Steffen, J. H. | Search bioRxiv

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2022

Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B

ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. Instead, an increased interaction between MBD5 and the core protein was observed that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

biophysics↗