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Radiopharmaceutical development hinges on the affinity and selectivity of the biological component for the intended target. An analogue of the neuropeptide Substance P (SP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[Thi 8 ,Met(O 2 ) 11 ]-SP (DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP), in the theranostic pair [ 68 Ga]Ga-/ [ 213 Bi]Bi-DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP has shown promising clinical results in the treatment of inoperable glioblastoma. As the theranostic targeting component, modifications to SP that affect the selectivity of the resulting analogue for the intended target (neurokinin-1 receptor [NK1R]) could be detrimental to its therapeutic potential. In addition to other closely related tachykinin receptors (neurokinin-2 receptor [NK2R] and neurokinin-3 receptor [NK3R]), SP can activate a mast cell expressed receptor Mas-related G protein-coupled receptor subtype 2 (MRGPRX2), which has been implicated in allergic-type reactions. Therefore, activation of these receptors by SP analogues has severe implications for their therapeutic potential. Here, the receptor selectivity of DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP was examined using inositol phosphate accumulation assay in HEK293-T cells expressing NK1R, NK2R, NK3R or MRGPRX2. DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP had similar efficacy and potency as native SP at NK1R, but displayed greater NK1R selectivity. DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP was unable to elicit significant activation of the other tachykinin receptors nor MRGPRX2 at high concentrations nor did it display antagonistic behaviour at these receptors. DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP, therefore has high potency and selectivity for NK1R, supporting its potential for targeted theranostic use in glioblastoma multiforme and other conditions characterised by NK1R overexpression.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.