by a torus-shaped, polycyclic framework, which is equipped with two hydrophilic phosphate groups. Cationic amino acid residues are bound by the synergistic effect of disperse, hydrophobic, and electrostatic interactions in a kinetically fast reversible process. Interactions of the same kind play a key role in numerous protein-protein interactions, as well as in pathologic protein aggregation. Therefore, these particular MTs show a high potential to disrupt such events, and indeed inhibit misfolding and self-assembly of amyloidogenic polypeptides without toxic side effects. The mini-review provides insight into the unique binding mode of MTs both toward peptides and aggregating proteins. It presents the synthesis of the lead compound CLR01 and its control, CLR03. Different biophysical experiments are explained which elucidate and help to better understand their mechanism of action. Specifically, we show how toxic aggregates of oligomeric and fibrillar protein species are dissolved and redirected to form amorphous, benign assemblies. Importantly, these new chemical tools are shown to be essentially non-toxic in vivo. Due to their reversible moderately tight binding, these agents are not protein-, but rather process-specific, which suggests a broad range of applications in protein misfolding events. Thus, MTs are highly promising candidates for disease-modifying therapy in early stages of neurodegenerative diseases. This is an outstanding example in the evolution of supramolecular concepts toward biological application.

4.
Biochim Biophys Acta Mol Basis Dis ; 1865(11): 165513, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319154

RESUMO

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.

5.
ACS Chem Biol ; 14(6): 1363-1379, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31046227

RESUMO

Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer's disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced ( Despres et al. ( 2017 ) Proc. Natl. Acad. Sci. U.S.A. , 114 , 9080 - 9085 ). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure-activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.


Assuntos
Heparina/farmacologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Animais , Humanos , Fosforilação , Ratos , Proteínas tau/antagonistas & inibidores
6.
J Biol Chem ; 294(10): 3501-3513, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30602569

RESUMO

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Mutação , Organofosfatos/farmacologia , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Sequência de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Sítios de Ligação , Peso Corporal/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Modelos Animais de Doenças , Camundongos , Força Muscular/efeitos dos fármacos , Organofosfatos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Superóxido Dismutase-1/metabolismo , Análise de Sobrevida
7.
J Am Soc Mass Spectrom ; 30(1): 16-23, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30062477

RESUMO

Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer's disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. Graphical Abstract ᅟ.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Organofosfatos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Proteínas tau/química , Proteínas tau/metabolismo , Sítios de Ligação , Hidrocarbonetos Aromáticos com Pontes/química , Concentração de Íons de Hidrogênio , Organofosfatos/química , Fosforilação
8.
Chemistry ; 24(44): 11332-11343, 2018 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-30015416

RESUMO

A new synthetic access to molecular tweezers with one or two aliphatic phosphate ester groups in the central benzene spacer-unit is presented. Alkynyl ester groups offer the prospect to attach additional functional units by click chemistry and greatly broaden the scope of these tools for chemical biology. We present two alternative strategies: the trichloroacetonitrile method involves activation of only one OH group of each phosphoric acid substituent by way of trichloroacetimidate intermediates and subsequent introduction of an aliphatic ester alcohol moiety. The method is versatile, robust and combines simple workup with high yields. Mono- and disubstituted novel host structures are thus accessible in a convenient way. Alternatively, the phosphoramidite strategy activates the hydroquinone precursor by way of phosphoramidite intermediates and couples the desired ester alcohols followed by mild oxidation to the desired phosphate esters. Each step of the synthesis is carried out at very mild conditions and allows to combine sensitive host candidates and recognition elements. After neutralization of the phosphoric acids to water-soluble tri- and tetra-anions the cavities of the new tweezer derivatives are open to bind lysine and arginine as well as peptidic guests. The concept of introducing clickable alkynyl phosphates to free OH groups may be transferred to other major macrocyclic host classes to introduce additional recognition elements, biomolecules or fluorescence labels.

9.
Methods Mol Biol ; 1777: 369-386, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29744849

RESUMO

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.


