Mixed squaramide thioesters as phosphate group analogues for non-competitive antagonists of the phospholipid-sensing G protein-coupled receptor GPR55.

High-affinity inhibitors of specific receptors are a valuable tool to elucidate cellular signaling, control biological systems and develop therapeutic drugs. Here, we report mixed squaramide thioesters as a novel electrophilic handle, demonstrating an optimal balance between nucleophile accessibility and hydrolytic stability in aqueous environments. Density function theory calculations show that the energy of the lowest unoccupied molecular orbital (LUMO) of mixed squaramide thioesters is lower compared to mixed squaramide esters, rendering them more reactive to nucleophilic attack. Synthetic access to various lysophosphatidylglucoside (LysoPtdGlc) analogues incorporating mixed squaramide thioesters as phosphate bioisosteres was readily established by condensation of mixed squaramide esters with the corresponding thiols. Next, we characterised the inhibitory activity of these analogues in biological assays of axon growth cone chemotropism in cultured primary nociceptive neurons. These synthetic analogues induce both acute and sustained (more than 12 h) inhibition of the GPR55/LysoPtdGlc signaling. This inhibition resulted in sustained antagonistic attenuation of GPR55-mediated axon chemotropism while preserving growth cone sensitivity to other GPR55-independent chemotropic signaling molecules. Our findings demonstrate the potential of thiosquaramide-based phosphate bioisosteres as highly specific inhibitors with well controlled reactivity, expanding the repertoire of modulators for lipid-sensing GPCRs.