Verteporfin inhibits TGF-ß signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-ß (TGF-ß) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-ß signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-ß signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-ß signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-ß-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-ß signaling pathway for treatments of related diseases.

Phase separation of Axin organizes the ß-catenin destruction complex.

In Wnt/ß-catenin signaling, the ß-catenin protein level is deliberately controlled by the assembly of the multiprotein ß-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3ß (GSK3ß), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid-liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3ß, CK1α, and ß-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates ß-catenin phosphorylation by GSK3ß and is critical for the regulation of ß-catenin protein stability and thus Wnt/ß-catenin signaling.

Identification of Interferon Receptor IFNAR2 As a Novel HCV Entry Factor by Using Chemical Probes.

Upon sensing pathogen-associated patterns and secreting interferons (IFNs) into the environment, host cells perceive extracellular type I IFNs by the IFNα/ß receptors IFNAR1 and IFNAR2 to stimulate downstream innate immune signaling cascades. Through the use of chemical probes, we demonstrated that IFNAR2 facilitates hepatitis C virus (HCV) entry. Silencing of IFNAR2 significantly attenuated HCV proliferation. IFNAR2 binds infectious HCV virions through a direct interaction of its D2 domain with the C-terminal end of apolipoprotein E (apoE) on the viral envelope and facilitates virus entry into host cells. The antibody against the IFNAR2 D2 domain attenuates IFNAR2-apoE interaction and impairs HCV infection. The recombinant IFNAR2 protein and the chemical probe potently inhibit major HCV genotypes in various human liver cells in vitro. Moreover, the impact of a chemical probe on HCV genotype 2a is also documented in immune-compromised humanized transgenic mice. Our results not only expand the understanding of the biology of HCV entry and the virus-host relationship but also reveal a new target for the development of anti-HCV entry inhibitors.

Resistance analysis and characterization of NITD008 as an adenosine analog inhibitor against hepatitis C virus.

Hepatitis disease caused by hepatitis C virus (HCV) is a severe threat to global public health, affecting approximately 3% of the world's population. Sofosbuvir (PSI-7977), a uridine nucleotide analog inhibitor targeting the HCV NS5B polymerase, was approved by FDA at the end of 2013 and represents a key step towards a new era in the management of HCV infection. Previous study identified NITD008, an adenosine nucleoside analog, as the specific inhibitor against dengue virus and showed good antiviral effect on other flaviviruses or enteroviruses. In this report, we systematically analyzed the anti-HCV profile of NITD008, which was discovered to effectively suppress the replication of different strains of HCV in human hepatoma cells with a low nanomolar activity. On genotype 2a virus, or 2a, 1a, and 1b replicon cells, EC50 values were 8.7 nM, 93.3 nM, 60.0 nM and 67.2 nM, and selective index values were >2298.9, >214.4, >333.3, >298.5 respectively. We demonstrated that resistance to NITD008 was conferred by mutation in NS5B (S282T) in the HCV infectious virus genotype 2a (JFH-1). Then, we compared the resistant profiles of NITD008 and PSI-7977, and found that the folds change of EC50 of NITD008 to full replicon cells containing mutation S282T was much bigger than PSI-7977(folds 76.50 vs. 4.52). Analysis of NITD008 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of NITD008 was not completely similar to PSI-7977, and meanwhile, S282T resistant mutation to NITD008 emerge more easily in cell culture than PSI-7977. Interestingly, NITD008 displayed significant synergistic effects with the NS5B polymerase inhibitor PSI-7977, however, only additive effects with alpha interferon (IFNα-2b), ribavirin, and an NS3 protease inhibitor. These results verify that NITD008 is an effective analog inhibitor against hepatitis C virus and a good research tool as a supplement to other types of nucleoside analogs.

Identification, synthesis and pharmacological evaluation of novel anti-EV71 agents via cyclophilin A inhibition.

In this work, the relationship between cyclophilin A (CypA) and EV71 prompted us to screen a series of small molecular CypA inhibitors which were previously reported by our group. Among them, compounds 1 and 2 were discovered as non-immunosuppressive anti-EV71 agents with an EC50 values of 1.07±0.17µM and 3.36±0.45µM in virus assay, respectively, which were desirably for the further study. The subsequent chemical modifications derived a novel class of molecules, among which compound 11 demonstrated the most potent anti-EV71 activity in virus assay (EC50=0.37±0.17µM), and low cytotoxicity (CC50>25µM). The following CypA enzyme inhibition studies indicated that there was not only the enzyme inhibition activity, undoubtedly important, functioning in the antiviral process, but also some unknown mechanisms worked in combination, and the further study is underway in our laboratory. Nevertheless, to the best of our knowledge, compound 11 was probably the most potent small molecular anti-EV71 agent via CypA inhibitory mechanism to date. Consequently, our study provided a new potential small molecule for curing EV71 infection.