RESUMO
Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.
Assuntos
Envelhecimento , Metilação de DNA , Telomerase , Telomerase/metabolismo , Telomerase/genética , Humanos , Animais , Camundongos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Senescência Celular , Regiões Promotoras Genéticas , DNA Metiltransferase 3B , Encéfalo/metabolismo , Telômero/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Fator de Transcrição AP-1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , NeurogêneseRESUMO
Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ß strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.
Assuntos
Diversidade de Anticorpos , Bovinos/imunologia , Regiões Determinantes de Complementaridade , Imunoglobulina G/genética , Imunoglobulina M/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Cisteína/análise , Cisteína/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Camundongos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.
Assuntos
Células Epiteliais Alveolares , Diabetes Mellitus Tipo 2 , Animais , Camundongos , Células Epiteliais Alveolares/metabolismo , Dipeptidil Peptidase 4/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pulmão/metabolismo , Modelos Animais de DoençasRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.
Assuntos
Antivirais/análise , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , COVID-19 , Linhagem Celular , Inibidores de Cisteína Proteinase/análise , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Morfolinas/análise , Morfolinas/farmacologia , Pandemias , Pirimidinas , Reprodutibilidade dos Testes , SARS-CoV-2 , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Triazinas/análise , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.
Assuntos
Cisteína , Fator 2 Relacionado a NF-E2 , Proteína 1 Associada a ECH Semelhante a Kelch/química , Fator 2 Relacionado a NF-E2/metabolismo , Cisteína/metabolismo , Transdução de Sinais , Estresse OxidativoRESUMO
Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.
Assuntos
Qualidade de Vida , Cicatrização , Humanos , Animais , Suínos , Cicatrização/fisiologia , Pele/lesões , Queratinócitos/metabolismo , Administração CutâneaRESUMO
The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.
Assuntos
Aminoácidos/química , Código Genético , Engenharia de Proteínas , Aminoacil-tRNA Sintetases/química , Animais , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , RNA de Transferência/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.
Assuntos
Adenilil Ciclases/metabolismo , Colesterol/metabolismo , AMP Cíclico/metabolismo , Mycobacterium tuberculosis/genética , Animais , Proteínas de Bactérias/metabolismo , Camundongos Endogâmicos BALB C , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologiaRESUMO
Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.
Assuntos
Glicólise , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Elementos de Resposta Antioxidante/genética , Arginina/química , Arginina/metabolismo , Linhagem Celular , Cisteína/química , Cisteína/metabolismo , Citoproteção , Glicólise/efeitos dos fármacos , Humanos , Imidazóis/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/agonistas , Fosfoglicerato Quinase/antagonistas & inibidores , Multimerização Proteica , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica , UbiquitinaçãoRESUMO
Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.
Assuntos
Parede Celular/química , Bacilos Gram-Positivos/química , Peptidoglicano/química , Aminoácidos/química , Aminoácidos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Bacilos Gram-Positivos/citologia , Bacilos Gram-Positivos/crescimento & desenvolvimento , Bacilos Gram-Positivos/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , FenótipoRESUMO
Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
Assuntos
Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Fases de Leitura Aberta , Peptídeos/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulação da Expressão Gênica , Loci Gênicos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Camundongos , Pan troglodytes , Peptídeos/metabolismo , Ligação Proteica/efeitos da radiação , Mapeamento de Interação de Proteínas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos da radiação , Transgenes , Raios UltravioletaRESUMO
Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Multimerização Proteica/fisiologia , Fatores de Transcrição TFII/metabolismo , Ativação Transcricional/fisiologia , Linhagem Celular Tumoral , Humanos , Microscopia de Interferência , Ligação Proteica , RNA Polimerase II/metabolismo , Deleção de Sequência , Fatores de TempoRESUMO
The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anexina A2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/metabolismo , Administração Tópica , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Anexina A2/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Proteínas de Sinalização YAPRESUMO
The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.
Assuntos
Aminoácidos/genética , Diferenciação Celular/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/fisiologia , Engenharia de Proteínas/métodos , Animais , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Código Genético , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Plasmídeos/genética , Cultura Primária de Células/métodos , Transfecção/métodos , Quimeras de Transplante , Transplante Heterólogo/métodosRESUMO
To direct checkpoint inhibition to the tumor microenvironment, while avoiding systemic immune activation, we have synthesized a bispecific antibody [norleucine4, d-Phe7]-melanocyte stimulating hormone (NDP-MSH)-antiprogrammed cell death-ligand 1 antibody (αPD-L1) by conjugating a melanocyte stimulating hormone (α-MSH) analog to the antiprogrammed cell death-ligand 1 to (αPD-L1) antibody avelumab. This bispecific antibody can bind to both the melanocortin-1 receptor (MC1R) and to PD-L1 expressed on melanoma cells and shows enhanced specific antitumor efficacy in a syngeneic B16-SIY melanoma mouse model compared with the parental antibody at a 5 mg/kg dose. Moreover, the bispecific antibody showed increased infiltrated T cells in the tumor microenvironment. These results suggest that a tumor-targeted PD-L1-blocking bispecific antibody could have a therapeutic advantage in vivo, especially when used in combination with other checkpoint inhibitors.
Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Células HEK293 , Humanos , Melanoma Experimental/patologia , Camundongos , Peptídeos/química , alfa-MSH/análogos & derivados , alfa-MSH/químicaRESUMO
Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Animais , Astrócitos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Glioblastoma , Xenoenxertos , Ensaios de Triagem em Larga Escala , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.
Assuntos
Antioxidantes/farmacologia , Descoberta de Drogas , Tiofenos/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3-SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.