NMR Based Screening Assay for Novel Chemotypes against influenza A virus
Of the current available drugs against influenza A virus, two target the M2 proton channel . These are the adamantane-based compounds Amantadine and its close analogue Rimantadine. The M2 protein includes a 24-residue N-terminal extracellular domain, a 19-residue hydrophobic transmembrane domain, and a 54-residue cytoplasmic tail. A number of biophysical and computational studies strongly more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: R03MH082366
Assay Provider: Dr. Maurizio Pellecchia, Sanford-Burnham Medical Research Institute
Of the current available drugs against influenza A virus, two target the M2 proton channel . These are the adamantane-based compounds Amantadine and its close analogue Rimantadine. The M2 protein includes a 24-residue N-terminal extracellular domain, a 19-residue hydrophobic transmembrane domain, and a 54-residue cytoplasmic tail. A number of biophysical and computational studies strongly suggest that M2 forms a homotetrameric transmembrane alpha-helical bundle that acts as a proton channel. Here, the channel s activated at lower values (pH 6.2) and it is closed at physiological pH. This function has been attributed to the H37 residue located in the channel pore. As anticipated above, this pH regulation by the M2 proton channel is fundamental to the virus life cycle. Following virus endocytosis and endosome the M2 activation induces acidification that weakens the bonds between the viral RNA and the virus capsid stimulating its release. Conversely, during virus exit, the M2 channel prevents acidification of the exosome which would cause premature and irreversible conformational changes of the viral hemagglutinin. Hence, the development of molecules capable of blocking the M2 channel represent an effective and attractive strategy for anti-influenza drugs. However, the use of currently available drugs against the M2 receptors, Amantadine (1-aminoadamantane) and Rimantadine rather limited since the 1960s, due to several side effects, due to the inhibition of the human NMDA receptor, and the appearance of amantadine-resistant viral strains that contain single amino acid substitutions in the M2 protein. Two of the most recurring mutations that conferred amantadine resistance are localized to the transmembrane domain of the protein, the site of binding of amantadine, are S31N and V27A. The first has been shown to reduce the size of the pore thereby preventing the binding of Amantadine. The second has been shown to weaken the binding of Amantadine by reducing the interaction interface between the drug and the interior of the pore. The scope of this project was to screen for small molecules capable of binding to the M2 V27A proton channel in an in vitro binding assay with a reconstituted channel in lipids and to subsequently verify that this binding results in inhibition of viral replication. Unfortunately, no viable hits were identified
1. De Clercq, E. (2006). Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 5, 1015-1025.
1. Sample preparation
The TM domain (22-46) of influenza A virus M2 ion channel was synthesized by Anaspec with purity >95%. The peptide was dissolved in DPC (at a peptide/DPC concentration ratio of 1:100). The final test samples were dilute with 100mM phosphate buffer (%95/5% H2O/D2O, pH 8.15) to obtain 25 uM of the tetrameric M2 channel. The sequence of the peptide used was 22-SSDPLAVAASIIGILHLILWILDRL -46 (MW 2700), hence including the V27A mutation that is amantadine resistant.
2. Compound Library Screened by NMR
We have assembled a scaffold library composed of ~ 4,000 compounds. The compounds have been selected based on their anticipated use as building blocks or scaffolds components of further optimized molecules. The scaffold library has been acquired from three different sources and the chemical structures of the library have been deposited into PubChem. In line with the general NIH Molecular Libraries Screening Centers Network (MLSCN) library, we have also included a collection of 602 Natural Products (MicroSource) that could be screened by NMR
3. NMR Based Screening description
A. Ligand binding was monitored by comparing side chain amino proton peak of the tryptophan residue (~10.5ppm) of a 25 uM M2 channel solution (100 mM sodium phosphate buffer at pH 8.15 containing 95%/5% H2O/D2O; T= 300 K) in the presence and in absence of compounds tested at a final concentration of 1000 uM. Compounds were initially tested at mixtures of 20, and then individual compounds for those mixtures that caused significant perturbations of the tryptophan peak were further deconvoluted. No significant hits were reported by this screen and compounds are with an estimated Kd > 4000 uM.
The score is an estimate that reflects the binding affinity of the compound for the target and the accuracy of the method used to measure the dissociation constants. Compounds with an estimated Kd > 4000 uM are assigned a Kd of 4001 uM. Compounds for which we do not have a complete titration curve are scored on a scale from 0 to 40 according to the following scale:
Kd > 4000 uM Assigned Score 0
1000 < Kd < 4000 uM Assigned Score 10
500 < Kd < 1000 uM Assigned Score 20
100 < Kd < 500 uM Assigned Score 30
Kd < 100 uM Assigned Score 40
Data Table (Concise)