Hypoxia-activated prodrugs (HAPs) have drawn increasing attention for improving the antitumor effects while minimizing side effects. However, the heterogeneous distribution of the hypoxic region in tumors severely impedes the curative effect of HAPs. Additionally, most HAPs are not amenable to optical imaging, and it is difficult to precisely trace them in tissues. Herein, we carefully designed and synthesized a multifunctional therapeutic BAC prodrug by connecting the chemotherapeutic drug camptothecin (CPT) and the fluorescent photothermal agent boron dipyrromethene (BODIPY) via hypoxia-responsive azobenzene linkers. To enhance the solubility and tumor accumulation, the prepared BAC was further encapsulated into a human serum albumin (HSA)-based drug delivery system to form HSA@BAC nanoparticles. Since the CPT was caged by a BODIPY-based molecule at the active site, the BAC exhibited excellent biosafety. Importantly, the activated CPT could be quickly released from BAC and could perform chemotherapy in hypoxic cancer cells, which was ascribed to the cleavage of the azobenzene linker by overexpressed azoreductase. After irradiation with a 730 nm laser, HSA@BAC can efficiently generate hyperthermia to achieve irreversible cancer cell death by oxygen-independent photothermal therapy. Under fluorescence imaging-guided local irradiation, both in vitro and in vivo studies demonstrated that HSA@BAC exhibited superior antitumor effects with minimal side effects.
The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an ‘off-switch’ regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.
Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus , with IC 50 values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus . Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.
The cyclic oligoadenylates (cOAs) act as second messengers of type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an off-switch regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the CRISPR-associated ring nuclease 1 (Crn1) from Saccharolobus solfataricus (Sso) 2081 in its apo or bound to cA4 in both pre-cleavage and transient intermediate states. Sso2081 harbors a unique helical insert that encloses cA4 in the central cavity. Two free phosphates symmetrically bind the catalytic site of apo Sso2081 and overlap with the two scissile phosphates of cA4, supporting a bilaterally symmetrical cleavage. The structure of transient intermediate state captured by Ser11Ala mutation immediately illustrates a stepwise cleavage of cA4 by Sso2081. Our study establishes atomic mechanisms of cA4 recognition and degradation by the type III CRISPR ring nuclease Crn1/Sso2081.
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