Anti-Angiogenetic and Anti-Lymphangiogenic Effects of a Novel 2-Aminobenzimidazole Derivative, MFB.

Background and Purpose: Benzimidazoles have attracted much attention over the last few decades due to their broad-spectrum pharmacological properties. Increasing evidence is showing the potential use of benzimidazoles as anti-angiogenic agents, although the mechanisms that impact angiogenesis remain to be fully defined. In this study, we aim to investigate the anti-angiogenic mechanisms of MFB, a novel 2-aminobenzimidazole derivative, to develop a novel angiogenesis inhibitor. Experimental Approach: MTT, BrdU, migration and invasion assays, and immunoblotting were employed to examine MFB's effects on vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration, invasion, as well as signaling molecules activation. The anti-angiogenic effects of MFB were analyzed by tube formation, aorta ring sprouting, and matrigel plug assays. We also used a mouse model of lung metastasis to determine the MFB's anti-metastatic effects. Key Results: MFB suppressed cell proliferation, migration, invasion, and endothelial tube formation of VEGF-A-stimulated human umbilical vascular endothelial cells (HUVECs) or VEGF-C-stimulated lymphatic endothelial cells (LECs). MFB suppressed VEGF-A and VEGF-C signaling in HUVECs or LECs. In addition, MFB reduced VEGF-A- or tumor cells-induced neovascularization in vivo. MFB also diminished B16F10 melanoma lung metastasis. The molecular docking results further showed that MFB may bind to VEGFR-2 rather than VEGF-A with high affinity. Conclusions and Implications: These observations indicated that MFB may target VEGF/VEGFR signaling to suppress angiogenesis and lymphangiogenesis. It also supports the role of MFB as a potential lead in developing novel agents for the treatment of angiogenesis- or lymphangiogenesis-associated diseases and cancer.

A Structural Comparison of SARS-CoV-2 Main Protease and Animal Coronaviral Main Protease Reveals Species-Specific Ligand Binding and Dimerization Mechanism.

Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (Mpro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV Mpro and can thermodynamically stabilize its folding. The structure of CCoV Mpro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV Mpro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV Mpro and other animal CoV Mpros with SARS-CoV-2 Mpro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral Mpros, SARS-CoV-2 Mpro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46-49 in animal coronaviral Mpros has been replaced by a stable α-helix in SARS-CoV-2 Mpro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV Mpros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV Mpros among different species.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

Synthetic Tryptanthrin Derivatives Induce Cell Cycle Arrest and Apoptosis via Akt and MAPKs in Human Hepatocellular Carcinoma Cells.

Trytanthrin, found in Ban-Lan-Gen, is a natural product containing an indoloquinazoline moiety and has been shown to possess anti-inflammatory and anti-viral activities. Chronic inflammation and hepatitis B are known to be associated with the progression of hepatocellular carcinoma (HCC). In this study, a series of tryptanthrin derivatives were synthesized to generate potent anti-tumor agents against HCC. This effort yielded two compounds, A1 and A6, that exhibited multi-fold higher cytotoxicity in HCC cells than the parent compound. Flow cytometric analysis demonstrated that A1 and A6 caused S-phase arrest and downregulated the expression of cyclin A1, B1, CDK2, and p-CDC2. In addition to inducing caspase-dependent apoptosis, A1 and A6 exhibited similar regulation of the phosphorylation or expression of multiple signaling targets, including Akt, NF-κB, and mitogen-activated protein kinases. The anti-tumor activities of A1 and A6 were also attributable to the generation of reactive oxygen species, accompanied by an increase in p-p53 levels. Therefore, A1 and A6 have potential clinical applications since they target diverse aspects of cancer cell growth in HCC.

Development of Novel Rhodacyanine-Based Heat Shock Protein 70 Inhibitors.

