Design, Synthesis And Biological Evaluation Of Clathrin Inhibitors

More than half of the major current epidemic diseases are caused by the viral infection. There are hundreds of different kinds of viruses that cause diseases in human, however, the approved antiviral therapies are less than 10 classes. Most clinical frontline antiviral drugs are designed to act at the specific enzymes of viruses, which is called direct acting antivirals(DAAs). However, because of the variation of virus in the evolution and the tolerance to the existing antiviral drugs, it is hard to implement the broad-spectrum, efficient and safe treatment like antibiotics, which makes the development of broad-spectrum antiviral drugs more necessary and ambitious. It is known that most of the viruses enter to the host cell through the Clathrin-mediated endocytosis, such as dengue virus, hepatitis C virus, ebola virus and many other viruses. Because the function and mechanism that the viruses enter the cells do not rely on the virus itself, the clathrin-mediated endocytosis has been the potential target for broad-spectrum antiviral drugs in the recent years.It has been reported that the compound pitstop 2 could block the virus entry into cell by selectively inhibiting the binding site between the clathrin terminal domain(CTD) and other ligand or protein. Based on the binding mode of pitstop 2 and Clathrin terminal domain, clathrin inhibitors have been designed according to the three parts of pitstop2 by using the pitstop 2 as lead compound and retaining its essential groups. So far, 28 target compounds have been synthesized for the first time. The structures of all the 28 compounds were identified by methods of MS, 1H-NMR. Pharmacodynamic evaluation of the synthesized target compounds was conducted at the cellular level, using Vero and RD cell, to assess their activity to inhibit EV71, and their toxicity of Vero and RD cell were evaluated as well. We also examine whether our compounds can affect clathrin-mediated endocytosis(CME) and Clathrin-independent endocytosis(CIE) in living cells by using Immunofluorescence, in order to figure out the antiviral mechanism of compounds.According to the anti-EV71 results, compound 3,4,5,6,8 and 14 are well active on anti-EV71 activity. Among them, compound 3 performs equal antiviral activity and less cytotoxicity than positive drug. The immunofluorescence results show that compound 3 at 10μM concentration significantly inhibited clathrin-mediated endocytosis pathway, and the effect is reversible. Besides, the compound 3 does not inhibit clathrin-independent endocytosis, which indicates that the compounds can be specific for clathrin-mediated endocytic pathway. Primary structure-activity relationships of these target compounds were identified based on the biological activity assessment. The hydroxyl groups on the phenyl ring may have a great effect on the activity of the compounds. Besides, the activity of thiazole ring connected to hydrophobic group is better, and naphthalene ring is the essential group of the compounds. These results indicated that the compound 3 can be used as lead compound of the broad-spectrum antivirus agents for the further study.