Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance

SSFLNet: A Novel Fault Diagnosis Method for Double Shield TBM Tool System

In tunnel boring projects, wear and tear in the tooling system can have significant consequences, such as decreased boring efficiency, heightened maintenance costs, and potential safety hazards. In this paper, a fault diagnosis method for TBM tooling systems based on SAV−SVDD failure location (SSFL) is proposed. The aim of this method is to detect faults caused by disk cutter wear during the boring process, which diminishes the boring efficiency and is challenging to detect during construction. This paper uses SolidWorks to create a complete three−dimensional model of the TBM hydraulic thrust system and tool system. Then, dynamic simulations are performed with Adams. This helps us understand how the load on the propulsion hydraulic cylinder changes as the TBM tunneling tool wears to different degrees during construction. The hydraulic propulsion system was modeled and simulated using AMESIM software. Utilizing the load on the hydraulic propulsion cylinder as an input signal, pressure signals from the two chambers of the hydraulic cylinder and the system’s flow signal were acquired. This enabled an in−depth exploration of the correlation between these acquired signals and the extent of the tooling system failure. Following this analysis, a collection of normal sample data and sample data representing different degrees of disk cutter abrasions was amassed for further study. Next, an SSFL network model for locating the failure area of the cutter was established. Fault sample data were used as the input, and the accuracy of the fault diagnosis model was tested. The test results show that the performance of the SSFL network model is better than that of the SAE−SVM and SVDD network models. The SSFL model achieves 90% accuracy in determining the failure area of the cutter head. The model effectively identifies the failure regions, enabling timely tool replacement to avoid decreased boring efficiency under wear conditions. The experimental findings validate the feasibility of this approach

Comparison of key NA-ligand interactions.

<p>All residues are N2 numbered and distances are given in Å. The 4-guanidino group of zanamivir, laninamivir and CS-8958 is abbreviated as ‘4-guan’. Bond distances are based on the distances between oxygen and nitrogen atoms and do not include hydrogen atoms, which cannot be directly observed using X-ray diffraction. Distances are given for molecule A in the asymmetric unit of each structure and are highly consistent between molecules. The distance of the unique hydrogen bond between the laninamivir octanoate 9-ester-O and p09N1 Asn294 is given for both molecules A and B as it varies significantly. Laninamivir octanoate is listed as CS-8958 to save space.</p><p>*The Asp151 side chain carboxy hydrogen bonds with the 4-N of all the ligands used in this study.</p><p>**The Asp151 and Trp178 backbone carbonyl groups both hydrogen bond with the 4-guanidino group of zanamivir, laninamivir and laninamivir octanoate.</p><p>***The laninamivir octanoate 9-ester-carbonyl forms a hydrogen bond with N2 Arg224.</p

Binding of laninamivir and zanamivir to p57N2, p09N1 and N5.

<p>In each panel, zanamivir appears as the same color as the respective NA active site and laninamivir appears as turquoise. Acidic and basic side chains of key residues are colored red and blue, respectively. The 4-guanidino group of laninamivir and zanamivir is buried deep beneath the 150-loop where it engages many key interactions with NA residues (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002249#ppat-1002249-t002" target="_blank">Table 2</a>). Although the binding modes of laninamivir and zanamivir are highly similar, the accessibility of the 4-guanidino to its binding site is lowest in p57N2, with a 147–150 salt bridge in its closed 150-loop (A - green) and highest in group 1 N5, which contains a 150-cavity in its uncomplexed structure (C - yellow). Inhibition by zanamivir and laninamivir are highest for N5, and lowest for p57N2. p09N1, with its unique 150-loop characteristics (B - magenta), has intermediate laninamivir inhibition.</p

The chemical structures of influenza NA inhibitors used in this study.

<p>1, Neu5Ac2en (NA transition state analogue); 2, zanamivir; 3, laninamivir; 4, laninamivir octanoate (CS-8958); and 5, oseltamivir.</p

IC₅₀ values and 95% CIs for the inhibition of p57N2, 09N1 and N5.

<p>Laninamivir octanoate is listed as CS-8958 to save space.</p

Crystallographic X-ray diffraction and refinement statistics.

<p>Laninamivir octanoate is listed as CS-8958 to save space.</p