The amantadine-resistant S31N mutant of the influenza A M2 proton channel has become prevalent in currently circulating viruses. Here we have solved an X-ray crystal structure of M2(22-46) S31N that contains two distinct conformational states within its asymmetric unit. This structure reveals the mechanism of adamantane resistance in both conformational states of the M2 channel. In the Inward(open) conformation, the mutant Asn31 side chain faces the channel pore and sterically blocks the adamantane binding site. In the Inward(closed) conformation, Asn31 forms hydrogen bonds with carbonyls at the monomer-monomer interface, which twists the monomer helices and constricts the channel pore at the drug binding site. We also examine M2(19-49) WT and S31N using solution NMR spectroscopy, and show that distribution of the two conformational states is dependent on both detergent choice and experimental pH.

The FDA-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive towards amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics (MD) simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N-terminus of the transmembrane residues 22-31 that ultimately broadened the drug binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared to its complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism towards AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high profile antiviral drug target as more than 95% of current circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using one of our previously developed AM2 S31N channel blocker as a chemical probe, we, for the first time, identified a novel resistant mutant L46P. The L46P mutant located outside of the drug binding site. It was shown by molecular dynamics simulations that the L46P causes a dilation of drug binding site between residues 22-31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor, amantadine.

Immunoassays with ultra-high sensitivity for the rapid detection of chemical contaminants in food are urgently required. However, conventional enzyme-linked immunosorbent assay (ELISA) usually suffer from the moderate sensitivity. Herein, we aim to improve the sensitivity of conventional ELISA by employing the fluorescent carbon dots (CDs) as the signal probes based on the principle of inner filter effect (IFE). In this strategy, the enzymatically formed products of horseradish peroxidase/alkaline phosphatase efficiently quenched the CDs via the IFE. The absorption signal of the conventional ELISA was converted into the fluorescence signal. The fluorescent immunoassay was successfully developed and used to detect amantadine residues in chicken, achieving a limit of detection of 0.02 ng mL-1. The fluorescent immunoassay is a straightforward, extendable and general strategy and exhibits potential in detecting trace amounts of chemical contaminants in foodstuff.

N-(p-amylcinnamoyl) anthranilic acid (ACA) is a blocker of transient receptor potential melastatin-2 (TRPM2) which is a non-selective, Ca2+-permeable and oxidative stress sensor cation channel. Intracerebroventricular (ICV) streptozotocin (STZ) induction successfully generates spatial memory deficits in rats. The purpose of this study was to investigate effects of ACA on a rat model of STZ-induced learning and memory deficits. A total of 60 Wistar rats randomly divided into six groups; (1) control, (2) sham-operated, (3) ICV-STZ administered, (4) ICV-STZ + memantine (5 mg/kg i.p.), (5) ICV-STZ + ACA (25 mg/kg i.p.) and (6) a combination therapy group, ICV-STZ + ACA (25 mg/kg) + memantine (5 mg/kg). Effects of the drugs on spatial memory deficits were appraised in Morris water maze (MWM) apparatus. Anxiety-like behavior of the rats were also assessed by using both the elevated plus maze (EPM) and open field maze (OFM) apparatuses. Western blot analysis of hippocampal tissues revealed TRPM2-L channel protein expression levels. Serum levels of tumor necrosis factor alpha (TNF-α) and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA) kits. Memantine treatment ameliorated the spatial memory deficits induced, as evidenced by the MWM tests. However, ACA treatment did not provide any improvement, instead positive effects of memantine were attenuated by ACA treatment. Western blot analysis in hippocampal tissues showed that TRPM2-L protein expression was markedly suppressed in ICV-STZ administered group. The ACA treatment reversed that suppression. Surprisingly, the memantine treatment resulted in overexpression of TRPM2-L, to a certain extent. Examination of the rats in EPM and OFM apparatuses, as a display of anxiety-like behavior, did not reveal any marked difference among groups. Serum levels of TNF-α and MDA also did not vary significantly among groups, as well. Conclusively, our findings showed for the first time that TRPM2-L protein expression was significantly suppressed in the ICV-STZ induced memory deficit model. Even though ACA reversed this suppression, no improvement in spatial memory was observed following ACA treatment.

A series of new matrinic derivatives with an 11-adamantyl group were designed, synthesized and evaluated for their anti-influenza A H3N2 activities, based on the privileged structure strategy.SAR analysis indicated that introduction of an 11-adamantyl by ester linker might be helpful for the activity. Among them, compound 7b exhibited promising anti-H3N2 activities with IC50 value of 5.14 μM, slightly better than that of amantadine. Its activity was further confirmed at the protein level. In primary mechanism, compound 7b could inhibit virus replication cycle at early stage by targeting M2 protein, consistent with that of amantadine. This study represents a successful application of combined strategy of privileged amantadine segment for further structural optimization and development of a new class of anti-influenza agents.

