Photoswitchable ORG25543 Congener Enables Optical Control of Glycine Transporter 2

Mostyn, Shannon N.; Sarker, Subhodeep; Muthuraman, Parthasarathy; Raja, Arun; Shimmon, Susan; Rawling, Tristan; Cioffi, Christopher L.; Vandenberg, Robert J.

doi:10.1021/acschemneuro.9b00655

Cited by 8 publications

(11 citation statements)

References 29 publications

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“…As expected, no inhibition decay was observed for ORG25543 according to its irreversibility. A second piece of evidence came from experiments using the A223C mutant of GlyT2 that contains an exogenous cysteine in EL1, a region sensitive to the different conformations of the transporter induced by the substrates during the transport cycle . As reported previously, the MTSEA-biotin labeling of the A223C mutant is protected by glycine in a buffer containing NaCl but not in a medium containing choline chloride, indicating the accessibility of the cysteine is reduced when sodium is bound (Figure B,C).…”

Section: Resultsmentioning

confidence: 51%

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Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543

Benito-Muñoz

Perona

Felipe

et al. 2021

ACS Chem. Neurosci.

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The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O -[(2-benzyloxyphenyl-3-fluorophenyl)methyl]- l -serine (ALX1393) and N -[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster . Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

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Section: Resultsmentioning

confidence: 51%

“…Interestingly, a photoswitchable derivative of this compound made by substituting the benzyl phenyl ether moiety (that substitutes R6 in ORG25543) by an azobenzene in the trans configuration, to which it is structurally homologous, still maintains the reversibility and the noncompetitive inhibition. 44 …”

Section: Resultsmentioning

confidence: 99%

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543

Benito-Muñoz

Perona

Felipe

et al. 2021

ACS Chem. Neurosci.

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“…GlyT2 is expressed by presynaptic neurons and is also responsible for replenishing presynaptic glycine concentrations to maintain glycinergic neurotransmission. Inhibitors of GlyT2 slow the reuptake of glycine to prolong glycine neurotransmission ( 5 ) and have been developed as potential therapeutics in the treatment of chronic pain ( 6 , 7 , 8 , 9 , 10 , 11 ).…”

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confidence: 99%

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

Wilson

Mostyn

Frangos

et al. 2021

Journal of Biological Chemistry

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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.

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“…First, regarding carbon double bond configuration, only lipids with carboncarbon double bonds in the cis configuration are active and lipids with double bonds in the trans configuration are inactive (Mostyn et al, 2017). Isomerization is also important for small molecule inhibitors of GlyT2, albeit to a lesser extent with only subtle differences in potency between isomers (Mostyn et al, 2020). The contrast in activity of different tail configurations suggests that the binding site of these lipids is conformationally restricted.…”

Section: Lipid Modulation Of Glyt2mentioning

confidence: 99%

Glycine Transporter 2: Mechanism and Allosteric Modulation

Frangos

Chater

Vandenberg

2021

Front. Mol. Biosci.

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Neurotransmitter sodium symporters (NSS) are a subfamily of SLC6 transporters responsible for regulating neurotransmitter signalling. They are a major target for psychoactive substances including antidepressants and drugs of abuse, prompting substantial research into their modulation and structure-function dynamics. Recently, a series of allosteric transport inhibitors have been identified, which may reduce side effect profiles, compared to orthosteric inhibitors. Allosteric inhibitors are also likely to provide different clearance kinetics compared to competitive inhibitors and potentially better clinical outcomes. Crystal structures and homology models have identified several allosteric modulatory sites on NSS including the vestibule allosteric site (VAS), lipid allosteric site (LAS) and cholesterol binding site (CHOL1). Whilst the architecture of eukaryotic NSS is generally well conserved there are differences in regions that form the VAS, LAS, and CHOL1. Here, we describe ligand-protein interactions that stabilize binding in each allosteric site and explore how differences between transporters could be exploited to generate NSS specific compounds with an emphasis on GlyT2 modulation.

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Photoswitchable ORG25543 Congener Enables Optical Control of Glycine Transporter 2

Cited by 8 publications

References 29 publications

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543

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

Glycine Transporter 2: Mechanism and Allosteric Modulation

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