RESUMO
Nervous system disorders affect millions of people around the world, through a very broad range of diseases. Here we describe our contribution to find a treatment for patients suffering from three of those diseases. The first one, autism spectrum disorder (ASD), affects approximately one in every 59 children in US. The second one, spinal muscular atrophy (SMA) is a rare disease affecting 1 in 10000 live births worldwide, often leading to death if untreated. The third one, Alzheimer's disease (AD) is a very well known devastating disease with an estimated 50 million people living with AD and other dementia, a number expected to triple by 2050. Our strategy to address those diseases was directed towards the discovery of a selective vasopressin 1a (V1a) antagonist for ASD, a splicing modifier of the survival of motor neuron 2 (SMN2) for SMA, and finally a γ-secretase modulator (GSM) for AD. In the frame of our GSM project, we also reported the discovery of a bridge piperidine moiety as a bioisostere for a phenyl moiety with an improved drug-like profile.
Assuntos
Transtorno do Espectro Autista , Atrofia Muscular Espinal , Doenças Neurodegenerativas , Transtorno do Espectro Autista/tratamento farmacológico , Criança , Humanos , Atrofia Muscular Espinal/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológicoRESUMO
Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.
Assuntos
Atrofia Muscular Espinal , Processamento Alternativo , Animais , Modelos Animais de Doenças , Progressão da Doença , Éxons , Feminino , Humanos , Masculino , Camundongos , Atrofia Muscular Espinal/metabolismo , Splicing de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
Not too long ago, the concept of selectively targeting mRNA with small molecules was perceived as a formidable scientific challenge. The discovery of small molecule splicing modifiers and the development of risdiplam for the treatment of spinal muscular atrophy (SMA) have firmly established proof of concept for this exciting new platform and transformed a scientific curiosity into a viable technology to target disease. Today, several approaches to target mRNA with small molecules, supported by biophysical and screening methods, are in place to deliver new drugs with high therapeutic relevance.
RESUMO
γ-Secretase (GS) is a key target for the potential treatment of Alzheimer's disease. While inhibiting GS led to serious side effects, its modulation holds a lot of potential to deliver a safe treatment. Herein, we report the discovery of a potent and selective gamma secretase modulator (GSM) (S)-3 (RO7185876), belonging to a novel chemical class, the triazolo-azepines. This compound demonstrates an excellent in vitro and in vivo DMPK profile. Furthermore, based on its in vivo efficacy in a pharmacodynamic mouse model and the outcome of the dose range finding (DRF) toxicological studies in two species, this compound was selected to undergo entry in human enabling studies (e.g., GLP toxicology and scale up activities).
RESUMO
We recently reported the discovery of a potent, selective, and brain-penetrant V1a receptor antagonist, which was not suitable for full development. Nevertheless, this compound was found to improve surrogates of social behavior in adults with autism spectrum disorder in an exploratory proof-of-mechanism study. Here we describe scaffold hopping that gave rise to triazolobenzodiazepines with improved pharmacokinetic properties. The key to balancing potency and selectivity while minimizing P-gp mediated efflux was fine-tuning of hydrogen bond acceptor basicity. Ascertaining a V1a antagonist specific brain activity pattern by pharmacological magnetic resonance imaging in the rat played a seminal role in guiding optimization efforts, culminating in the discovery of balovaptan (RG7314, RO5285119) 1. In a 12-week clinical phase 2 study in adults with autism spectrum disorder balovaptan demonstrated improvements in Vineland-II Adaptive Behavior Scales, a secondary end point comprising communication, socialization, and daily living skills. Balovaptan entered phase 3 clinical development in August 2018.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Transtorno do Espectro Autista/tratamento farmacológico , Benzodiazepinas/uso terapêutico , Piridinas/uso terapêutico , Receptores de Vasopressinas/metabolismo , Triazóis/uso terapêutico , Adolescente , Adulto , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/síntese química , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacocinética , Transtorno do Espectro Autista/metabolismo , Benzodiazepinas/síntese química , Benzodiazepinas/farmacocinética , Encéfalo/metabolismo , Criança , Ensaios Clínicos como Assunto , Descoberta de Drogas , Feminino , Humanos , Masculino , Mamíferos , Piridinas/síntese química , Piridinas/farmacocinética , Triazóis/síntese química , Triazóis/farmacocinéticaRESUMO
Splicing modifiers promoting SMN2 exon 7 inclusion have the potential to treat spinal muscular atrophy, the leading genetic cause of infantile death. These small molecules are SMN2 exon 7 selective and act during the early stages of spliceosome assembly. Here, we show at atomic resolution how the drug selectively promotes the recognition of the weak 5' splice site of SMN2 exon 7 by U1 snRNP. The solution structure of the RNA duplex formed following 5' splice site recognition in the presence of the splicing modifier revealed that the drug specifically stabilizes a bulged adenine at this exon-intron junction and converts the weak 5' splice site of SMN2 exon 7 into a stronger one. The small molecule acts as a specific splicing enhancer cooperatively with the splicing regulatory network. Our investigations uncovered a novel concept for gene-specific alternative splicing correction that we coined 5' splice site bulge repair.
