0000-0002-9245-7846 (De La Cruz, Daniel)

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Purpose: Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) is a small peptide with numerous neuro-modulatory impacts beyond its role within the neuroendocrine system. TRH's broad central nervous system (CNS) effects, acting as a neurotransmitter and neuromodulator, emphasize a great potential to treat many neurological and psychological disorders. However, its pharmacological applications remain unrealized due to brain delivery shortcomings following its systemic administration. Previously, our laboratory's novel prodrug design, relying on two highly brain-expressed enzymes for prodrug metabolism to TRH, successfully delivered the metabolically highly unstable peptide into the brain. Consequently, an in vivo therapeutic safety assessment was conducted to further validate our prodrug approach through a comparative study that capitalizes on TRH's stimulatory release of thyroid hormones. Likewise, TRH's ability to trigger acetylcholine release is also well documented, and here, this neurochemical marker has been utilized to determine the extent to which TRH is delivered to the CNS via our prodrug approach. Moreover, our laboratory has recently identified pGlu-βGlu-Pro-NH2 ([βGlu2]TRH) as the first functional antagonist of the central cholinergic actions of TRH, and as such, we have explored the receptor-associated mechanism responsible for this antagonism by utilizing a human TRH receptor (hTRH-R) homology model. Ultimately, our extensive computational chemistry-based studies revealed a novel receptor allosteric site that exhibits a selective and high-affinity binding for [βGlu2 ]TRH, while also demonstrating our prodrug's inability to bind and activate this hTRH-R. Methods: Lead TRH prodrugs and various TRH analogues were designed in silico for docking experiments with the hTRH-R using SeeSAR and AutoDock Vina software. A TRH challenge in CD-1 mice, utilizing systemically administered TRH and an equimolar concentration of a TRH prodrug, measured downstream effector levels of thyroid hormones at several subsequent time points analyzed by LC-MS/MS. Microdialysis studies, in the frontal cortex of SD rats, compared each animal's baseline acetylcholine concentration to subsequent levels, following the perfusion of a TRH prodrug and TRH, as a positive control, at equimolar concentrations. This neurochemical survey quantifies acetylcholine turnover using LC-MS/MS, as a surrogate measure of the extent to which TRH is delivered into the brain via our prodrug approach. Results: Compared to TRH, prodrugs were unable to dock to the hTRH-R's active site, and when systemically administered, the TRH prodrug failed to elicit a thyroid response while simultaneously triggering a profound release of acetylcholine in the brain. Conclusions: The inability of TRH prodrugs to elicit a thyroid response was predicted by its in vitro metabolic stability, as well as computational chemistry studies, that demonstrate TRH prodrugs exhibit physiochemical properties that prohibit the direct activation of the hTRH-R. Furthermore, based on [βGlu2]TRH as a template, the design of novel hTRH-R inhibitors will be conducted in a follow-up study to further substantiate our prodrug approach and aid the elucidation of TRH activity and pathways.