Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC₅₀ values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC₅₀ for HEK-293/IC₅₀ for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.

Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit βG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial βG activity in nude mice. In vitro cell-based assays showed that the detection limit is 10⁴ colony-forming units/well of βG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for βG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial βG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to deplete intestinal bacteria, the FDGlcU-based images showed that the βG activity was decreased by 8.5-fold on day 4 and then gradually increased after treatment stopped. The results suggested that FDGlcU-based imaging revealed the in vitro and in vivo activity of intestinal bacterial βG, which would facilitate pharmacodynamic studies of specific bacterial βG inhibitors in animal studies.

The tumor microenvironment is mainly flooded with immunosuppressive cells and inhibitory cytokines, resulting in the inability of effective immune cells to infiltrate and recognize tumors and even the loss of anti-cancer ability. We propose a novel HDAC6/HSP90 dual inhibitory strategy as well as a chemoimmunotherapeutic agent that does not only kill tumor cells but also destroys the tumor microenvironment and enhances anti-cancer immunity. A hybrid scaffold construction approach was leveraged to furnish a series of rationally designed resorcinol-based hydroxamates as dual selective HDAC6/HSP90 inhibitors. The drug design campaign commenced with a fragment recruitment process to pinpoint validated structural units to inhibit HDAC6 and HSP90, followed by their installation in flexible HDAC inhibitory templates via an efficient and facile multistep synthetic route. Subsequent evaluations identified a strikingly potent selective HDAC6/HSP90 dual inhibitor (compound 17) via molecular and biological analysis in vitro and in vivo. Compound 17 exhibited not only direct cytotoxicity to cancer cells but also downregulated immune checkpoints (PD-L1 and IDO) expression in tumors via the inhibition of STAT1 pathway and degradation of oncogene proteins (Src, AKT, Rb, and FAK), leading to in vivo tumor growth inhibition. These multiple effects enabled the effector T cells to largely infiltrate into the tumor region and release granzyme B to kill cancer cells. In addition, compound 17 also decreased TGF-β secretion from normal cells, resulting in the systemic reduction of immunosuppressive regulatory T cells. Delightfully, a cocktail treatment of compound 17 and anti-PD-1 antibodies demonstrated synergistic efficacy to eliminate solid tumors with 83.9% of tumor growth inhibition. In summary, the impressive activity profile of compound 17, as an effective anticancer agent and a potential immunosensitizer, forecasts the application of HDAC6/HSP90 dual inhibitory strategy to overcome the immunosuppressive tumor microenvironment.

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.

The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5–23 times and 1.2–6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-α sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.