Assuntos
Splicing de RNA , RNA/química , Conformação Molecular , Atrofia Muscular Espinal/metabolismo , Ribonucleoproteína Nuclear Pequena U1/químicaRESUMO
Targeting RNA drastically expands our target space to therapeutically modulate numerous cellular processes implicated in human diseases. Of particular interest, drugging pre-mRNA splicing appears a very viable strategy; to control levels of splicing product by promoting the inclusion or exclusion of exons. After describing the concept of "splicing modulation", this chapter will cover the outstanding progress achieved in this field, by highlighting the breakthrough accomplished recently for the treatment of spinal muscular atrophy using two therapeutic modalities: splice switching oligonucleotides and small molecules. This review discusses the vital but feasible requirement for such drugs to deliver selectivity, and critical safety aspects are highlighted. Transformational medicines such as those developed to treat SMA are likely just the beginning of this story.
Assuntos
Atrofia Muscular Espinal/patologia , Compostos Azo/química , Compostos Azo/uso terapêutico , Descoberta de Drogas , Fluorbenzenos/química , Fluorbenzenos/uso terapêutico , Humanos , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Splicing de RNA , Proteínas do Complexo SMN/genética , Proteínas do Complexo SMN/metabolismoRESUMO
AIMS: Risdiplam (RG7916, RO7034067) is an orally administered, centrally and peripherally distributed, survival of motor neuron 2 (SMN2) mRNA splicing modifier for the treatment of spinal muscular atrophy (SMA). The objectives of this entry-into-human study were to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of risdiplam, and the effect of the strong CYP3A inhibitor itraconazole on the PK of risdiplam in healthy male volunteers. METHODS: Part 1 had a randomized, double-blind, adaptive design with 25 subjects receiving single ascending oral doses of risdiplam (ranging from 0.6-18.0 mg, n = 18) or placebo (n = 7). A Bayesian framework was applied to estimate risdiplam's effect on SMN2 mRNA. The effect of multiple doses of itraconazole on the PK of risdiplam was also assessed using a two-period cross-over design (n = 8). RESULTS: Risdiplam in the fasted or fed state was well tolerated. Risdiplam exhibited linear PK over the dose range with a multi-phasic decline with a mean terminal half-life of 40-69 h. Food had no relevant effect, and itraconazole had only a minor effect on plasma PK indicating a low fraction of risdiplam metabolized by CYP3A. The highest tested dose of 18.0 mg risdiplam led to approximately 41% (95% confidence interval 27-55%) of the estimated maximum increase in SMN2 mRNA. CONCLUSIONS: Risdiplam was well tolerated and proof of mechanism was demonstrated by the intended shift in SMN2 splicing towards full-length SMN2 mRNA. Based on these data, Phase 2/3 studies of risdiplam in patients with SMA are now ongoing.
Assuntos
Compostos Azo/administração & dosagem , Fármacos Neuromusculares/administração & dosagem , Pirimidinas/administração & dosagem , Splicing de RNA/efeitos dos fármacos , Administração Oral , Adolescente , Adulto , Área Sob a Curva , Compostos Azo/efeitos adversos , Compostos Azo/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Método Duplo-Cego , Interações Medicamentosas , Voluntários Saudáveis , Humanos , Itraconazol/farmacocinética , Masculino , Pessoa de Meia-Idade , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Fármacos Neuromusculares/efeitos adversos , Fármacos Neuromusculares/farmacocinética , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética , RNA Mensageiro/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Adulto JovemRESUMO
Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.
