Copper Complexes as Influenza Antivirals: Reduced Zebrafish Toxicity

Abstract

Copper complexes have previously been developed to target His37 in influenza M2 and are effective blockers of both the wild type (WT) and the amantadineresistant M2S31N. Here, we report that the complexes were much less toxic to zebrafish than CuCl 2. In addition, we characterized albumin binding, mutagenicity, and virus resistance formation of these metal complexes, and employed steered molecular dynamics simulations to explore whether the complexes would fit in M2. We also examined their anti-viral efficacy in a multi-generation cell culture assay to extend the previous work with an initial-infection assay, discovering that this is complicated by cell culture medium components. The number of copper ions binding to bovine serum albumin (BSA) correlates well with the number of surface histidines and BSA binding affinity is low compared to M2. No mutagenicity of the complexes was observed when compared to sodium azide. After 10 passages of virus in MDCK culture, the EC 50 was unchanged for each of the complexes, i.e. resistance did not develop. The simulations revealed that the compounds fit well in the M2 channel, much like amantadine.

Mechanism and kinetics of copper complexes binding to the influenza A M2 channel

“…In summary, the ITC and computational chemistry results provide insight into the ability of the copper complexes to fit in the M2 channel near the His37 cluster and deliver the copper to the imidazole nitrogen. This provides an explanation for the two-stage M2 current block in the electrophysiology results, and the invulnerability of the complexes to resistance formation (17).…”

“…Considering that IDA ligation of the copper substantially reduces its toxicity in zebrafish embryos (17), it would be interesting to examine toxicity and ADME properties of the complexes in higher animal models. Table 4: Copper complexes binding imidazole N δ in implicit water solvent.…”

“…In vitro, Cu(CO-IDA) and had low cytotoxicity and was potent at sub-micromolar concentrations against A/Calif/07/2009 H1N1, which contains M2 S31N (17). In an evaluation of M2 resistance development against these copper complexes, 10 passages were insufficient for resistance development in contrast to resistance to amantadine, which develops after only a few passages (17). However, there is another naturally occurring mutation, G34E, that could interfere with binding of the copper complexes because of the proximity to the binding site and added bulk of the glutamate side chains.…”

An Overview of Antivirals for Treating Lower Respiratory Tract Infections

“…In addition, there are currently in vitro, in vivo, and clinical studies of other promising anti-influenza compounds; some are based on oseltamivir: NA inhibitors such Laninamivir octanoate (23) [50,57,59], HA protein inhibitors BMY-27709 (24) and MBX-2546 (25), and viral replication inhibitors favipiravir (26) and pimodivir (27) [60]. Some compounds that have shown in vitro antiviral activity (as M2 channel inhibitors) are copper based compounds 28 and 29, and cobalt compound (30) [61]. Amantadine (31), previously considered as a useful M2 ion channel inhibitor, was found ineffective against influenza B viruses.…”

Mechanism and Kinetics of Copper Complexes Binding to the Influenza A M2 S31N and S31N/G34E Channels

“…In vitro, Cu(CO-IDA) and had low cytotoxicity and was potent at submicromolar concentrations against A/Calif/07/ 2009 H1N1, which contains M2 S31N (17). In an evaluation of M2 resistance development against these copper complexes, 10 passages were insufficient for resistance development, in contrast to resistance to amantadine, which develops after only a few passages (17).…”