RESUMEN
Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.
Asunto(s)
Proteínas que Contienen Bromodominio , Quelantes , Células Secretoras de Insulina , Zinc , Animales , Humanos , Masculino , Ratones , Azepinas/farmacología , Azepinas/química , Proteínas que Contienen Bromodominio/antagonistas & inhibidores , Proteínas que Contienen Bromodominio/química , Quelantes/farmacología , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos C57BL , Proteínas Nucleares , Factores de Transcripción/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Triazoles/química , Zinc/química , Zinc/farmacología , Zinc/metabolismoRESUMEN
The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, â¼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.
Asunto(s)
Proteínas de Unión al ADN , Mutación Missense , Neoplasias , Dominios Proteicos , Factores de Transcripción , Humanos , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Ligandos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.
Asunto(s)
Descubrimiento de Drogas , Proteínas , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Descubrimiento de Drogas/métodos , Espectroscopía de Resonancia Magnética , Sitios de Unión , LigandosRESUMEN
PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 µM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.
Asunto(s)
Histonas , Neoplasias de la Próstata , Masculino , Humanos , Histonas/metabolismo , Dominios Proteicos , Cromatina , Neoplasias de la Próstata/tratamiento farmacológico , Carcinógenos , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Durante un período de dos meses se realizó un estudio prospectivo, longitudinal y observacional, de 1,032 venoclisis instaladas en 342 niños internados en cuatro servicios generales de un hospital pediátrico. En 903 venoclisis se utilizó aguja de mariposa de acero inoxidable y en 129, cateteres de teflón. Las principales complicaciones observadas fueron flebitis (55.2%), cuerda residual (28.5%), infiltración (28.1%) y obstrucción (19.5%). Doce casos (1.2%) presentaron necrosis dérmica y dos (0.2%) flebitis supurada. Se observó incremento del riesgo de flebitis a mayor tiempo de permanencia de la venoclisis, así como mayor duración de la fase aguda de la inflamación venosa, cuando ocurrió retraso en el retiro de la cánula instalada. Se evidenció así mismo mayor riesgo de flebitis en pacientes infectados, con dermatosis, en aquellos que recibieron dicloxacilina y con venoclisis instaladas en el pie