Seasonal influenza viruses infect 5-10% of adults and 20-30% of children each year, yet only two classes of antiviral drugs are available so far, one targeting the influenza A M2 (AM2) protein and the other targeting neuraminidase (NA). AM2 forms a proton channel in the virus envelope that is crucial for the virus lifecycle; blockage of the AM2 pore curbs virus infection. Unfortunately, in recent years 99% of flu strains have become resistant to the FDA-approved AM2 channel blockers, amantadine and rimantadine. Recently, new antiviral drugs effective against amantadine-resistant AM2 proteins have been reported, but their mechanisms of action have remained elusive. Water plays an important role in proton conduction through the AM2 pore, but it is not understood how the new AM2 inhibitors affect water dynamics in the channel. Elucidating channel water dynamics should provide insights into the mechanism of action of anti-influenza drugs. I propose to develop and employ solid-state nuclear magnetic resonance (SSNMR) spectroscopy to investigate the dynamic interactions of AM2 with water, with drugs, and with another virus protein, matrix protein 1 (AM1). Specifically, I will develop SSNMR techniques that correlate the dynamically sensitive 2H quadrupolar coupling with 13C chemical shifts to provide site-resolved information about small-molecule dynamics and protein dynamics.
In Aim 1, I will measure the effects of antiviral drugs on channel hydration and on water dynamics in the AM2 pore. Both 1H-13C and 2H- 13C correlation experiments will be conducted to detect the dynamics of protonated and deuterated channel water, respectively, giving complementary information. The results will provide a detailed molecular understanding of the mechanism of action of the novel drugs and facilitate future design of better antiviral compounds.
In Aim 2, I will measure the conformational dynamics of AM2 as influenced by drugs, to address how the transmembrane (TM) domain dynamics affect antiviral drug activity and whether the protein dynamics is related to channel-water dynamics. These studies will shed light on the yet unkown binding site of recently developed antiviral drugs. 2H, 13C-labeled proteins will be used in this study. Secondly, I will characterize AM2 interaction with AM1. Biochemical evidence suggests that this interaction is important for virus assembly and budding, thus elucidating the structural basis for this interaction may help the design of alternative antiviral drugs. I will measure changes in the AM2 cytoplasmic domain?s dynamics and chemical shifts upon binding to AM1, to understand whether binding immobilizes AM2 and changes protein structure. I have obtained extensive preliminary data that demonstrate the feasibility of the 2H-13C correlation techniques and have successfully expressed and purified 13C,2H,15N-labeled proteins. It will also provide first-time information about the importance of molecular motion on the function of an important biomedical target.
The influenza A M2 (AM2) protein is one of only two antiviral drug targets for seasonal and pandemic flu. Unfortunately, mutations in the transmembrane domain of this membrane protein have caused widespread resistance to the FDA-approved drugs. This proposal seeks to design novel solid-state NMR techniques to obtain structural and dynamical information that reveal the mechanism of action of new drugs against drug-resistant AM2 proteins, and to understand the interaction between AM2 and matrix protein 1 (AM1), which may act as an alternative drug target.
