RESUMEN
For a long time, the development of bromodomain (BD) inhibitors (BDi) was almost exclusively related to the BET family. More recently, BDi for BDs outside the BET family have also been developed. Here we present a novel pan-BDi with micromolar affinities to various BDs, and nanomolar affinities to representatives of BD families I, II (Bromodomain and Extra-Terminal Domain (BET) family), III, and IV. The inhibitor shows a broad activity profile with nanomolar growth inhibition (GI50) values on various cancer cell lines. Subsequently, we were able to control the selectivity of the inhibitor by simple modifications and turned it into a highly selective BRD9 inhibitor.
Asunto(s)
Diseño de Fármacos , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Dominios Proteicos , Línea Celular , Epigénesis GenéticaRESUMEN
NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0â Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.
Asunto(s)
Complejo I de Transporte de Electrón/antagonistas & inhibidores , Inhibidores Enzimáticos/química , NAD/análogos & derivados , Aquifex/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , NAD/química , NAD/metabolismo , NAD/farmacología , Unión ProteicaRESUMEN
Objective: The human intestinal microbiome plays an important role in inflammatory bowel disease (IBD) and colorectal cancer (CRC) development. One of the first discovered bacterial mediators involves Bacteroides fragilis toxin (BFT, also named as fragilysin), a metalloprotease encoded by enterotoxigenic Bacteroides fragilis (ETBF) that causes barrier disruption and inflammation of the colon, leads to tumorigenesis in susceptible mice, and is enriched in the mucosa of IBD and CRC patients. Thus, targeted inhibition of BFT may benefit ETBF carrying patients. Design: By applying two complementary in silico drug design techniques, drug repositioning and molecular docking, we predicted potential BFT inhibitory compounds. Top candidates were tested in vitro on the CRC epithelial cell line HT29/c1 for their potential to inhibit key aspects of BFT activity, being epithelial morphology changes, E-cadherin cleavage (a marker for barrier function) and increased IL-8 secretion. Results: The primary bile acid and existing drug chenodeoxycholic acid (CDCA), currently used for treating gallstones, cerebrotendinous xanthomatosis, and constipation, was found to significantly inhibit all evaluated cell responses to BFT exposure. The inhibition of BFT resulted from a direct interaction between CDCA and BFT, as confirmed by an increase in the melting temperature of the BFT protein in the presence of CDCA. Conclusion: Together, our results show the potential of in silico drug discovery to combat harmful human and microbiome-derived proteins and more specifically suggests a potential for retargeting CDCA to inhibit the pro-oncogenic toxin BFT.
Asunto(s)
Carcinógenos/metabolismo , Transformación Celular Neoplásica , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Microbioma Gastrointestinal , Toxinas Biológicas , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/efectos de los fármacos , Endotoxinas/química , Endotoxinas/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Metaloendopeptidasas/química , Metaloendopeptidasas/farmacología , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Relación Estructura-Actividad , Toxinas Biológicas/efectos adversos , Toxinas Biológicas/biosíntesisRESUMEN
With the bromodomain (BRD) inhibitor JQ1, a remarkable success story of BRD4 as a novel drug target has been set off that yielded many anti-cancer drugs that are now in clinical trials. But not all of the great prospects of BRDs as drug targets may become true. First evaluations of ongoing clinical trials revealed that treatment with BET-inhibitors can be accompanied with significant toxic side effects and the validation of the therapeutic benefit of BET-inhibitors compared to existing therapies is still pending. New strategies that may overcome possible obstacles in BRD drug discovery include combination therapies with other agents, dual target inhibitors, and proteolysis targeting chimeras (PROTACs). Furthermore, non-BET proteins seem promising drug targets as well. Most recently, BRDs have been identified as putative targets to treat parasitic diseases such as malaria. Milestones in BRD drug discovery are reviewed and promising new developments are evaluated.