Discovery Of Enterovirus71(Ev71)3C Protease Inhibitors And Their Functional Studies

HFMD (Hand foot and mouth disease) caused by intestinal virus is a kind of childhood infectious disease, also known as Eruptive Eesicular Stomatitis with hand, foot and mouth mucosal herpes or ulceration after rupture as the main clinical symptoms. There are20kinds of intestinal viruses can cause HFMD, among which Coxsackievirus A16(Cox A16) and Enterovirus71(EV71) are the most common ones. In recent years, EV71has been considered as the highest death rate, contagious intestinal virus.EV713Cpro (EV713C protease) is very important during the EV71virus replication,which is essentially involved in cutting all proteins.Besides,it can also inhibit the host immune system, shut down the host cell protein synthesis system, induce programmed host cell apoptosis and so on,thus, EV713Cpr0is considered as antiviral target, and screening compounds inhibiting the activity of EV713Cpro for the treatment of HFMD has a very important significance.In this article, EV713Cpro prokaryotic expression is carried out through a series of steps,such as transformation, picking a single colony, shaking bacteria at37degree, transferring, hypothermia inducement;the protease was purified by the principle of nickel column;EV713Cpro activity platform was established by FRET (fluorescence resonance energy transfer) principle, and the substrate is a polypeptide (Dabcyl-RTATVQGPSLDFE-Edans) which was synthesized with EV71self-cleavage sites3B-3C (with QG connection) with a fluorophore at both ends; We used the SPR(surface plasmon resonance) method and virtual screening method for screening EV713Cpro ligands from7164small molecule compounds, then, verified that whether the screened compounds are the inhibitors of the EV713Cpro with AG7088(Rupintrivir) as positive inhibitor, and the result is that we screened3401EV713Cpro ligands from7031compounds by SPR, but only Compound1478has inhibitory effect and133EV713Cpro ligands from SPACE Compound Libraries with virtual screening method,on one hand,83compounds of which contain α,β-unsaturated double bond and five of them(compound2,39,72,75,99) have inhibitory effect on EV713Cpro,on the other hand,50 compounds of which do not contain such a double bond and only compound90has inhibitory effect; we verified antiviral effects of those7inhibitors by EV71C4virus infected RD cell(Malignant embryonic rhabdomyosarcoma cells) administration of drugs and only Compound90has some antiviral effect; at last, we may provide new idea for the development of antiviral drugs by studying the co-crystallized structure of Compound90and EV713Cpr0and at the same time, we have transformated the structure of Compound90aiming to discover new EV71inhibitors with high activity.AG7088which has a broad spectrum of anti-viral effect on3C protease contains a special structure, α,β-unsaturated double bond,and we found compounds containing such an unsaturated double bond are generally highly toxic at the cellular level.However, Compound90with the highest non-toxic to cells at concentrations up to180uM and the IC50value of antiviral effect at150uM does not contain the α,β-unsaturated double bond,thus,it is possible to provide new sources for the development of drugs against the EV71.