Measuring, in solution, multiple-fluorophore labeling by combining Fluorescence Correlation Spectroscopy and photobleaching

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (@ 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other.Comment: 38 pages (avec les figures

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

Molecular Mechanism of AMPA Receptor Noncompetitive Antagonism

10 páginas, 6 figuras.AMPA-type glutamate receptors are specifically inhibited by the noncompetitive antagonists GYKI-53655 and CP-465,022, which act through sites and mechanisms that are not understood. Using receptor mutagenesis, we found that these antagonists bind at the interface between the S1 and S2 glutamate binding core and channel transmembrane domains, specifically interacting with S1-M1 and S2-M4 linkers, thereby disrupting the transduction of agonist binding into channel opening. We also found that the antagonists' affinity is higher for agonist-unbound receptors than for activated nondesensitized receptors, further depending on the level of S1 and S2 domain closure. These results provide evidence for substantial conformational changes in the S1-M1 and S2-M4 linkers following agonist binding and channel opening, offering a conceptual frame to account for noncompetitive antagonism of AMPA receptors.This work was supported by grants from the Israel Science Foundation (Y.S.-B.), the Israeli Ministry of Health (Y.S.-B.), the Spanish Ministry of Education and Science (J.L.), and by an Israeli-Spanish Scientific Cooperation grant from the Israeli Ministry of Science and the Spanish Ministry of Foreign Affairs (Y.S.-B. and J.L.). V.B. is a recipient of a scholarship from the Bernard Katz Minerva Center for Cell Biophysics.Peer reviewe

Discovery of Novel Dual Inhibitors of the Wild-Type and the Most Prevalent Drug-Resistant Mutant, S31N, of the M2 Proton Channel from Influenza A Virus

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (44) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections. [Image: see text

3-Azatetracyclo[5.2.1.1 5,8

We have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane scaffold designed as analogs of amantadine, an inhibitor of the M2 proton channel of influenza A virus. Inhibition of the wild-type (wt) M2 channel and the amantadine-resistant A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Most of the novel compounds inhibited the wt ion channel in the low micromolar range. Of note, several compounds inhibited the A/M2 V27A mutant ion channel, one of them with submicromolar IC(50). None of the compounds was found to inhibit the S31N mutant ion channel. The antiviral activity of three novel dual wt and A/M2-V27A channels inhibitors was confirmed by influenza virus yield assays

Perampanel Inhibition of AMPA Receptor Currents in Cultured Hippocampal Neurons

Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM), concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M-1 s-1 and 0.58 s-1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM) was similar at all kainate concentrations (3-100 µM), demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics