2-Aminoethoxydiphenyl borate (2-APB), a commonly used blocker of IP₃-induced calcium ion release and of store-operated channels, inhibits gap junction conductance when applied to cultured cells. The character and pharmacology of this inhibition was explored using 1) hemichannels composed of connexin32 (Cx32) and/or connexin26 (Cx26) purified from native sources and from transfected HeLa cells in which the connexin had a cleavable C-terminal epitope tag and 2) the corresponding junctional channels. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay (TSF), 2-APB reversibly inhibited homomeric Cx32 and heteromeric Cx26/Cx32 channels from native tissue and their tagged forms from HeLa cells. The IC_50-TSF value of the inhibition was ∼47 μM at pH 6.5. 2-APB did not inhibit tagged homomeric Cx26 channels even after tag cleavage (leaving several amino acids at the carboxyl terminus). Protonated 2-APB is the inhibitory agent, but channel sensitivity to 2-APB also increases as pH is lowered. To help define the chemical requirements for inhibition, the effects of four structural analogs of 2-APB were determined. The inhibitory action of 2-APB was shown to be distinct from that of aminosulfonates. 2-APB and its analogs, except phenytoin, inhibited dye-coupling through junctional channels formed by all the tagged channel forms except Cx26, consistent with the TSF studies. However 2-APB significantly inhibited dye coupling between cells expressing untagged Cx26, suggesting that an unmodified C terminus is required for action on Cx26 channels. These results show that protonated 2-APB directly and reversibly inhibits connexin channels composed of Cx26 and/or Cx32 and suggest involvement of the carboxyl-terminal domain.

Drug–targeted therapies are promising approaches to treating tumors, and research on receptor–ligand interactions for discovering high-affinity targeted drugs has been accelerating drug development. This study presents a mechanism-driven deep learning-based computational model to learn double drug sequences, protein sequences, and drug graphs to project drug–target affinities (DTAs), which was termed the DoubleSG-DTA. We deployed lightweight graph isomorphism networks to aggregate drug graph representations and discriminate between molecular structures, and stacked multilayer squeeze-and-excitation networks to selectively enhance spatial features of drug and protein sequences. What is more, cross-multi-head attentions were constructed to further model the non-covalent molecular docking behavior. The multiple cross-validation experimental evaluations on various datasets indicated that DoubleSG-DTA consistently outperformed all previously reported works. To showcase the value of DoubleSG-DTA, we applied it to generate promising hit compounds of Non-Small Cell Lung Cancer harboring $EGF{R}^{T790M}$ mutation from natural products, which were consistent with reported laboratory studies. Afterward, we further investigated the interpretability of the graph-based “black box” model and highlighted the active structures that contributed the most. DoubleSG-DTA thus provides a powerful and interpretable framework that extrapolates for potential chemicals to modulate the systemic response to disease.

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18‐27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA‐34a. Additionally, these effects of microRNA‐34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.

Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear. The expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice. Our study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression. The present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.