Epigenetic deregulation plays an essential role in colorectal cancer progression. Bromodomains are epigenetic “readers” of histone acetylation. Bromodomain-containing protein 4 (BRD4) plays a pivotal role in transcriptional regulation and is a feasible drug target in cancer cells. Disease-specific elevation of nucleoporin, a component of the nuclear pore complex (NPC), is a determinant of cancer malignancy, but BRD4-driven changes of NPC composition remain poorly understood. Here, we developed novel aminocyclopropenones and investigated their biological effects on cancer cell growth and BRD4 functions. Among 21 compounds developed here, we identified aminocyclopropenone 1n (ACP-1n) with the strongest inhibitory effects on the growth of the cancer cell line HCT116. ACP-1n blocked BRD4 functions by preventing its phase separation ability both in vitro and in vivo, attenuating the expression levels of BRD4-driven MYC. Notably, ACP-1n significantly reduced the nuclear size with concomitant suppression of the level of the NPC protein nucleoporin NUP210. Furthermore, NUP210 is in a BRD4-dependent manner and silencing of NUP210 was sufficient to decrease nucleus size and cellular growth. In conclusion, our findings highlighted an aminocyclopropenone compound as a novel therapeutic drug blocking BRD4 assembly, thereby preventing BRD4-driven oncogenic functions in cancer cells. This study facilitates the development of the next generation of effective and potent inhibitors of epigenetic bromodomains and extra-terminal (BET) protein family.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.