RESUMEN
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Asunto(s)
Antineoplásicos , Descubrimiento de Drogas , Inhibidores Enzimáticos , Inestabilidad de Microsatélites , Neoplasias , Mutaciones Letales Sintéticas , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Ratones , Administración Oral , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Dominios Proteicos , Reproducibilidad de los Resultados , Supresión Genética , Mutaciones Letales Sintéticas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.
Asunto(s)
Interleucina-17/inmunología , Multimerización de Proteína/inmunología , Receptores de Interleucina-17/inmunología , Transducción de Señal/inmunología , Unión Competitiva , Cristalografía por Rayos X , Células HEK293 , Humanos , Interleucina-17/química , Interleucina-17/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de Interleucina-17/química , Receptores de Interleucina-17/metabolismoRESUMEN
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
Asunto(s)
Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patologíaRESUMEN
MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.
Asunto(s)
Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Regulación de la Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Linfocitos/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/ultraestructura , FN-kappa B/metabolismo , Proteínas de Neoplasias , Transducción de SeñalRESUMEN
Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.
Asunto(s)
Aminopiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Peroxidasa/antagonistas & inhibidores , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Disponibilidad Biológica , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.
Asunto(s)
Antineoplásicos/farmacología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Células Jurkat , Microsomas/efectos de los fármacos , Estructura Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Piperidinas/síntesis química , Piperidinas/química , Proteolisis/efectos de los fármacos , Ratas , Relación Estructura-ActividadRESUMEN
Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarinâ A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarinâ A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarinâ A binds a dimeric PfAp3Aase and prevents the formation of the enzymeâ substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarinâ A in Mycobacterium tuberculosis. Thus, cyclomarinâ A is a rare example of a natural product with two distinct and specific modes of action.
Asunto(s)
Productos Biológicos/química , Oligopéptidos/química , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.
Asunto(s)
ADN Helicasas , Tiadiazoles , Urea , ADN Helicasas/metabolismo , Helicasa del Síndrome de Werner/metabolismoRESUMEN
Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.
Asunto(s)
Interleucina-1beta , Humanos , Descubrimiento de Drogas , Interleucina-1beta/metabolismo , Ligandos , Receptores Tipo I de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , ADN/química , Biblioteca de GenesRESUMEN
WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes. It is also a prominent target for pharmacological inhibition in diseases such as cancer, aging, and neurodegenerative disorders. Interactions between WDR5 and various partners are essential for sustaining its function. Most drug discovery efforts center on the WIN (WDR5 interaction motif) site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM (WDR5 binding motif) pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization, and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Neoplasias , Humanos , Unión Proteica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cromatina , Descubrimiento de DrogasRESUMEN
Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.
Asunto(s)
Citocinas , Delgadez , Humanos , Interleucina-1beta , Peso Molecular , Sitios de Unión , BiofisicaRESUMEN
Inappropriate activation of TLR7 and TLR8 is linked to several autoimmune diseases, such as lupus erythematosus. Here we report on the efficient structure-based optimization of the inhibition of TLR8, starting from a co-crystal structure of a small screening hit. Further optimization of the physicochemical properties for cellular potency and expansion of the structure-activity relationship for dual potency finally resulted in a highly potent TLR7/8 antagonist with demonstrated in vivo efficacy after oral dosing.
RESUMEN
MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.
Asunto(s)
Inhibidores de Caspasas/uso terapéutico , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Urea/análogos & derivados , Urea/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/farmacología , Descubrimiento de Drogas , Femenino , Humanos , Inmunidad Humoral/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Urea/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This article summarizes the evolution of the screening deck at the Novartis Institutes for BioMedical Research (NIBR). Historically, the screening deck was an assembly of all available compounds. In 2015, we designed a first deck to facilitate access to diverse subsets with optimized properties. We allocated the compounds as plated subsets on a 2D grid with property based ranking in one dimension and increasing structural redundancy in the other. The learnings from the 2015 screening deck were applied to the design of a next generation in 2019. We found that using traditional leadlikeness criteria (mainly MW, clogP) reduces the hit rates of attractive chemical starting points in subset screening. Consequently, the 2019 deck relies on solubility and permeability to select preferred compounds. The 2019 design also uses NIBR's experimental assay data and inferred biological activity profiles in addition to structural diversity to define redundancy across the compound sets.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Asunto(s)
Lupus Eritematoso Sistémico/tratamiento farmacológico , Piridonas/química , Piridonas/farmacología , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Animales , Células Cultivadas , Descubrimiento de Drogas , Humanos , Indazoles/química , Indazoles/farmacocinética , Indazoles/farmacología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Piridonas/farmacocinética , Ratas Sprague-Dawley , Receptor Toll-Like 7/química , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/química , Receptor Toll-Like 8/metabolismoRESUMEN
FGF19 signaling through the FGFR4/ß-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.
Asunto(s)
Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisteína/química , Perros , Diseño de Fármacos , Semivida , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the FGF19/FGFR4 axis has been implicated in HCC. Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3, and all other kinases in the kinome. FGF401 has excellent drug-like properties showing a robust pharmacokinetic/pharmacodynamics/efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has remarkable antitumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft models that are positive for FGF19, FGFR4, and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a phase I/II study is currently ongoing in HCC and other solid malignancies.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Transducción de SeñalRESUMEN
A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addition, the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.
RESUMEN
This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.
Asunto(s)
Polarización de Fluorescencia/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Quinasas/análisis , Bioensayo/métodos , Evaluación Preclínica de Medicamentos , Tamaño de la Partícula , Péptidos/química , Especificidad por SustratoRESUMEN
Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 A resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 A, alpha = beta = 90, gamma = 120 degrees and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.