Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Small molecule inhibitors modulate the glycine transporter 2 by interacting at an allosteric site (#103)

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 and related compounds.

Two potential binding sites have been proposed for ORG25543. The first is an allosteric lipid inhibitor binding site comprised of transmembrane domains (TMs) 5, 7 and 8 and extracellular loop (EL) 42. The second site is based on the cryo-EM structure of s-citalopram bound to the human serotonin transporter, a homologue of GlyT23. This site, known as the vestibule allosteric site, is formed by TMs 1b, 6a, 10 and 11. We have identified several key residues in the allosteric lipid binding site that modulate the potency and reversibility of ORG25543. The mutation Y550L in EL4 results in reversible inhibition by ORG25543 but does not alter potency. Mutations W563L (TM 8) (IC50= 65.7 nM) and V432A (TM 5) (IC50= 245 nM) decrease the potency of ORG25543. W563L maintains ORG25543 irreversibility however V432A induces slow reversibility. We propose that the cyclopentane functional group of ORG25543 interacts with Y550 and is the major determinant of reversibility. At the alternate vestibule site, the mutations D469A (TM6a), Q473A (TM6a) and T634A (TM10) did not alter the potency of ORG25543. 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 TM53. We propose that ORG25543 inhibits GlyT2 by preventing the unwinding of TM5 and slows the transition to the inward facing state.

  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. 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.
  3. Coleman JA, Green EM, & Gouaux E (2016). X-ray structures and mechanism of the human serotonin transporter. Nature 532: 334-339.