Assuntos
Compostos Azo/farmacocinética , Atrofia Muscular Espinal/tratamento farmacológico , Fármacos Neuromusculares/farmacocinética , Pirimidinas/farmacocinética , Splicing de RNA/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Compostos Azo/líquido cefalorraquidiano , Compostos Azo/farmacologia , Compostos Azo/uso terapêutico , Encéfalo/metabolismo , Encéfalo/patologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Cães , Avaliação Pré-Clínica de Medicamentos , Éxons/efeitos dos fármacos , Éxons/genética , Feminino , Humanos , Macaca fascicularis , Células Madin Darby de Rim Canino , Masculino , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Fármacos Neuromusculares/líquido cefalorraquidiano , Fármacos Neuromusculares/farmacologia , Fármacos Neuromusculares/uso terapêutico , Pirimidinas/líquido cefalorraquidiano , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Ratos Wistar , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Suínos , Distribuição TecidualRESUMO
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 , SegurançaRESUMO
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
Assuntos
Vias Biossintéticas/genética , Evolução Molecular , Variação Genética , Família Multigênica , Bioengenharia , Policetídeo Sintases/genética , Sirolimo/química , Sirolimo/metabolismoRESUMO
Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.
Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Piperazinas/farmacologia , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Biflavonoides/farmacologia , Sistema Livre de Células , Biologia Computacional , Compostos de Epóxi/farmacologia , Éxons/genética , Fibroblastos , Células HEK293 , Células HeLa , Humanos , Ligantes , Macrolídeos/farmacologia , Atrofia Muscular Espinal/genética , Piperazinas/síntese química , Ligação Proteica , Estrutura Quaternária de Proteína , Proteômica/métodos , Precursores de RNA/genética , RNA Mensageiro/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
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 farmacológicoRESUMO
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 terapêuticoRESUMO
Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a â¼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.
Assuntos
Isocumarinas/administração & dosagem , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Piperazinas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacocinética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons/genética , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/sangue , Atrofia Muscular Espinal/patologia , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Pele/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Proteína 2 de Sobrevivência do Neurônio Motor/sangueRESUMO
Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.
Assuntos
Folistatina/farmacologia , Fatores Imunológicos/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Splicing de RNA/efeitos dos fármacos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/agonistas , Adolescente , Adulto , Idade de Início , Animais , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Miostatina/antagonistas & inibidores , Miostatina/genética , Miostatina/metabolismo , Fenótipo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Encéfalo/metabolismo , Genômica/métodos , Indóis/farmacologia , Prurido/tratamento farmacológico , Receptores de Vasopressinas/metabolismo , Compostos de Espiro/farmacologia , Vasopressinas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/química , Transtorno Autístico/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Indóis/química , Masculino , Camundongos , Estrutura Molecular , Prurido/induzido quimicamente , Receptores de Vasopressinas/química , Receptores de Vasopressinas/genética , Compostos de Espiro/química , Vasoconstritores/metabolismoRESUMO
The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3.
Assuntos
Carbamatos/farmacologia , Neurotransmissores/farmacologia , Piperidinas/farmacologia , Receptores da Neurocinina-3/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antipsicóticos/farmacologia , Sítios de Ligação , Carbamatos/farmacocinética , Fármacos do Sistema Nervoso Central/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/fisiologia , Feminino , Gerbillinae , Cobaias , Células HEK293 , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neurotransmissores/farmacocinética , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/fisiologia , Fragmentos de Peptídeos/farmacologia , Piperidinas/farmacocinética , Receptores da Neurocinina-2/antagonistas & inibidores , Receptores da Neurocinina-2/metabolismo , Receptores da Neurocinina-3/agonistas , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/metabolismo , Substância P/análogos & derivados , Substância P/farmacologia , Técnicas de Cultura de TecidosRESUMO
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.