BACKGROUND: A growing body of evidence suggests that Hsp70, which is overexpressed in human breast tumors, plays a role in tumorigenesis and tumor progression in breast cancer as well as in its aggressive phenotypes. Hsp70 constitutes a potential therapeutic target in the treatment of this disease. METHODS: We developed a new series of rhodacyanine-based Hsp70 inhibitors, represented by compounds 1 and 6, in which the cationic pyridin-1-ium or thiazol-3-ium ring of existing Hsp70 inhibitors (e.g., JG-40 and JG-98) was replaced by a corresponding benzo- fused N-heterocycle. RESULTS: Several lines of evidence suggest that these benzo-fused derivatives may exert their antitumor activities, in part, by targeting Hsp70. These putative inhibitors displayed differential antiproliferative efficacy against breast cancer cells (IC50 as low as 0.25 µM) versus nontumorigenic MCF-10A breast epithelial cells (IC50 ≥ 5 µM). This was correlated with the corresponding Hsp70 expression levels. Using a protein refolding assay, we confirmed that these agents effectively inhibited the chaperone activity of Hsp70. Moreover, these inhibitors effectively suppressed the expression of well-known oncogenic client proteins of Hsp70's, including FoxM1, HuR, and Akt, which paralleled their antiproliferative efficacy. Supporting the established role of Hsp70 in regulating protein refolding, these derivatives induced autophagy, as manifested by the conversion of LC3B-I to LC3B-II. Notably, these putative Hsp70 inhibitors did not cause a compensatory elevation in Hsp90 expression, contrasting with the previously reported effects of Hsp90 inhibitors on Hsp70 upregulation. CONCLUSION: Together with the finding that compounds 1 and 6 showed improved microsomal stability, these results suggest the translational potential of these putative Hsp70 inhibitors to foster new strategies for cancer therapy. However, whether these benzo-fused rhodacyanines act on kinases or other targets remains unclear. It is currently under investigation.

Correlation between mitochondrial TRAP-1 expression and lymph node metastasis in colorectal cancer.

AIM: To evaluate the effect of mitochondrial tumor necrosis factor receptor-associated protein-1 (TRAP-1) on the lymph node metastasis (LNM) in Chinese colorectal cancer (CRC) patients, and develop potential LNM-associated biomarkers for CRC using quantitative real-time polymerase chain reaction (RT-PCR) analysis. METHODS: Differences in mitochondrial TRAP-1 gene expression between primary CRC with LNM (LNM CRC) and without LNM (non-LNM CRC) were assessed in 96 Chinese colorectal carcinoma samples using quantitative RT-PCR analysis, Western blotting, and confirmed with immunohistochemical assay. The relationship between clinicopathological parameters and potential diagnostic biomarkers was also examined. RESULTS: TRAP-1 was significantly upregulated in LNM CRC compared with non-LNM CRC, which was confirmed by RT-PCR, Western blotting and immunohistochemical assay. The expression of TRAP-1 in two different metastatic potential human colorectal cancer cell lines, LoVo and HT29, was analyzed with Western blotting. The expression level of TRAP-1 was dramatically higher in LoVo than in HT29. Overexpression of TRAP-1 was significantly associated with LNM (90.2% in LNM group vs 22% in non-LNM group, P < 0.001), the advanced tumor node metastasis stage (89.1% in LNM group vs 26.9% in non-LNM group, P < 0.001), the increased 5-year recurrence rate (82.7% in LNM group vs 22.6% in non-LNM group, P < 0.001) and the decreased 5-year overall survival rate (48.4% in LNM vs 83.2% in non-LNM group, P < 0.001). Univariate and multivariate analyses indicated that TRAP-1 expression was an independent prognostic factor for recurrence and survival of CRC patients (Hazard ratio of 2.445 in recurrence, P = 0.017; 2.867 in survival, P = 0.028). CONCLUSION: Mitochondria TRAP-1 affects the lymph node metastasis in CRC, and may be a potential biomarker for LNM and a prognostic factor in CRC. Over-expression of TRAP-1 is a predictive factor for the poor outcome of colorectal cancer patients.