How to overcome resistance of influenza A viruses against adamantane derivatives.

We tested two approaches to overcoming resistance of influenza A viruses against adamantane derivatives. First, adamantane derivatives that interfere with the ion channel function of the variant M2 protein of amantadine-resistant viruses may prevent drug resistance, if they are used in mixture with amantadine. Second, amantadine acts on the M2 protein (at low concentrations) and indirectly on the hemagglutinin (at concentrations at least 100 times higher). Identifying and using a drug that reacted with both targets at the same concentration might reduce development of resistance, since, in this case, two mutations, one in each target protein would be necessary at once. Such a double mutation is assumed to be a rare event. We evaluated forty adamantane derivatives and two related compounds to determine whether they interfered with plaque formation by influenza A strains, including A/Singapore/1/57 (H2N2). Variants resistant to drugs that interfered at low concentrations (approximately 1 microg/ml; e.g. amantadine) were cross-resistant with each other, but were sensitive to those agents effective at high concentrations (8 microg/ml; e.g. memantine). The former group of compounds act on the ion channel; the corresponding escape mutants tested had amino acid replacements at positions 27, 30 or 31 of the M2 protein. Hemagglutinin was the indirect target of the latter group of compounds. Variants resistant to these agents lacked amino acid replacements within the ion channel of the M2 protein and the mutants tested had amino acid replacements in the hemagglutinin. Although we failed to identify compounds that interacted with the ion channel of amantadine-resistant variants and inhibited their replication, we were able to construct at least two compounds that interfered with both the ion channel and the hemagglutinin at about the same concentration. After passage in the presence of these compounds, we either failed to obtain any drug-resistant mutants or those obtained had amino acid replacements in the ion channel of the M2 protein and the hemagglutinin.