The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity

Abstract

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric-binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here, we use a combination of medicinal chemistry, electrophysiology, and computational modeling to develop a rational structure–activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length, and double-bond position promote enhanced inhibition. Overall, the l -stereoisomer is generally a better inhibitor than the d -stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric-binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment.

Glycine Transporter 2: Mechanism and Allosteric Modulation

“…In contrast to the lysine analogues where potency is slightly reduced, the D-tryptophan isomer is inactive (Mostyn et al, 2019a). In simulations, the tail of C18 ω9 D-tryptophan fails to properly extend into the hydrophobic groove, instead projecting towards EL4 (Wilson et al, 2021). Shallower penetration of the C18 ω9 D-lysine tail may be mitigated by stacking between Y550 and W563, resulting in a reduction in potency rather than the complete loss of activity observed with C18 ω9 D-tryptophan.…”

“…Y550 coordinates C18 ω9 L-lysine above the double bond whereas changing the stereochemistry of the lipid headgroup to D-lysine shifts this interaction below the double bond (Wilson et al, 2021). This shift allows Y550 to hydrogen bond with W563, locking the tail of C18 ω9 D-lysine between these two residues (Wilson et al, 2021). However, the same trend is not observed when modelling the change from C18 ω9 L-tryptophan to C18 ω9 D-tryptophan.…”

“…Simulations of compounds docked in this region of a GlyT2 homology model have been complemented with mutagenesis studies to define the LAS (Subramanian et al, 2016;Mostyn et al, 2019b;Wilson et al, 2021). During the simulations the lipids manoeuvre themselves away from their docked position such that the tail extends into a hydrophobic groove between TMs 5, 7, and 8 (Figure 5) (Mostyn et al, 2019b).…”

“…In initial simulations Y550 (EL4) interacts with the lipid headgroup of all compounds suggesting it interacts with a conserved element of the amino acids (Mostyn et al, 2019b). However, subsequent simulations have shown that Y550 interacts with the acyl tail double bond, and have helped explain the structure activity relationships of the bioactive lipids (Wilson et al, 2021). Y550 coordinates C18 ω9 L-lysine above the double bond whereas changing the stereochemistry of the lipid headgroup to D-lysine shifts this interaction below the double bond (Wilson et al, 2021).…”

“…However, subsequent simulations have shown that Y550 interacts with the acyl tail double bond, and have helped explain the structure activity relationships of the bioactive lipids (Wilson et al, 2021). Y550 coordinates C18 ω9 L-lysine above the double bond whereas changing the stereochemistry of the lipid headgroup to D-lysine shifts this interaction below the double bond (Wilson et al, 2021). This shift allows Y550 to hydrogen bond with W563, locking the tail of C18 ω9 D-lysine between these two residues (Wilson et al, 2021).…”