RESUMO
Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.
Assuntos
Disautonomia Familiar , Doenças Neurodegenerativas , Degeneração Retiniana , Camundongos , Animais , Disautonomia Familiar/genética , Cinetina , Marcha Atáxica , Administração OralRESUMO
The concept of using small molecules to therapeutically modulate pre-mRNA splicing was validated with the US Food and Drug Administration (FDA) approval of Evrysdi® (risdiplam) in 2020. Since then, efforts have continued unabated toward the discovery of new splicing-modulating drugs. However, the drug development world has evolved in the 10 years since risdiplam precursors were first identified in high-throughput screening (HTS). Now, new mechanistic insights into RNA-processing pathways and regulatory networks afford increasingly feasible targeted approaches. In this review, organized into classes of biological target, we compile and summarize small molecules discovered, devised, and developed since 2020 to alter pre-mRNA splicing.
Assuntos
Precursores de RNA , Splicing de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Compostos Azo , Pirimidinas , Processamento AlternativoRESUMO
Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.
Assuntos
Aprendizado Profundo , Marcação de Genes/métodos , Splicing de RNA , Animais , Biologia Computacional , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Éxons , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteína 1 Homóloga a MutL/genética , Mutação , Fenetilaminas/administração & dosagem , Piridazinas/administração & dosagem , Esterol Esterase/genética , Transcriptoma , Proteínas tau/genéticaRESUMO
Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.
Assuntos
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Splicing de RNA , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Humanos , Doença de Huntington/metabolismo , Camundongos , Splicing de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacosRESUMO
There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.
Assuntos
Antibacterianos/farmacologia , DNA Topoisomerases Tipo II/química , Descoberta de Drogas , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Sepse/tratamento farmacológico , Inibidores da Topoisomerase II/farmacologia , Animais , Antibacterianos/química , Feminino , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Piridinas/química , Sepse/microbiologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/químicaRESUMO
We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe --> F(5)-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F(5)-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl-aryl or perfluoroaryl-perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 --> F(5)-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by approximately 1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe --> F(5)-Phe mutations offer the possibility of greater tertiary structural stability from side chain-side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.
Assuntos
Flúor/química , Proteínas de Neurofilamentos/química , Fragmentos de Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Animais , Galinhas , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neurofilamentos/genética , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/genética , Fenilalanina/análogos & derivados , Estrutura Secundária de Proteína , Soluções , TermodinâmicaRESUMO
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
The underlying cause of spinal muscular atrophy (SMA) is a deficiency of the survival motor neuron (SMN) protein. Starting from hits identified in a high-throughput screening campaign and through structure-activity relationship investigations, we have developed small molecules that potently shift the alternative splicing of the SMN2 exon 7, resulting in increased production of the full-length SMN mRNA and protein. Three novel chemical series, represented by compounds 9, 14, and 20, have been optimized to increase the level of SMN protein by >50% in SMA patient-derived fibroblasts at concentrations of <160 nM. Daily administration of these compounds to severe SMA Δ7 mice results in an increased production of SMN protein in disease-relevant tissues and a significant increase in median survival time in a dose-dependent manner. Our work supports the development of an orally administered small molecule for the treatment of patients with SMA.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Atrofia Muscular Espinal/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Descoberta de Drogas , Éxons/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos Knockout , Atrofia Muscular Espinal/genética , RNA Mensageiro/genética , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
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/metabolismoRESUMO
Cationic organic intermediates participate in a wide variety of useful synthetic transformations, but their high reactivity can render selectivity in competing pathways difficult to control. Here, we describe a strategy for inducing enantioselectivity in reactions of protio-iminium ions, wherein a chiral catalyst interacts with the highly reactive intermediate through a network of noncovalent interactions. This interaction leads to an attenuation of the reactivity of the iminium ion and allows high enantioselectivity in cycloadditions with electron-rich alkenes (the Povarov reaction). A detailed experimental and computational analysis of this catalyst system has revealed the precise nature of the catalyst-substrate interactions and the likely basis for enantioinduction.
Assuntos
Ácidos/química , Alcenos/química , Benzenossulfonatos/química , Catálise , Iminas/química , Ureia/análogos & derivados , Ureia/química , Fenômenos Químicos , Espectroscopia de Ressonância Magnética , Prótons , Estereoisomerismo , Tioureia/análogos & derivados , Tioureia/químicaRESUMO
We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.
Assuntos
Mutação/genética , Fenilalanina/metabolismo , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Fenilalanina/genética , Estrutura Terciária de Proteína , Proteínas/genética , Alinhamento de SequênciaRESUMO
An amide bond has been replaced by a thioester in bovine pancreatic polypeptide (bPP) to allow rapid and reversible (dynamic) exchange of the alpha-helical segment with other thiols in solution. We have begun to study the higher order structural stability of bPP by measuring the equilibrium constant of the "backbone thioester exchange" (BTE) reaction. The extent to which the equilibrium (KBTE) favors one set of peptides over the other, which can be easily measured, can be directly correlated to the energy gained from favorable noncovalent interactions that occur between peptide segments on either side of the thioester bond (Kfold).
Assuntos
Polipeptídeo Pancreático/química , Sequência de Aminoácidos , Animais , Bovinos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Termodinâmica , Tirosina/químicaRESUMO
A stereoselective synthetic route is reported for the introduction of side chains at the 3-position of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically defined fashion, which should facilitate the design of beta-peptides for biological applications.