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1.
Nature ; 441(7091): 375-7, 2006 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-16710424

RESUMO

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.


Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/enzimologia , Archaea/genética , Sítios de Ligação , Cátions/metabolismo , Endorribonucleases/genética , Células Eucarióticas/metabolismo , Modelos Biológicos , Mutação/genética , Precursores de RNA/química , Sítios de Splice de RNA , Splicing de RNA , RNA de Transferência/química , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
J Med Chem ; 61(15): 6501-6517, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30044619

RESUMO

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.


Assuntos
Compostos Azo/farmacologia , Descoberta de Drogas , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Pirimidinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Compostos Azo/efeitos adversos , Compostos Azo/uso terapêutico , Humanos , Pirimidinas/efeitos adversos , Pirimidinas/uso terapêutico , Segurança
3.
J Med Chem ; 60(10): 4444-4457, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28441483

RESUMO

Spinal muscular atrophy (SMA) is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene, resulting in low levels of functional SMN protein. We have reported recently the identification of small molecules (coumarins, iso-coumarins and pyrido-pyrimidinones) that modify the alternative splicing of SMN2, a paralogous gene to SMN1, restoring the survival motor neuron (SMN) protein level in mouse models of SMA. Herein, we report our efforts to identify a novel chemotype as one strategy to potentially circumvent safety concerns from earlier derivatives such as in vitro phototoxicity and in vitro mutagenicity associated with compounds 1 and 2 or the in vivo retinal findings observed in a long-term chronic tox study with 3 at high exposures only. Optimized representative compounds modify the alternative splicing of SMN2, increase the production of full length SMN2 mRNA, and therefore levels of full length SMN protein upon oral administration in two mouse models of SMA.


Assuntos
Benzamidas/química , Benzamidas/farmacologia , Atrofia Muscular Espinal/genética , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Benzamidas/farmacocinética , Desenho de Fármacos , Camundongos , Modelos Moleculares , Atrofia Muscular Espinal/tratamento farmacológico
4.
PLoS One ; 11(12): e0167077, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27907033

RESUMO

INTRODUCTION AND OBJECTIVE: Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the SMN1 gene and retention of the SMN2 gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers. METHODS: SMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from SMN2 or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90. RESULTS: Of the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO. CONCLUSION: This study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.


Assuntos
Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/genética , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Regulação da Expressão Gênica , Teste de Complementação Genética , Humanos , Injeções Intraventriculares , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Oligonucleotídeos Antissenso/metabolismo , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
5.
J Med Chem ; 59(13): 6086-100, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27299419

RESUMO

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Atrofia Muscular Espinal/tratamento farmacológico , Pirimidinonas/química , Pirimidinonas/farmacologia , RNA Mensageiro/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Éxons/efeitos dos fármacos , Humanos , Camundongos , Atrofia Muscular Espinal/genética , Pirimidinonas/farmacocinética , Pirimidinonas/uso terapêutico
6.
Science ; 345(6197): 688-93, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-25104390

RESUMO

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Cumarínicos/administração & dosagem , Isocumarinas/administração & dosagem , Longevidade/efeitos dos fármacos , Atrofia Muscular Espinal/tratamento farmacológico , Pirimidinonas/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Administração Oral , Animais , Células Cultivadas , Cumarínicos/química , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Isocumarinas/química , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Pirimidinonas/química , RNA Mensageiro/genética , Deleção de Sequência , Bibliotecas de Moléculas Pequenas/química , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
7.
PLoS One ; 7(4): e33572, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558076

RESUMO

BACKGROUND: The universal presence of a gene (SMN2) nearly identical to the mutated SMN1 gene responsible for Spinal Muscular Atrophy (SMA) has proved an enticing incentive to therapeutics development. Early disappointments from putative SMN-enhancing agent clinical trials have increased interest in improving the assessment of SMN expression in blood as an early "biomarker" of treatment effect. METHODS: A cross-sectional, single visit, multi-center design assessed SMN transcript and protein in 108 SMA and 22 age and gender-matched healthy control subjects, while motor function was assessed by the Modified Hammersmith Functional Motor Scale (MHFMS). Enrollment selectively targeted a broad range of SMA subjects that would permit maximum power to distinguish the relative influence of SMN2 copy number, SMA type, present motor function, and age. RESULTS: SMN2 copy number and levels of full-length SMN2 transcripts correlated with SMA type, and like SMN protein levels, were lower in SMA subjects compared to controls. No measure of SMN expression correlated strongly with MHFMS. A key finding is that SMN2 copy number, levels of transcript and protein showed no correlation with each other. CONCLUSION: This is a prospective study that uses the most advanced techniques of SMN transcript and protein measurement in a large selectively-recruited cohort of individuals with SMA. There is a relationship between measures of SMN expression in blood and SMA type, but not a strong correlation to motor function as measured by the MHFMS. Low SMN transcript and protein levels in the SMA subjects relative to controls suggest that these measures of SMN in accessible tissues may be amenable to an "early look" for target engagement in clinical trials of putative SMN-enhancing agents. Full length SMN transcript abundance may provide insight into the molecular mechanism of phenotypic variation as a function of SMN2 copy number. TRIAL REGISTRY: Clinicaltrials.gov NCT00756821.


Assuntos
Biomarcadores/metabolismo , Variações do Número de Cópias de DNA/fisiologia , Atrofia Muscular Espinal/metabolismo , Fatores Etários , Análise de Variância , Estudos de Casos e Controles , Estudos Transversais , Variações do Número de Cópias de DNA/genética , Primers do DNA/genética , Feminino , Humanos , Masculino , Atividade Motora/fisiologia , Atrofia Muscular Espinal/genética , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
8.
Cell ; 117(3): 311-21, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-15109492

RESUMO

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.


Assuntos
Endonucleases/química , Endonucleases/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Sequência Consenso , Sequência Conservada , Endonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Células HeLa , Humanos , Íntrons , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos
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