Structural basis for identification of the interface of the N-terminal domain of CoV N protein dimer as a target for drug development

• Main Authors: Jia-Ning Hsu, 許家寧
• Other Authors: Ming-Hon Hou
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/xj63nm

碩士 === 國立中興大學 === 生命科學系所 === 106 === 　　In 1973, the coronavirus was isolated for the first time. The coronavirus are known to cause the respiratory and gastrointestinal diseases in humans and vertebrates. The more severe infections could cause the death. In 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered in Saudi Arabia, with a mortality rate of 35% followed by the number of infections in South Korea too. Currently, there are no effective therapeutic as well as prophylactic treatments available against the coronavirus. So understanding the pathways of infection of coronaviruses and developing antiviral drugs has become a hot topic among countries. 　　Coronavirus is a positive single-stranded RNA virus with high variability and can easily become drug resistant. However, specific structural proteins are expressed in the life cycle of the coronavirus, and these protein sequences are highly conserved. During replication, the nucleocapsid protein (NP) binds to the RNA genome to form a long chain helical ribonucleoprotein complex (RNP). It has been found that the RNP complex plays an important role in the transcription and reproduction of virus life cycle. In the current study, we focus on utilizing the MERS-CoV N-NTD as a target for anti-viral drug development. Initially, the structure-based molecular docking using the ZINC database was performed to identify two P3 derivative compounds namely P3-1 and P3-2 respectively. To understand the possible mechanism of binding of these compounds to the N-NTD, we solve the crystal structure of these compounds with N-NTD protein. The previous studies has shown that the tryptophan 43 (W43) is an important residue at the ligand binding site. In the P3-1 complex with N-NTD, we found that M38 prior to N39 occupies the ligand binding pocket which makes the drug unable to penetrate deeply into the ligand binding site. Although in the P3-2 complex M38 was not found to interfere at binding site, however, there was no interaction of P3-2 with the W43. 　　In the fluorescence spectra measurement, the intrinsic fluorescence of N protein and N-NTD showed the light shift in the presence of the compound P3-2. In addition, the P3-2 can also enhance the oligomerization of N protein by small angle X-ray scattering (SAXS) assay. The Rg value was increased by 5.59 Å compared to that of the control, but the effect of MERS-CoV NP polymerization was not ideal to that of the original P3 compounds. Thus to retain the P3 ligand in the indoline, we then identify four new derivatives of P3 called as P4 series of small molecules. The crystal structures of P4 series of compounds with N-NTD showed that the M38 occupied the ligand binding pocket in all structures. However, the P4-2 complex showed the π-π interaction with W43, and hydrogen bond with T40, which made P4-2 deeper than dimer interface in the N-NTD to stabilize the structure of the MERS-CoV N-NTD dimer. In addition, the P4-2 compound can also enhance the oligomerization of N protein by small angle scattering assay. The Rg value was increased by 7.15 Å compared with the control which confirms that the P4-2 has the oligomerization properties of MERS-CoV N protein. Although the W43 non-RNA binding site, we consider that P3-2 and P4-2 by allosteric modulation of the ligand binding site to influence the function of MERS-CoV NP and its oligomerization properties. We provide a new method for screening antiviral drugs by dimer interface of HCoV N-NTD