RESUMEN
Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Pirofosfatasas , Humanos , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/metabolismo , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Relación Estructura-Actividad , Cristalografía por Rayos X , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Pirazoles/metabolismo , Piperidinas/farmacología , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/síntesis química , Descubrimiento de Drogas , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Adenina/análogos & derivados , Adenina/química , Adenina/farmacología , Adenina/metabolismo , Modelos Moleculares , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis químicaRESUMEN
Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme's substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.
RESUMEN
Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.
Asunto(s)
Cordoma/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Proteínas Fetales/genética , Histona Demetilasas/antagonistas & inhibidores , Proteínas de Dominio T Box/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cordoma/genética , Cordoma/patología , Cromatina/genética , Cromatina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Epigénesis Genética , Proteínas Fetales/metabolismo , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Terapia Molecular Dirigida , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Dominio T Box/metabolismoRESUMEN
Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Electrones , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Peso Molecular , Conformación Proteica , Factores de TiempoRESUMEN
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas F-Box/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
An efficient and convenient solution-phase synthesis of a 1H-1,2,4-triazole library with potential agrochemical activity is reported employing microwave-assisted organic synthesis (MAOS) and continuous-flow microfluidic synthetic methods starting from commercially available 3,5-dibromo-1H-1,2,4-triazole (1). MAOS was used for the synthesis of 5-amino-3-bromo-1,2,4-triazole analogs 3 and for their 3-aryl derivatives 4 via Suzuki-Miyaura coupling with polymer-supported catalyst. A microfluidic hydrogenation reactor integrated into an automated parallel synthesis platform was built and utilized for the reductive dehalogenation reactions providing 5-aminotriazoles (5).
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Microfluídica/métodos , Microondas , Reología , Triazoles/química , Triazoles/síntesis química , Aminas/química , Bromuros/química , Espectroscopía de Resonancia Magnética , Peso Molecular , Solventes/químicaRESUMEN
The authors describe an innovative approach for designing novel inhibitors. This approach effectively integrates the emerging chemogenomics concept of target-family-based drug discovery with bioanalogous design strategies, including privileged structures, molecular frameworks as well as bioisosteric and bioanalogous/isofunctional modifications. The authors applied this method in the design of selective inhibitors of matrix metalloproteases (MMPs), also referred to as matrixins, on the basis of a unique analysis of the ligand-target knowledge base, the 'matrixinome'. For this analysis, the authors created an annotated MMP database containing â¼ 300 inhibitors with their published activity profile. The ligand space was then arranged into a lead evolution tree, where the substructural transformations in each virtual step led to marked changes in the activity pattern. This allowed subtype-specific privileged fragments to be extracted as well as modifications, which improve activity and/or selectivity. Furthermore, the compounds with the preferred activity profile were correlated with sequence homology as well as binding site similarity within the target family, thereby leading to the identification of substructural modifications that turn non-selective, biohomologous structures into selective inhibitors. The matrixinomic application of the authors' approach, therefore, provides an example of how the combination of ligand space knowledge with sequence-related data can radically improve the outcome of the lead optimisation process to achieve higher selectivity within a given target family.