Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

BMC Structural Biology

Blaine HM Mooers¹  Wayne J Brouillette²  Gillian M Air¹  Gundurao Kolavi²  Eric S Johnson²  Lalitha Venkatramani¹ 

[1] Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA;Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA

关键词: Pyrrolidinone;    Benzoic acid;    Influenza virus;    Glycan structure;    Glycoprotein;    Structure-based drug design;    Antiviral;    Enzyme-ligand complex;    Influenza neuraminidase inhibitor;   

Others  :  1092347
DOI  :  10.1186/1472-6807-12-7

 received in 2012-02-24, accepted in 2012-04-13,  发布年份 2012

【 摘 要 】

Background

Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite.

Results

Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC₅₀ for compound 2 remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit.

Conclusions

We designed inhibitor 2 with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in 2 is tilted relative to 1 and the number of contacts increased, but one hydrophobic branch makes no contacts, perhaps explaining why the IC₅₀ did not decrease. Future design efforts will include branches of unequal length so that both branches may be accommodated in the C6-subsite without conformational disorder. The high-mannose glycan attached to Asn200 makes several inter-subunit contacts and appears to stabilize the tetramer.

【 授权许可】

   
2012 Venkatramani et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 1.

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Figure 5.

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