Two Birds with One Stone? Possible Dual-Targeting H1N1 Inhibitors from Traditional Chinese Medicine

The H1N1 influenza pandemic of 2009 has claimed over 18,000 lives. During this pandemic, development of drug resistance further complicated efforts to control and treat the widespread illness. This research utilizes traditional Chinese medicine Database@Taiwan (TCM Database@Taiwan) to screen for compounds that simultaneously target H1 and N1 to overcome current difficulties with virus mutations. The top three candidates were de novo derivatives of xylopine and rosmaricine. Bioactivity of the de novo derivatives against N1 were validated by multiple machine learning prediction models. Ability of the de novo compounds to maintain CoMFA/CoMSIA contour and form key interactions implied bioactivity within H1 as well. Addition of a pyridinium fragment was critical to form stable interactions in H1 and N1 as supported by molecular dynamics (MD) simulation. Results from MD, hydrophobic interactions, and torsion angles are consistent and support the findings of docking. Multiple anchors and lack of binding to residues prone to mutation suggest that the TCM de novo derivatives may be resistant to drug resistance and are advantageous over conventional H1N1 treatments such as oseltamivir. These results suggest that the TCM de novo derivatives may be suitable candidates of dual-targeting drugs for influenza.National Science Council of Taiwan (NSC 99-2221-E-039-013-)Committee on Chinese Medicine and Pharmacy (CCMP100-RD-030)China Medical University and Asia University (CMU98-TCM)China Medical University and Asia University (CMU99-TCM)China Medical University and Asia University (CMU99-S-02)China Medical University and Asia University (CMU99-ASIA-25)China Medical University and Asia University (CMU99-ASIA-26)China Medical University and Asia University (CMU99-ASIA-27)China Medical University and Asia University (CMU99-ASIA-28)Taiwan Department of Health. Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004)Taiwan Department of Health. Cancer Research Center of Excellence (DOH100-TD-C-111-005

Kinetics of Proton Transport into Influenza Virions by the Viral M2 Channel

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pKm of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer

Study on Phylogenetic Relationships, Variability, and Correlated Mutations in M2 Proteins of Influenza Virus A

M2 channel, an influenza virus transmembrane protein, serves as an important target for antiviral drug design. There are still discordances concerning the role of some residues involved in proton transfer as well as the mechanism of inhibition by commercial drugs. The viral M2 proteins show high conservativity; about 3/4 of the positions are occupied by one residue in over 95%. Nine M2 proteins from the H3N2 strain and possibly two proteins from H2N2 strains make a phylogenic cluster closely related to 2RLF. The variability range is limited to 4 residues/position with one exception. The 2RLF protein stands out by the presence of 2 serines at the positions 19 and 50, which are in most other M2 proteins occupied by cysteines. The study of correlated mutations shows that there are several positions with significant mutational correlation that have not been described so far as functionally important. That there are 5 more residues potentially involved in the M2 mechanism of action. The original software used in this work (Consensus Constructor, SSSSg, Corm, Talana) is freely accessible as stand-alone offline applications upon request to the authors. The other software used in this work is freely available online for noncommercial purposes at public services on bioinformatics such as ExPASy or NCBI. The study on mutational variability, evolutionary relationship, and correlated mutation presented in this paper is a potential way to explain more completely the role of significant factors in proton channel action and to clarify the inhibition mechanism by specific drugs

Novel Inhibitor Design for Hemagglutinin against H1N1 Influenza Virus by Core Hopping Method

The worldwide spread of H1N1 avian influenza and the increasing reports about its resistance to the current drugs have made a high priority for developing new anti-influenza drugs. Owing to its unique function in assisting viruses to bind the cellular surface, a key step for them to subsequently penetrate into the infected cell, hemagglutinin (HA) has become one of the main targets for drug design against influenza virus. To develop potent HA inhibitors, the ZINC fragment database was searched for finding the optimal compound with the core hopping technique. As a result, the Neo6 compound was obtained. It has been shown through the subsequent molecular docking studies and molecular dynamic simulations that Neo6 not only assumes more favorable conformation at the binding pocket of HA but also has stronger binding interaction with its receptor. Accordingly, Neo6 may become a promising candidate for developing new and more powerful drugs for treating influenza. Or at the very least, the findings reported here may provide useful insights to stimulate new strategy in this area

Identification of a novel splice variant form of the influenza a virus m2 ion channel with an antigenically distinct ectodomain

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle

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

Analysis and Prediction of the Metabolic Stability of Proteins Based on Their Sequential Features, Subcellular Locations and Interaction Networks

The metabolic stability is a very important idiosyncracy of proteins that is related to their global flexibility, intramolecular fluctuations, various internal dynamic processes, as well as many marvelous biological functions. Determination of protein's metabolic stability would provide us with useful information for in-depth understanding of the dynamic action mechanisms of proteins. Although several experimental methods have been developed to measure protein's metabolic stability, they are time-consuming and more expensive. Reported in this paper is a computational method, which is featured by (1) integrating various properties of proteins, such as biochemical and physicochemical properties, subcellular locations, network properties and protein complex property, (2) using the mRMR (Maximum Relevance & Minimum Redundancy) principle and the IFS (Incremental Feature Selection) procedure to optimize the prediction engine, and (3) being able to identify proteins among the four types: “short”, “medium”, “long”, and “extra-long” half-life spans. It was revealed through our analysis that the following seven characters played major roles in determining the stability of proteins: (1) KEGG enrichment scores of the protein and its neighbors in network, (2) subcellular locations, (3) polarity, (4) amino acids composition, (5) hydrophobicity, (6) secondary structure propensity, and (7) the number of protein complexes the protein involved. It was observed that there was an intriguing correlation between the predicted metabolic stability of some proteins and the real half-life of the drugs designed to target them. These findings might provide useful insights for designing protein-stability-relevant drugs. The computational method can also be used as a large-scale tool for annotating the metabolic stability for the avalanche of protein sequences generated in the post-genomic age