RESUMEN
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.
Asunto(s)
Envejecimiento , Metilación de ADN , Telomerasa , Telomerasa/metabolismo , Telomerasa/genética , Humanos , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Senescencia Celular , Regiones Promotoras Genéticas , ADN Metiltransferasa 3B , Encéfalo/metabolismo , Telómero/metabolismo , Ratones Endogámicos C57BL , Masculino , Factor de Transcripción AP-1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , NeurogénesisRESUMEN
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.
Asunto(s)
Diversidad de Anticuerpos , Bovinos/inmunología , Regiones Determinantes de Complementariedad , Inmunoglobulina G/genética , Inmunoglobulina M/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Cisteína/análisis , Cisteína/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulina G/química , Inmunoglobulina M/química , Ratones , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Células Epiteliales Alveolares , Diabetes Mellitus Tipo 2 , Animales , Ratones , Células Epiteliales Alveolares/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pulmón/metabolismo , Modelos Animales de EnfermedadRESUMEN
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.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Línea Celular , Inhibidores de Cisteína Proteinasa/análisis , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazonas , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Morfolinas/análisis , Morfolinas/farmacología , Pandemias , Pirimidinas , Reproducibilidad de los Resultados , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Triazinas/análisis , Triazinas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
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.
Asunto(s)
Cisteína , Factor 2 Relacionado con NF-E2 , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/metabolismo , Cisteína/metabolismo , Transducción de Señal , Estrés OxidativoRESUMEN
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.
Asunto(s)
Calidad de Vida , Cicatrización de Heridas , Humanos , Animales , Porcinos , Cicatrización de Heridas/fisiología , Piel/lesiones , Queratinocitos/metabolismo , Administración CutáneaRESUMEN
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.
Asunto(s)
Aminoácidos/química , Código Genético , Ingeniería de Proteínas , Aminoacil-ARNt Sintetasas/química , Animales , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , ARN de Transferencia/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
ConspectusChemical synthesis as a tool to control the structure and properties of matter is at the heart of chemistryâfrom the synthesis of fine chemicals and polymers to drugs and solid-state materials. But as the field evolves to tackle larger and larger molecules and molecular complexes, the traditional tools of synthetic chemistry become limiting. In contrast, Mother Nature has developed very different strategies to create the macromolecules and molecular systems that make up the living cell. Our focus has been to ask whether we can use the synthetic strategies and machinery of Mother Nature, together with modern chemical tools, to create new macromolecules, and even whole organisms with properties not existing in nature. One such example involves reprogramming the complex, multicomponent machinery of ribosomal protein synthesis to add new building blocks to the genetic code, overcoming a billion-year constraint on the chemical nature of proteins. This methodology exploits the concept of bioorthogonality to add unique codons, tRNAs and aminoacyl-tRNA synthetases to cells to encode amino acids with physical, chemical and biological properties not found in nature. As a result, we can make precise changes to the structures of proteins, much like those made by chemists to small molecules and beyond those possible by biological approaches alone. This technology has made it possible to probe protein structure and function in vitro and in vivo in ways heretofore not possible, and to make therapeutic proteins with enhanced pharmacology. A second example involves exploiting the molecular diversity of the humoral immune system together with synthetic transition state analogues to make catalytic antibodies, and then expanding this diversity-based strategy (new to chemists at the time) to drug discovery and materials science. This work ushered in a new nature-inspired synthetic strategy in which large libraries of natural or synthetic molecules are designed and then rationally selected or screened for new function, increasing the efficiency by which we can explore chemical space for new physical, chemical and biological properties. A final example is the use of large chemical libraries, robotics and high throughput phenotypic cellular screens to identify small synthetic molecules that can be used to probe and manipulate the complex biology of the cell, exemplified by druglike molecules that control cell fate. This approach provides new insights into complex biology that complements genomic approaches and can lead to new drugs that act by novel mechanisms of action, for example to selectively regenerate tissues. These and other advances have been made possible by using our knowledge of molecular structure and reactivity hand in hand with our understanding of and ability to manipulate the complex machinery of living cells, opening a new frontier in synthesis. This Account overviews the work in my lab and with our collaborators, from our early days to the present, that revolves around this central theme.
RESUMEN
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.
Asunto(s)
Adenilil Ciclasas/metabolismo , Colesterol/metabolismo , AMP Cíclico/metabolismo , Mycobacterium tuberculosis/genética , Animales , Proteínas Bacterianas/metabolismo , Ratones Endogámicos BALB C , Transducción de Señal/fisiología , Activación Transcripcional/fisiologíaRESUMEN
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.
Asunto(s)
Glucólisis , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Animales , Elementos de Respuesta Antioxidante/genética , Arginina/química , Arginina/metabolismo , Línea Celular , Cisteína/química , Cisteína/metabolismo , Citoprotección , Glucólisis/efectos de los fármacos , Humanos , Imidazoles/química , Masculino , Ratones , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/agonistas , Fosfoglicerato Quinasa/antagonistas & inhibidores , Multimerización de Proteína , Piruvaldehído/química , Piruvaldehído/metabolismo , Piruvaldehído/farmacología , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/genética , Transcripción Genética , UbiquitinaciónRESUMEN
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.
Asunto(s)
Pared Celular/química , Bacilos Grampositivos/química , Peptidoglicano/química , Aminoácidos/química , Aminoácidos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/citología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Pared Celular/metabolismo , Bacterias Gramnegativas/química , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismo , Bacilos Grampositivos/citología , Bacilos Grampositivos/crecimiento & desarrollo , Bacilos Grampositivos/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , FenotipoRESUMEN
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.
Asunto(s)
Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Sistemas de Lectura Abierta , Péptidos/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulación de la Expresión Génica , Sitios Genéticos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Ratones , Pan troglodytes , Péptidos/metabolismo , Unión Proteica/efectos de la radiación , Mapeo de Interacción de Proteínas , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de la radiación , Transgenes , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Regiones Promotoras Genéticas/fisiología , Multimerización de Proteína/fisiología , Factores de Transcripción TFII/metabolismo , Activación Transcripcional/fisiología , Línea Celular Tumoral , Humanos , Microscopía de Interferencia , Unión Proteica , ARN Polimerasa II/metabolismo , Eliminación de Secuencia , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anexina A2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Administración Tópica , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/metabolismo , Animales , Anexina A2/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Proteínas Señalizadoras YAPRESUMEN
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.
Asunto(s)
Aminoácidos/genética , Diferenciación Celular/genética , Vectores Genéticos/genética , Células Madre Hematopoyéticas/fisiología , Ingeniería de Proteínas/métodos , Animales , Sangre Fetal/citología , Técnicas de Transferencia de Gen , Código Genético , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Herpesvirus Humano 4/genética , Humanos , Ratones , Ratones Endogámicos NOD , Plásmidos/genética , Cultivo Primario de Células/métodos , Transfección/métodos , Quimera por Trasplante , Trasplante Heterólogo/métodosRESUMEN
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.
Asunto(s)
Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Animales , Células HEK293 , Humanos , Melanoma Experimental/patología , Ratones , Péptidos/química , alfa-MSH/análogos & derivados , alfa-MSH/químicaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Animales , Astrocitos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Femenino , Glioblastoma , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Piroptosis/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
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.