Assuntos
Proteínas Amiloidogênicas/química , Modelos Moleculares , Multimerização Proteica , Proteínas/química , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Estrutura Molecular , Agregados Proteicos , Agregação Patológica de Proteínas
10.
J Am Heart Assoc ; 6(8)2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28862927

RESUMO

BACKGROUND: Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid ß-protein, tau, and α-synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation-prone proteins and thereby disrupting aggregate formation. However, the effects of CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated αB-crystallin (CryABR120G). METHODS AND RESULTS: A proteotoxic model of desmin-related cardiomyopathy caused by cardiomyocyte-specific expression of CryABR120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild-type CryAB or CryABR120G. Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryABR120G expression in a dose-dependent manner, whereas CLR03 had no effect. Ubiquitin-proteasome system function was analyzed using a ubiquitin-proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein. CLR01 improved proteasomal function in CryABR120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryABR120G transgenic mice. CONCLUSIONS: CLR01 can inhibit CryABR120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress, presumably through clearance of the misfolded protein via increased proteasomal function. CLR01 or related compounds may be therapeutically useful in treating the pathogenic sequelae resulting from proteotoxic heart disease.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Organofosfatos/farmacologia , Agregação Patológica de Proteínas , Cadeia B de alfa-Cristalina/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Vetores Genéticos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Compostos Organofosforados/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteólise , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdução Genética , Transfecção , Ubiquitinação , Cadeia B de alfa-Cristalina/genética
11.
Neurotherapeutics ; 14(4): 1107-1119, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28585223

RESUMO

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.


Assuntos
Encéfalo/metabolismo , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Atividade Motora/efeitos dos fármacos , Organofosfatos/administração & dosagem , alfa-Sinucleína/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Humanos , Injeções Intraventriculares , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tirosina 3-Mono-Oxigenase/metabolismo
12.
J Am Chem Soc ; 139(16): 5640-5643, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28406616

RESUMO

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Proteína Huntingtina/antagonistas & inibidores , Organofosfatos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/química , Éxons , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Estrutura Molecular , Organofosfatos/química , Agregados Proteicos/efeitos dos fármacos
13.
Angew Chem Int Ed Engl ; 56(14): 3751-3752, 2017 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-28276615

RESUMO

Fritz Vögtle, Professor Emeritus of Organic Chemistry and former Director of the Kekulé Institute of Organic Chemistry and Biochemistry at the University of Bonn passed away on January 3, 2017. He was significantly involved in the development of supramolecular chemistry as a new and seminal research field, and his outstanding achievements, including dendrimer, catenane, rotaxane, and knot synthesis, brought this area to both national and international attention.

14.
Chem Commun (Camb) ; 52(76): 11318-34, 2016 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-27546596

RESUMO

Molecular tweezers represent the first class of artificial receptor molecules that have made the way from a supramolecular host to a drug candidate with promising results in animal tests. Due to their unique structure, only lysine and arginine are well complexed with exquisite selectivity by a threading mechanism, which unites electrostatic, hydrophobic and dispersive attraction. However, tweezer design must avoid self-dimerization, self-inclusion and external guest binding. Moderate affinities of molecular tweezers towards sterically well accessible basic amino acids with fast on and off rates protect normal proteins from potential interference with their biological function. However, the early stages of abnormal Aß, α-synuclein, and TTR assembly are redirected upon tweezer binding towards the generation of amorphous non-toxic materials that can be degraded by the intracellular and extracellular clearance mechanisms. Thus, specific host-guest chemistry between aggregation-prone proteins and lysine/arginine binders rescues cell viability and restores animal health in models of AD, PD, and TTR amyloidosis.


Assuntos
Arginina/química , Lisina/química , Agregação Patológica de Proteínas/tratamento farmacológico , Receptores Artificiais/química , Animais , Humanos , Agregação Patológica de Proteínas/metabolismo
15.
Environ Health Perspect ; 124(11): 1766-1775, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27301718

RESUMO

BACKGROUND: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood. OBJECTIVE: We studied the mechanisms of ziram's (a DTC fungicide) neurotoxicity in vivo. METHODS: Zebrafish (ZF) embryos were utilized to determine ziram's effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity. RESULTS: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram's DA toxicity. CONCLUSIONS: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. Citation: Lulla A, Barnhill L, Bitan G, Ivanova MI, Nguyen B, O'Donnell K, Stahl MC, Yamashiro C, Klärner FG, Schrader T, Sagasti A, Bronstein JM. 2016. Neurotoxicity of the Parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos. Environ Health Perspect 124:1766-1775; http://dx.doi.org/10.1289/EHP141.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Neurotoxinas/toxicidade , Doença de Parkinson/etiologia , Sinucleínas/fisiologia , Peixe-Zebra/embriologia , Ziram/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Embrião não Mamífero/metabolismo , Sinucleínas/genética , Sinucleínas/metabolismo
16.
J Org Chem ; 81(6): 2572-80, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26919259

