Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

The glycine transporter 2 allosteric inhibitor ORG25543 has different molecular determinants of potency and reversibility (#309)

Ryan Cantwell Chater 1 , Katie Wilson 2 , Megan O'Mara 2 , Christopher Cioffi 3 , Robert Vandenberg 1
  1. Molecular Bioscience, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia
  2. Research School of Chemistry, College of Science, The Australian National University, Canberra, ACT, Australia
  3. Basic and Clinical Sciences and Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA

Neuropathic pain is caused, in part, by disrupted glycinergic neurotransmission. It is hypothesised that inhibitors of the glycine transporter 2 (GlyT2) will transiently increase glycine concentrations to restore nociceptive control. ORG25543 (IC50= 18.0 nM) is a selective, irreversible and non-competitive inhibitor of GlyT2 that has analgesics effects in animal models of pain1. We have used site directed mutagenesis to characterise the binging site and mechanism of action of ORG25543.

It has been proposed that ORG25543 binds to an extracellular vestibule site of GlyT22. This site, known as the vestibule allosteric site, is formed by transmembrane domains (TMs) 1b, 6a, 10 and 11. We have found that ORG25543 does not bind to this site, but to an allosteric lipid inhibitor binding site comprised of TMs 5, 7 and 8 and extracellular loop (EL) 43. Contrary to previous studies, we have found that mutations in the vestibule allosteric site do not significantly alter the potency of ORG255432. We have identified several key residues in the allosteric lipid binding site that modulate the potency ORG25543, particularly in TM5. The transition from an outward-open confirmation to an inward-open conformation is a crucial process in the transport cycle and is associated with partial unwinding of TM54. We propose that ORG25543 inhibits GlyT2 by preventing the unwinding of TM5 and slows the transition to the inward facing state. Reversibility of the compound is impacted, to different extents, by mutations in the allosteric lipid binding site, the vestibule allosteric site and the substrate site. The mutations that have the greatest impact on potency do not have the greatest impact on reversibility. We suggest that ORG25543 binds to the allosteric lipid binding site, with reversibility being determined by the conformational flexibility of the transporter.

  1. Mingorance-Le Meur A, Ghisdal P, Mullier B, De Ron P, Downey P, Van Der Perren C, et al. (2013). Reversible inhibition of the glycine transporter GlyT2 circumvents acute toxicity while preserving efficacy in the treatment of pain. Br J Pharmacol 170: 10531063.
  2. Benito-Muñoz, C., Perona, A., Felipe, R., Pérez-Siles, G., Núñez, E., Aragón, C. and López-Corcuera, B., 2021. Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543. ACS Chemical Neuroscience.
  3. Mostyn SN, Wilson KA, Schumann-Gillett A, Frangos ZJ, Shimmon S, Rawling T, et al. (2019). Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics. Elife 8.
  4. Wilson, K.A., Mostyn, S.N., Frangos, Z.J., Shimmon, S., Rawling, T., Vandenberg, R.J. and O’Mara, M.L., 2021. The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity. Journal of Biological Chemistry, 296.