Structure Function Relationship Study Of SARS Main Proteinase And Spike Protein And The Drug Design Of SARS Inhibitors By Kewen Zheng

Since its broke out, Severe Acute Respiratory Syndrome (SARS) has lead to huge harm to human being and economic in the world, especially in China. Taking the advantage of our long-time experience in drug design, we carried out research work in anti-SARS drug design and immunity in the following aspects:1. The determination for SARS coronavirus RNA gene sequences of SARS was finished not long after its broke out, it was predicted that the main proteins coded by SARS coronavirus RNA gene sequences are RNA polymerization enzyme (ORF la and 1b), spike protein, envelope protein, nucleocapsid protein, 3C-like proteinase and etc, in which the main proteinase turns to be the focus of research work due to its important role in the replication of SARS coronavirus RNA gene sequence. Making use of the advanced bioinformatics analysis software and search in the known protein database, it was found that the protein sequences of transmissible gastroenteritis virus, TGEV and Human coronavirus, HCoV main proteinase share high homology with SARSmain proteinase, with the similarities of 60% and identities of 40%. Based on this, the 3D structure of SARS coronavirus main proteinase was homology modeled according to the 3D crystal structure of TGEV main proteinase, the structure and activity site were analyzed.2. It was predicted that the SARS main proteinase exists as dimer because that the TGEV and HcoVmain proteinases are all dimer in solvent and may be important to the activities of coronavirus main proteinase. The dimer of SARS coronavirus main proteinase was first constructed, using the dimer of TGEV and HCoV main proteinase as reference. It was discovered, in each dimer, the two monomers exhibit apparent electrostatic complementarity and the electrostatic interaction energies are very similar among the dimers. The solvent accessible surface model was supplied for the molecular surface and hydrophobicity. The hydrophobic distributions were similar forthe three proteinases. So, it is shown that SARS main proteinase exists as dimer in solution, which is in accordance with later experiment.3. The phenomenon that SARS Mpro dimer is the main functional form has been conformed by experiment. However, due to the absence of the structural information of monomer, the reason for this remains unknown. To investigate it, two molecular dynamics simulations in water for dimer and monomer model have been carried out, used the crystal structure of protomer A within dimer as the starting structure of monomer. During the MD simulation of dimer, three interest phenomena of protomer A has been observed: i) the active site, His41 and Cysl45, forms the Cysl45-S-...His41-H+ ions pair, which is important to attack the active site nucleophile on the carbonyl carbon of the scissile bond; ii) His 163 and Glul66 forms the "tooth" conformational properties, resulting in the specificity for glutamine at substrate PI site; and iii) the substrate-binding pocket formed by loop 140-146 and loop 184-197 is large enough to accommodate the substrate analog. But during the MD simulation of monomer complex, the three structural characteristics are all absent, which result directly in the inactivation of monomer. Throughout the MD simulation of dimer, the N-terminus of protomer B forms stable hydrogen bonds with Phel40 and Glul66, through which Hisl63, Glul66 and loop 140-146 are kept at active form. Furthermore, a water-bridge has been found between the N-terminus of protomer B and Glyl70, which stabilize His 172 and avoid it moving toward Tyrl61 to disrupt H-bond between Tyrl61 and Hisl63, stabilizing the conformation of His 163. The interactions between N-terminus and another monomer maintain the activity of dimer.4. Through the molecular dynamics simulation study of the binding of substrate anologue inhibitor CMK with SARSmain proteinase, it is shown that SARS main proteinase inhibitor should has following two characters: 1) it should be capable of forming stable H-bond with Hisl63 and Glul66 in main proteinase. 2) the group in inhibitors, that binds with the active pocket S2 hydrophobic subsite of SARS main proteinase, should be hydrophobic. Based on this, we proposed the3D-Pharmacophore of SARS coronavirus inhibitors and the pharmacophore was used to screen the MDL molecular database. The molecules up to the pharmacophore demand were obtained and was used for further screening by docking methods, after which a set of small molecules that bind well with SARS main proteinase had been got. At the same time, the modified molecules of HIV poly-protein inhibitors, based on the 3D-Pharmacophore, were used as inhibitors of SARS main proteinase. The modified molecules of HIV poly-protein inhibitors, which shows good binding property with SARS main proteinase, maybe become the leading compounds against SARS main proteinase.5. SARS coronavirus S protein can bind with the receptor in cell, causing the SARS virus fusion with cell and virus entry cell. So, cutting the binding of S protein with its functional recptor can prevent the intruding of SARS virus. Because of the limitation of experiment condition, the crystal structure of the S protein is still unknown. In this work, the 3D structure of S protein was obtained by homology method, and the key site and structure characters of S protein binding with its receptor were determined by studying the binding mode of Sprotein with its functional receptor ACE2 protein obtained by docking methods. All these work provide valuable clue in study of bacterin against SARS coronavirus.