Gepirone (Exxua^TM) is an agonist of the serotonin 5-HT_1A receptor and its metabolite 1-(2-pyrimidinyl)-piperazine (1-PP) is a potent antagonist of the α₂-adrenergic receptor. This medication, which is taken orally to treat major depressive disorder, already has been synthesized in 1986, however, several times been rejected by the US Food and Drug Administration (FDA) due to insufficient evidence of effectiveness. Finally, in 2023, it has been approved after a sustained release formulation was introduced. As typical for a small molecule drug, it shows a considerable number of side effects, such as dizziness, nausea, insomnia, abdominal pain, and dyspepsia.

Gepirone is a perfect example, how Linkerology® and conjugation to an appropriate carrier molecule can improve efficacy of a drug molecule and reduce side effects. A suitable targeting vehicle, e.g., a nanobody, will effectively transport this molecule to the point of action, shield it from interaction with undesired points of interaction and thus limit side effects, hence, improve the pharmacokinetic profile and boost its overall efficacy.

The tertiary amine of Gepirone (see chemical structure below) is the most nucleophilic position and can easily be alkylated to generate the quaternary ammonium derivative by reaction with the benzyl chloride function of an appropriate p-­aminobenzyl (PAB) linker. In this example, we selected the Val-Ala motif as cleavable part and decorated it with CliCr® (Tetramethylthiocycloheptyne-sulfoximine, TMTHSI), a highly reactive and very well water-soluble strained alkyne moiety for copper-free Click chemistry.

Alkylation of the cyclic tertiary amine in Gepirone by CliCr®-valyl-alanyl-benzyl chloride. This linker-Gepirone conjugate may be coupled in a Click reaction, e.g., to a nanobody, which carries p‑azidophenylalanine.

In contrast to monoclonal antibodies, which have slow blood clearance as well as slow and low tumor uptake, antibody fragments, such as affibodies, single-chain fragments (ScFv) or nanobodies, demonstrated superior behavior. Novel cell-free technologies allow the fast and easy synthesis of any fragment, including the incorporation of non-canonical amino acids, like p-azidophenylalanine (HAA2980), enabling the direct Click conjugation of any alkyne-terminated linker-payload construct. CliCr® is an ideal moiety for this purpose, as it is a small, polar, and highly reactive alkyne fragment for strain-promoted copper-free Click chemistry.

After internalization of the nanobody-payload construct, lysosomal C-terminal proteases, such as cathepsin B, hydrolyze the peptide bond, which leads to further decomposition and the traceless release of the payload Gepirone.

Lysosomal proteases, like Cathepsin B, hydrolyze the alanyl p-amidobenzyl bond, which results in the fragmentation and in the subsequent liberation of the cargo molecule Gepirone.

→ Interested in the vast possibilities of sophisticated linker technologies? Download our brochure Linkerology®

→ Do you want to learn more about CliCr®? Check-out our Click Chemistry Brochure or watch the recording of our online workshop about "Innovations in Click Chemistry"

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