RESUMO

The novel hydrocarbon propeller-shaped D3h-symmetric cyclophane (3), "anthraphane", was prepared through a revisited and optimized gram-scale synthesis of the key building block anthracene-1,8-ditriflate 7. Anthraphane has a high tendency to crystallize and single crystals in size ranges of 100-200 µm are easily obtained from different solvents. The crystallization behavior of 3 was extensively studied to unravel packing motifs and determine whether the packing can be steered into a desired direction, so to allow topochemical photopolymerization. SC-XRD shows that anthraphane packs in layers irrespective of the solvent used for crystallization. However, within the layers, intermolecular arrangements and π-π interactions of the anthracene units vary strongly. Four interaction motifs for the anthracene moieties are observed and discussed in detail: two types of exclusively edge-to-face (etf), a mixture of edge-to-face and face-to-face (ftf), and no anthracene-anthracene interaction at all. To elucidate why an exclusive ftf stacking was not observed, electrostatic potential surface (EPS) calculations with the semiempirical PM3 method were performed. They show qualitatively that the anthracene faces bear a strong negative surface potential, which may be the cause for this cyclophane to avoid ftf interactions. This combined crystallographic and computational study provides valuable insights on how to create all-ftf packings.

17.
Exp Neurol ; 278: 105-15, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26854933

RESUMO

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury.


Assuntos
Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Traumatismos da Medula Espinal/patologia , Sinucleínas/metabolismo , Análise de Variância , Animais , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Contagem de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Lampreias , Larva , Morfolinos/uso terapêutico , Neurônios/efeitos dos fármacos , Organofosfatos/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/mortalidade
18.
ACS Chem Neurosci ; 6(11): 1860-9, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26317327

RESUMO

Although the precise molecular factors linking amyloid ß-protein (Aß) to Alzheimer's disease (AD) have not been deciphered, interaction of Aß with cellular membranes has an important role in the disease. However, most therapeutic strategies targeting Aß have focused on interfering with Aß self-assembly rather than with its membrane interactions. Here, we studied the impact of three toxicity inhibitors on membrane interactions of Aß42, the longer form of Aß, which is associated most strongly with AD. The inhibitors included the four-residue C-terminal fragment Aß(39-42), the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and the lysine-specific molecular tweezer, CLR01, all of which previously were shown to disrupt different steps in Aß42 self-assembly. Biophysical experiments revealed that incubation of Aß42 with each of the three modulators affected membrane interactions in a distinct manner. Interestingly, EGCG and CLR01 were found to have significant interaction with membranes themselves. However, membrane bilayer disruption was reduced when the compounds were preincubated with Aß42, suggesting that binding of the assembly modulators to the peptide attenuated their membrane interactions. Importantly, our study reveals that even though the three tested compounds affect Aß42 assembly differently, membrane interactions were significantly inhibited upon incubation of each compound with Aß42, suggesting that preventing the interaction of Aß42 with the membrane contributes substantially to inhibition of its toxicity by each compound. The data suggest that interference with membrane interactions is an important factor for Aß42 toxicity inhibitors and should be taken into account in potential therapeutic strategies, in addition to disruption or remodeling of amyloid assembly.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Catequina/análogos & derivados , Bicamadas Lipídicas/química , Fármacos Neuroprotetores/farmacologia , Organofosfatos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos beta-Amiloides/química , Hidrocarbonetos Aromáticos com Pontes/química , Catequina/química , Catequina/farmacologia , Microscopia Crioeletrônica , Dimiristoilfosfatidilcolina/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fármacos Neuroprotetores/química , Organofosfatos/química , Fragmentos de Peptídeos/química , Fosfatidilgliceróis/química , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Lipossomas Unilamelares/química
19.
Elife ; 42015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26284498

RESUMO

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.


Assuntos
Amiloide/antagonistas & inibidores , Fármacos Anti-HIV/farmacologia , Antimetabólitos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Organofosfatos/farmacologia , Sêmen/efeitos dos fármacos , Transmissão de Doença Infecciosa/prevenção & controle , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Humanos , Masculino , Sêmen/química , Sêmen/virologia
20.
Biochemistry ; 54(24): 3729-38, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26030124

RESUMO

The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic ß-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of ß-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates.


Assuntos
Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Modelos Moleculares , Mutação , Organofosfatos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Proteína Supressora de Tumor p53/antagonistas & inibidores , Substituição de Aminoácidos , Antineoplásicos/química , Sítios de Ligação , Hidrocarbonetos Aromáticos com Pontes/química , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestrutura , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Organofosfatos/química , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Estabilidade Proteica/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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