Discovery and preclinical evaluations of TQB3616, a novel CDK4-biased inhibitor.

Among small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) approved for metastatic breast cancers, abemaciclib has a more tolerable adverse effects in clinic. This is attributable to preferential inhibition of CDK4 over CDK6. In our search for a biased CDK4 inhibitor, we discovered a series of pyrimidine-indazole inhibitors. SAR studies led us to TQB3616 as a preferential CDK4 inhibitor. TQB3616 exhibited improvements in both enzymatic and cellular proliferation inhibitory potency when tested side-by-side with the FDA approved palbociclib and abemaciclib. TQB3616 also possessed favorable PK profile in multiple species. These differentiated properties, together with excellent GLP safety profile warranted TQB3616 moving to clinic. TQB3616 entered into clinical development in 2019 and currently in phase III clinical trials (NCT05375461, NCT05365178).

Ethanolic extract of Arctium lappa leaves alleviates cerebral ischemia reperfusion-induced inflammatory injury via HDAC9-mediated NF-κB pathway.

BACKGROUND: Ischemic stroke (IS) is a major cause of mortality and disability worldwide. Inflammatory response is crucial in the pathogenesis of tissue injury in cerebral infarction. Arctium lappa leaves are traditionally used to treat IS. PURPOSES: To investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR). METHODS: Middle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways. RESULTS: ALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1ß, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1ß, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01). CONCLUSIONS: In conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.

Effect of palliative microwave ablation on metastatic osseous pain: a single-center retrospective study.

BACKGROUND: Bone is among the most common metastasis sites in patients with advanced cancer. Approximately two-thirds of bone metastasis results in pain, the majority of which is moderate to unbearable pain, which seriously affects the quality of life of patients. With the development of ablation techniques, microwave ablation (MWA) has great potential to eliminate the pain caused by bone metastasis. This study aimed to evaluate the efficacy and safety of image-guided (computed tomography-guided) percutaneous MWA for metastatic osseous pain. METHODS: This is a retrospective study involving 18 patients with cancer-related pain caused by osseous or soft tissue metastasis in the First Affiliated Hospital of Soochow University from June 2015 to October 2020. All patients (14 men and 4 women; mean age 60.2 years) underwent image-guided percutaneous palliative MWA. A paired-sample t-test was used to compare the changes in Numeric Rating Scale (NRS) score and dosage of morphine preoperatively and postoperatively (at 24 h, 3 days, and 14 days after MWA). In addition, we assessed the level of pain relief according to the patients' subjective feelings. RESULTS: The paired-samples t-test showed that the NRS score (6.83±0.92 vs. 1.67±0.97, P<0.05) and dosage of morphine (85.56±17.23 vs. 32.78±4.61, P<0.05) were significantly decreased at 3 days after MWA. At 14 days after MWA, the NRS score (6.83±0.92 vs. 0.94±0.87, P<0.05) and dosage of morphine (85.56±17.23 vs. 10.56±8.73, P<0.05) were also markedly decreased. Moreover, according to the patients' subjective feeling, 88.89% patients had pain relief postoperatively, while the remaining patients had no progress. CONCLUSIONS: Image-guided (Computed Tomography-guided) percutaneous MWA can effectively relieve pain, thus improving the quality of life in patients with osseous metastasis. MWA is a feasible, safe, and effective treatment for pain caused by bone metastasis.

Blocking MAPK/ERK pathway sensitizes hepatocellular carcinoma cells to temozolomide via downregulating MGMT expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fourth most common malignant tumor in China. Temozolomide (TMZ) is a common chemotherapy drug which can effectively kill HCC cells in vitro. However, it is possible that HCC cells possess intrinsic resistance to TMZ. A key mechanism of TMZ resistance is the overexpression of O6-methylguanine-DNA methyltransferase (MGMT). Studies have shown that MAPK may be related to MGMT expression, U0126 is a highly selective inhibitor of MEK1 and MEK2, which were crucial molecule in cascade of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway. Sorafenib was another widely applicated target drug in HCC which could inhibit multiple kinases including MAPK/ERK. This research was aimed to investigate the efficacy of MAPK/ERK inhibitor U0126 and sorafenib combine with TMZ in the treatment of HCC. METHODS: In HCC cells, MAPK/ERK signaling pathway was blocked by U0126 and sorafenib. The effect of blocking MAPK/ERK signaling pathway on TMZ-induced cytotoxicity was evaluated by MTT assay, flow cytometry and TUNEL assay. DNA damage protein and the expression of MGMT were detected by Western-blot. After the downregulation of MAPK/ERK signaling pathway, MGMT mRNA expression and the protein expression of MGMT were quantified by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence assay, respectively. HepG2 cells were transfected with an MGMT over expression plasmid. After transfection, the effect of U0126 on TMZ-induced cytotoxicity was evaluated by MTT and Western-Blot in MGMT OE cells. The influence of Sorafenib on TMZ-induced cytotoxicity to HCC cells was also detected by MTT assay. RESULTS: U0126 can enhance the chemosensitivity of HCC cells to TMZ. At the same time, we also found that U0126 increases the damage to DNA caused by TMZ in HepG2 cells. Moreover, the results from RT-qPCR and Western blot showed that U0126 downregulated MGMT mRNA and MGMT protein expression via blocking MAPK/ERK pathway. Furthermore, after transfection with an MGMT expression plasmid, overexpression of MGMT restored U0126-induced chemosensitivity to TMZ in HCC cells. Sorafenib can also increase the chemosensitivity of HCC cells to TMZ. CONCLUSIONS: Our studies suggest great clinical potential for the utilization of combined U0126 and TMZ in patients with advanced HCC.

Effect of microwave ablation treatment of hepatic malignancies on serum cytokine levels.

BACKGROUND: Microwave ablation (MWA) is widely used to treat unresectable primary and secondary malignancies of the liver, and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site but also an immunoreaction of the whole body. This study aimed to investigate the effects of MWA on cytokines in patients who underwent MWA for a hepatic malignancy. METHODS: Patients admitted to the Oncology Department in the First Affiliated Hospital of Soochow University between June 2015 and February 2019 were selected. Peripheral blood was collected from patients with a hepatic malignancy treated with MWA. The levels of cytokines (IL-2, IFN-γ, TNF-α, IL-12 p40, IL-12 p70, IL-4, IL-6, IL-8, IL-10, and vascular endothelial growth factor (VEGF)) were detected with a Milliplex® MAP Kit. The comparison times were as follows: before ablation, 24 h after ablation, 15 days after ablation, and 30 days after ablation. Data were analyzed using a paired sample t-tests and Spearman's correlation analysis. RESULTS: A total of 43 patients with hepatic malignancies were assessed. There were significant differences in IL-2, IL-12 p40, IL-12 p70, IL-1ß, IL-8, and TNF-α at 24 h after MWA. Significant increases (> 2-fold vs. before ablation) were observed in IL-2, IL-1ß, IL-6, IL-8, IL-10, and TNF-α after MWA. Elevated IL-2 and IL-6 levels after ablation were positively correlated with energy output during the MWA procedure. CONCLUSIONS: WA treatment for hepatic malignancies can alter the serum levels of several cytokines such as IL-2 and IL-6.

Effect of introducing disulfide bridges in C-terminal structure on the thermostability of xylanase XynZF-2 from Aspergillus niger.

In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA was improved from 45°C to 60°C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50°C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.0~7.0) of the mutant xylanase was increased to 3.0~9.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.