RESUMEN
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.
Asunto(s)
Muerte Celular Inmunogénica , Proteolisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Transducción de Señal , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Humanos , Animales , Ratones , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de los fármacos , Necroptosis/efectos de los fármacos , Necroptosis/inmunología , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Ratones Endogámicos C57BL , Antineoplásicos/farmacología , Inmunoterapia/métodosRESUMEN
Small molecules that induce protein degradation hold the potential to overcome several limitations of the currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activityâhow can we convert an inhibitor into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, we demonstrate that an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are tolerated, including both aromatic and nonaromatic groups. We used these findings to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and confirm the mechanism of degradation. These results suggest that general design principles can be generated for the development and optimization of monovalent degraders.
Asunto(s)
Ciclinas , Proteolisis , Puntos de Control del Ciclo Celular , Ciclinas/metabolismoRESUMEN
High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low µM to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Ligandos , Reproducibilidad de los Resultados , Espectroscopía de Protones por Resonancia Magnética , Bibliotecas de Moléculas Pequeñas/química , Unión ProteicaRESUMEN
B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.
Asunto(s)
Linfoma de Células B Grandes Difuso , Quinolonas , Animales , Humanos , Ratones , Línea Celular Tumoral , Modelos Animales de Enfermedad , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-6/química , Factores de TranscripciónRESUMEN
By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.
Asunto(s)
Linfoma de Células B Grandes Difuso , Humanos , Cristalografía por Rayos X , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Linfoma de Células B Grandes Difuso/patología , Diseño de Fármacos , LigandosRESUMEN
The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.
Asunto(s)
Carcinogénesis , Regulación Neoplásica de la Expresión Génica , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas c-bcl-6/metabolismoRESUMEN
To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.
Asunto(s)
Dominio BTB-POZ , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-6RESUMEN
We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Antineoplásicos/química , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Solubilidad , Relación Estructura-ActividadRESUMEN
Using a novel physiologically relevant in vitro human whole blood neutrophil shape change assay, an aminopyrazine series of selective PI3Kγ inhibitors was identified and prioritized for further optimization. Severe solubility limitations associated with the series leading to low oral bioavailability and poor exposures, especially at higher doses, were overcome by moving to an aminopyridine core. Compound 33, with the optimal balance of on-target activity, selectivity, and pharmacokinetic parameters, progressed into in vivo studies and demonstrated good efficacy (10 mg/kg) in a rat model of airway inflammation. Sufficient exposures were achieved at high doses to support toxicological studies, where unexpected inflammatory cell infiltrates in cardiovascular tissue prevented further compound development.
Asunto(s)
Aminopiridinas/uso terapéutico , Antiinflamatorios/uso terapéutico , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inflamación/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Aminopiridinas/toxicidad , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Antiinflamatorios/toxicidad , Femenino , Humanos , Estructura Molecular , Nivel sin Efectos Adversos Observados , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/toxicidad , Pirazinas/síntesis química , Pirazinas/farmacocinética , Pirazinas/uso terapéutico , Pirazinas/toxicidad , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.
Asunto(s)
Bencimidazoles/administración & dosificación , Bencimidazoles/química , Sistemas de Liberación de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Concomitant inhibition of anaplastic lymphoma kinase (ALK) and bromodomain-4 (BRD4) is a potential therapeutic strategy for targeting two key oncogenic drivers that co-segregate in a significant fraction of high-risk neuroblastoma patients, mutation of ALK and amplification of MYCN. Starting from known dual polo-like kinase (PLK)-1-BRD4 inhibitor BI-2536, we employed structure-based design to redesign this series toward compounds with a dual ALK-BRD4 profile. These efforts led to compound ( R)-2-((2-ethoxy-4-(1-methylpiperidin-4-yl)phenyl)amino)-7-ethyl-5-methyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5 H)-one (16k) demonstrating improved ALK activity and significantly reduced PLK-1 activity, while maintaining BRD4 activity and overall kinome selectivity. We demonstrate the compounds' on-target engagement with ALK and BRD4 in cells as well as favorable broad kinase and bromodomain selectivity.
Asunto(s)
Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
As part of an on-going lead optimisation effort, a cross screening exercise identified an aryl sulphonyl amide hit that was optimised to afford a highly potent series of ghrelin receptor agonists.
Asunto(s)
Química Farmacéutica/métodos , Ghrelina/química , Receptores de Ghrelina/antagonistas & inhibidores , Sulfonas/química , Administración Oral , Animales , Disponibilidad Biológica , Diseño de Fármacos , Hormona del Crecimiento/química , Masculino , Modelos Químicos , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Receptores de Ghrelina/química , Relación Estructura-ActividadRESUMEN
The optimisation of a tertiary sulfonamide high-throughput screening hit is described. A combination of high-throughput chemistry, pharmacophore analysis and in silico PK profiling resulted in the discovery of potent sulfonamide oxytocin receptor antagonists with oral exposure and good selectivity over vasopressin receptors.
Asunto(s)
Descubrimiento de Drogas , Oxitocina/antagonistas & inhibidores , Sulfonamidas/farmacología , Administración Oral , Modelos Moleculares , Sulfonamidas/administración & dosificación , Sulfonamidas/químicaRESUMEN
A novel oxytocin antagonist was identified by 'scaffold-hopping' using Cresset FieldScreen molecular field similarity searching. A single cycle of optimization driven by an understanding of the key pharmacophoric elements required for activity led to the discovery of a potent, selective and highly ligand-efficient oxytocin receptor antagonist. Selectivity over vasopressin receptors was rationalized based on differences in the structure of the natural ligands.
Asunto(s)
Diseño de Fármacos , Oxitocina/química , Receptores de Oxitocina/antagonistas & inhibidores , Receptores de Vasopresinas/química , Vasopresinas/química , Química Farmacéutica/métodos , Femenino , Humanos , Cinética , Ligandos , Modelos Químicos , Conformación Molecular , Trabajo de Parto Prematuro/tratamiento farmacológico , Embarazo , Vasotocina/análogos & derivados , Vasotocina/químicaRESUMEN
[structure: see text] We report the first theoretical studies on the asymmetric sulfonium ylide epoxidation reaction using a chiral sulfide that successfully reproduces the experimentally determined high enantiomeric excess. Calculations at the DFT level suggest that the transition states for the addition of the sulfonium ylide to benzaldehyde have energies which account for the observed enantioselectivity.
RESUMEN
Using a D-mannitol derived chiral sulfide, terminal epoxides are formed in up to 76% ee; the first example of double asymmetric induction in a sulfonium methylide epoxidation is reported and an improved method of generating sulfonium ylides is detailed.