Disorders of the central nervous system linked to dysregulation of dopamine D₂ receptors — including depression, drug addiction, Parkinson’s disease, and movement disorders — remain challenging to treat with selectivity. While most known allosteric modulators of G protein–coupled receptors are synthetic, the tripeptide melanostatin, MIF-1; Pro–Leu–Gly-NH₂, stands out as the only endogenous positive allosteric modulator, PAM, with specificity for the D₂ receptor. First isolated from hypothalamic extracts in 1971, MIF-1 potentiates dopamine agonist binding in an inverted-U concentration–response profile, yet its biosynthesis, bioactive conformation, and structural pharmacophore remain incompletely defined. Importantly, the l-proline residue in MIF-1 has long been suspected to play a critical role in adopting a type II β-turn conformation thought to underlie its activity.

To probe these structural determinants, researchers from the Sampaio-Dias Lab at the University of Porto, LAQV-REQUIMTE, in collaboration with colleagues from the University of Santiago de Compostela, published in ACS Medicinal Chemistry Letters, synthesized and evaluated a series of l-pipecolyl-based MIF-1 analogues, substituting proline with the six-membered pipecolic acid scaffold and introducing variations at the C-terminal functional group. The synthetic route employed standard peptide coupling, Boc deprotection, and amidation steps to yield a small focused library including compound 1, ipecolyl–Leu–Gly-NH₂, and compound 9, pipecolyl–Leu–Gly-OMe, with full characterization by NMR and high-resolution mass spectrometry.

When tested in functional assays at human D₂ receptors expressed in CHO cells, the analogues showed divergent effects on dopamine potency. At subnanomolar concentrations, 0.01 nM, compound 1 modestly increased dopamine potency, 1.8-fold, while compound 9 matched or exceeded the activity of MIF-1 itself, 4.1-fold. At 1 nM, MIF-1 activity declined and compound 1 lost activity altogether, but compound 9 retained robust PAM activity, shifting the dopamine EC50 more than four-fold without altering maximal efficacy. Control compounds containing carbamate substitutions were inactive, highlighting the importance of the amide-to-ester isosteric replacement in conferring activity.

Mechanistic studies confirmed that neither 1 nor 9 acted as intrinsic agonists, supporting a bona fide PAM mechanism. In silico calculations suggested that 1 preferentially adopts β-turn conformations, while 9 favors γ-turns due to the absence of a C-terminal carboxamide hydrogen bond donor. Cytotoxicity assays in differentiated SH-SY5Y neuroblastoma cells showed that both compounds were non-toxic at concentrations up to 100 μM, comparable to the parent peptide.

Taken together, this work demonstrates that homologation of proline with pipecolic acid is compatible with D₂ receptor PAM activity, and that an amide-to-ester substitution can not only be tolerated but also enhance potency. The findings challenge prior assumptions that β-turn conformations are strictly required for activity and instead point to alternative structural solutions, including γ-turn motifs. By revealing flexible pharmacophore requirements for MIF-1 derivatives, this study provides valuable structure–activity relationship insights and identifies compound 9 as a promising lead for the rational design of novel anti-Parkinson’s drug candidates.