Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay

Abstract

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.

Figure 1. Effect of M2 expression and amantadine on yeast growth.

Yeast strains containing an empty plasmid (A) or plasmid bearing WT M2 (B), S31N M2 (C) or V27A M2 (D) were distributed into 96-well plates and their growth was measured over time following transfer at 0h to medium containing galactose and addition of the indicated concentrations of amantadine.

Figure 2. Effect of a spiroadamantane amine on the growth of yeast expressing WT and mutated M2.

Yeast strains containing the indicated plasmids were distributed in 96-well plates in medium containing galactose and exposed to the indicated concentrations of the spiroadamantane amine (structure shown) for 40 h.

Figure 3. 96-well pilot screen results.

(A) Yeast expressing WT M2 were exposed to 1,440 screening chemicals (M2+ Drugs) with 8 untreated wells per plate as negative controls (M2, n = 144). Each plate also contained 8 positive controls not treated with drugs (Empty plasmid, n = 144). Growth was measured after 40 h and the % growth restoration was calculated as described in Materials and Methods. The arrow points to the single active compound found in this batch. (B) the active compound found in A was retested at different concentrations against yeast expressing WT M2 or bearing the empty plasmid and growth was measured after 40 h.

Figure 4. Structures, names and EC₅₀ of active compounds found in the screen.

Figure 5. Activity of hexamethylene amiloride and selected triazine analogs in the TEVC assay.

Each point is the mean and standard deviation of five to eight oocytes.

Figure 6. Antiviral activity and cytotoxicity of selected compounds.

The effect of the compounds on influenza A/Udorn/72 WT virus replication was evaluated by plaque formation in MDCK cells. Cytotoxicity towards MDCK cells was assayed after 48 h drug exposure with the MTT tetrazolium dye assay as described .