A glutamate/NMDA receptor (NMDA-R) antagonist, amantadine (AMA) exhibits a broad spectrum of clinically important properties, including antiviral, antiparkinsonian, neuroprotective, neuro-reparative and cognitive-enhancing effects. However, both clinical and pre-clinical studies have demonstrated that noncompetitive NMDA-R antagonists induce severe schizophrenia-like cognitive deficits. Therefore, this study aims to clarify the clinical discrepancy between AMA and noncompetitive NMDA-R antagonists by comparing the effects of AMA with those of a noncompetitive NMDA-R antagonist, MK801, on rat tripartite glutamatergic synaptic transmission using microdialysis and primary cultured astrocytes. Microdialysis study demonstrated that the stimulatory effects of AMA on L-glutamate release differed from those of MK801 in the globus pallidus, entorhinal cortex and entopeduncular nucleus. The stimulatory effect of AMA on L-glutamate release was modulated by activation of cystine/glutamate antiporter (Sxc). Primary cultured astrocytes study demonstrated that AMA also enhanced glutathione synthesis via Sxc activation. Furthermore, carbon-monoxide induced damage of the astroglial glutathione synthesis system was repaired by AMA but not MK801. Additionally, glutamate/AMPA receptor (AMPA-R) antagonist, perampanel enhanced the protective effects of AMA. The findings of microdialysis and cultured astrocyte studies suggest that a combination of Sxc activation with inhibitions of ionotropic glutamate receptors contributes to neuroprotective, neuro-reparative and cognitive-enhancing activities that can mitigate several neuropsychiatric disorders.

A new fluorescent "turn-on" probe-based immunosensor for detecting drug residues in foodstuffs was established by combining the mechanism of aggregation-induced emission (AIE) and an indirect competitive enzyme-linked immunosorbent assay (ELISA). In this study, a luminogen, with negligible fluorescence emission (TPE-HPro), aggregated in the presence of H2O2, and exhibited astrong yellow emission based on its AIE characteristics. This AIE process was further configured into an immunoassay for analyzing drug residues in foodstuffs. In this approach, glucose oxidase (GOx) was used as an enzyme label for the immunoassay and triggered GOx/glucose-mediated H2O2 generation, which caused oxidation of TPE-HPro and a "turn-on" fluorescence response at 540 nm. To quantitatively analyze the drug residues in foodstuffs, we used amantadine (AMD) as an assay model. By combining the AIE-active "turn-on" fluorescent signal generation mechanism with conventional ELISAs, quantifying AMD concentrations in chicken muscle samples was realized with an IC50 (50% inhibitory concentration) value of 0.38 ng/mL in buffer and a limited detection of 0.06 μg/kg in chicken samples. Overall, the conceptual integration of AIE with ELISA represents a potent and sensitive strategy that broadens the applicability of the AIE-based fluorometric assays.

Although the anti-influenza virus drug oseltamivir ameliorates the fever of influenza, adverse events related to its hypothermic effect have been reported. We found that oseltamivir causes dose-dependent hypothermia in normal mice, and have been studying the pharmacological mechanisms responsible for 12 years. Oseltamivir blocks nicotinic cholinergic transmission at sympathetic ganglia and reduces sympathetic modulation of brown adipose tissue (BAT), a heat generator. Oseltamivir was found to target the ion channels of nicotinic acetylcholine receptors, as demonstrated by patch clamp experiments with cells expressing the human α3β4 nicotinic receptor. Metabolized oseltamivir carboxylate, which inhibits the influenza virus neuraminidase, did not elicit hypothermia and ion channel suppression. Body temperature was decreased by intracerebroventricular administration of oseltamivir. Because this hypothermic effect was inhibited by dopamine D2 receptor blockade, it was suggested that oseltamivir centrally stimulates the D2 receptor. In Japan, the package inserts for oseltamivir and amantadine indicate very similar adverse neuropsychiatric reactions for the two drugs (abnormal behavior, consciousness disturbance, convulsion, delirium, delusion, hallucination). A literature search revealed that in some previous studies, oseltamivir and amantadine were shown to block the ion channel systems and activate the dopaminergic nervous system via several mechanisms. Therefore the similarity of the adverse reactions elicited by oseltamivir and amantadine was considered attributable to their similar pharmacological effects.