Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy (original) (raw)
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Proceedings of the National Academy of Sciences, 2004
This paper was submitted directly (Track II) to the PNAS office. Abbreviations: ALS, amyotrophic lateral sclerosis; FALS, familial ALS; SALS, sporadic ALS; SOD, superoxide dismutase; wtSOD, wild-type SOD; BSOD, bovine SOD; A4V, the Ala4Val mutant of SOD1; I113T, the Ile113Thr mutant of SOD1. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 1UXM (A4V) and 1UXL (I113T)].
The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation
Nature communications, 2018
Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is th...
Proceedings of the National Academy of Sciences of the United States of America, 2017
Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.
Proceedings of the National Academy of Sciences, 2010
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3-to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20°C and ∼45°C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies. mass spectrometry | thiol-disulfide I nnovative approaches are needed to combat neurodegenerative disease, among the most serious of which is amyotrophic lateral sclerosis (ALS), a disorder characterized by the death of both upper and lower motor neurons and by 3-to-5-yr median survival postdiagnosis. The only US Food and Drug Administrationapproved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life (1-3). Although the causes of sporadic neurodegenerative diseases remain a mystery, mutations causing familial forms of many of these diseases (e.g., Alzheimer's, Parkinson, and ALS) are known. For example, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for ∼20% of the familial ALS cases (fALS) and 2% of all ALS (4, 5). Two such mutations are G93A, which maintains wild-type-like enzymatic activity, and the metal-deficient G85R, which is essentially inactive. Posttranslational modifications of proteins involved in familial diseases have been invoked in the etiology of the corresponding sporadic diseases, for example, alpha-synuclein (6) and Parkin (7) modification in Parkinson, Abeta (8) and tau (9) modification in Alzheimer's, and TDP43 (10) and SOD1 (11-14) modification in ALS. The hope, therefore, is that strategies for treating familial diseases may translate to at least a subset of sporadic diseases. Both dominant inheritance of mutant SOD1 (15) and lack of symptoms in knockout mice (16) suggest a "gain of toxic function" as opposed to a loss of function (16-22). Aggregation propensity and loss of stability of SOD1 are synergistic risk fac
Two Approaches to Drug Discovery in SOD1-Mediated ALS
Journal of Biomolecular Screening, 2006
Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% of these cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). To date, 105 different mutations spanning all 5 exons have been identified in the SOD1 gene. Mutant SOD1-associated ALS is caused by a toxic gain of function of the mutated protein. Therefore, regardless of the specific mechanism whereby mutant SOD1 initiates motor neuron death, the authors hypothesize that measures that decrease levels of mutant SOD1 protein should ameliorate the phenotype in transgenic mice and potentially in patients with SOD1-mediated disease. They have designed 2 cell-based screening assays to identify small, brain-permeant molecules that inactivate expression of the SOD1 gene or increase the degradation of the SOD1 protein. Here they describe the development and optimization of these assays and the results of high-throughput screening using a variety of compound libraries, including a total of more than 116,000 compounds. The majority of the hit compounds identified that down-regulated SOD1 were shown to be toxic in a cell-based viability assay or were nonselective transcription inhibitors, but work is continuing on a number of nonspecific inhibitors of SOD1 expression. Ultimately, the authors believe that these 2 cell-based assays will provide powerful strategies to identify novel therapies for the treatment of inherited SOD1-associated forms of ALS. (Journal of Biomolecular Screening 2006:729-735)
2005
S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metaldeficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior. Over 100 different mutations in the superoxide dismutase 1(SOD1) 4 gene have been linked to the inherited form of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by progressive death of motor neurons and consequent paralysis. The individ-* This work was supported in part by Ministero Italiano dell'Universita' e della Ricerca
Journal of Molecular Biology, 2002
Mutations in human superoxide dismutase (HSOD) have been linked to the familial form of amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is one of the most common neurodegenerative disorders in humans. In ALS patients, selective killing of motor neurons leads to progressive paralysis and death within one to five years of onset. The most frequent FALS mutation in HSOD, Ala4 ! Val, is associated with the most rapid disease progression. Here we identify and characterize key differences in the stability between the A4V mutant protein and its thermostable parent (HSOD-AS), in which free cysteine residues were mutated to eliminate interferences from cysteine oxidation. Denaturation studies reveal that A4V unfolds at a guanidine-HCl concentration 1 M lower than HSOD-AS, revealing that A4V is significantly less stable than HSOD-AS. Determination and analysis of the crystallographic structures of A4V and HSOD-AS reveal structural features likely responsible for the loss of architectural stability of A4V observed in the denaturation experiments. The combined structural and biophysical results presented here argue that architectural destabilization of the HSOD protein may underlie the toxic function of the many HSOD FALS mutations.