Browsing by Author "De La Cruz, Daniel L."
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Item Advancing the Role of Thyrotropin-releasing Hormone as a Central Nervous System Agent(2022-05) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.; Forster, Michael J.Item Brain Delivery of Thyrotropin-Releasing Hormone via a Novel Prodrug Approach(MDPI, 2019-07-18) Prokai-Tatrai, Katalin; De La Cruz, Daniel L.; Nguyen, Vien; Ross, Benjamin P.; Toth, Istvan; Prokai, LaszloUsing thyrotropin-releasing hormone (TRH) as a model, we explored whether synergistic combination of lipoamino acid(s) and a linker cleaved by prolyl oligopeptidase (POP) can be used as a promoiety for prodrug design for the preferential brain delivery of the peptide. A representative prodrug based on this design principle was synthesized, and its membrane affinity and in vitro metabolic stability, with or without the presence of a POP inhibitor, were studied. The in vivo formation of TRH from the prodrug construct was probed by utilizing the antidepressant effect of the peptide, as well as its ability to increase acetylcholine (ACh) synthesis and release. We found that the prototype prodrug showed excellent membrane affinity and greatly increased metabolic stability in mouse blood and brain homogenate compared to the parent peptide, yet a POP inhibitor completely prevented prodrug metabolism in brain homogenate. In vivo, administration of the prodrug triggered antidepressant-like effect, and microdialysis sampling showed greatly increased ACh release that was also antagonized upon a POP inhibitor treatment. Altogether, the obtained promising exploratory data warrant further investigations on the utility of the prodrug approach introduced here for brain-enhanced delivery of small peptides with neurotherapeutic potential.Item Improving Brain Delivery of TRH: A Novel Prodrug Approach(2018-08-01) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.The goal of my research project was to validate a novel prodrug design concept for the brain-enhanced delivery of an important neuropeptide, thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2). TRH has a variety of clinically relevant central effects that cannot be utilized with the direct administration of TRH, due to intrinsic characteristics that give rise to metabolic instability and insufficient transport through the blood-brain barrier (BBB). Consequently, large doses are required to generate central effects, which concomitantly induces unwanted hormonal liabilities in the periphery. To overcome these caveats, the prodrug design described herein proposes a novel brain-targeting approach that synergistically utilizes two enzymes, both preferentially expressed in the brain, for the enhanced brain-delivery of TRH. The conjugation of a lipoamino acid (LAA) transport moiety to the N-terminus of a TRH progenitor sequence (Gln-His-Pro-NH2) via a prolyl oligopeptidase (POP)-sensitive linker allows the POP enzyme to release the TRH progenitor sequence at the site-of-action upon crossing the BBB, where it is further transformed by glutaminyl cyclase (QC). QC catalyzes Gln to form pGlu at the N-terminus of the progenitor sequence, thereby releasing TRH. I tested the hypothesis that a representative molecule, developed according to this prodrug design approach, would exhibit adequate drug-likeness for BBB penetration and efficacious release of TRH within the brain. Immobilized artificial membrane chromatography was used to predict the BBB penetration of this experimental prodrug, labeled “PRODRUG 1,” in addition to its calculated logP. Next, PRODRUG (1) was compared to TRH (the “parent” peptide) in an in vitro metabolic stability assessment, followed by an in vivo neuro-pharmacodynamical evaluation in rodents. The Porsolt swim test, PST, an established animal behavioral model that detects depressive-like behavior was used to confirm brain-delivery of prodrug-derived TRH after systemic administration of the prototype prodrug. Capitalizing on TRH’s antidepressant-like effect, the PST results were also used to validate the tail suspension test (TST), a new technique that I implemented in our laboratory for the evaluation of neuroactive compounds with potential antidepressant-like activity. My findings support the extension of the TRH progenitor sequence from the N-terminus, through the conjugation of two LAA residues (each with a 10-carbon sidechain) via a single proline POP-sensitive linker, as a successful means to increase penetration across the BBB and sufficiently bind with cleaving and activating enzymes, POP and QC, respectively, for efficacious TRH release in the brain. Lastly, molecular modeling was used to create a library of similarly designed prodrugs to computationally assess their bindings with POP, the cleaving enzyme, to further explore the customizable prodrug design concept described here. Ultimately, this adaptable prodrug delivery model demonstrates the effectiveness of increased lipophilicity and site-of-action targeting to facilitate brain-enhanced delivery of TRH.Item Proteomics-Based Retinal Target Engagement Analysis and Retina-Targeted Delivery of 17beta-Estradiol by the DHED Prodrug for Ocular Neurotherapy in Males(MDPI, 2021-09-02) Prokai-Tatrai, Katalin; Zaman, Khadiza; Nguyen, Vien; De La Cruz, Daniel L.; Prokai, LaszloWe examined the impact of 17beta-estradiol (E2) eye drops on the modulation of the proteome profile in the male rat retina. With discovery-driven proteomics, we have identified proteins that were regulated by our treatment. These proteins were assembled to several bioinformatics-based networks implicating E2's beneficial effects on the male rat retina in a broad context of ocular neuroprotection including the maintenance of retinal homeostasis, facilitation of efficient disposal of damaged proteins, and mitochondrial respiratory chain biogenesis. We have also shown for the first time that the hormone's beneficial effects on the male retina can be constrained to this target site by treatment with the bioprecursor prodrug, DHED. A large concentration of E2 was produced after DHED eye drops not only in male rat retinae but also in those of rabbits. However, DHED treatment did not increase circulating E2 levels, thereby ensuring therapeutic safety in males. Targeted proteomics focusing on selected biomarkers of E2's target engagement further confirmed the prodrug's metabolism to E2 in the male retina and indicated that the retinal impact of DHED treatment was identical to that of the direct E2 treatment. Altogether, our study shows the potential of topical DHED therapy for an efficacious and safe protection of the male retina without the unwanted hormonal side-effects associated with current estrogen therapies.Item Retina-Targeted Delivery of 17beta-Estradiol by the Topically Applied DHED Prodrug(MDPI, 2020-05-16) Prokai-Tatrai, Katalin; Nguyen, Vien; De La Cruz, Daniel L.; Guerra, Rebecca; Zaman, Khadiza; Rahlouni, Fatima; Prokai, LaszloThe purpose of this study was to explore retina-targeted delivery of 17beta-estradiol (E2), a powerful neuroprotectant, by its bioprecursor prodrug 10beta,17beta-dihydroxyestra-1,4-dien-3-one (DHED) administered as eye drops in animal models. Compared to the parent hormone, DHED displayed increased transcorneal flux ex vivo both with and without the presence of 2-hydroxypropyl-beta-cyclodextrin used as a penetration-enhancing excipient in rat, rabbit, and pig. In vitro, the prodrug also showed facile bioactivation to E2 in the retina but not in the cornea. After topical administration to rats and rabbits, peak DHED-derived E2 concentrations reached 13 +/- 5 ng/g and 18 +/- 7 ng/g in the retina of female rats and rabbits, respectively. However, the prodrug remained inert in the rest of the body and, therefore, did not cause increase in circulating hormone concentration, as well as wet uterine and anterior pituitary weights as typical markers of E2's endocrine impact. Altogether, our studies presented here have demonstrated the premise of topical retina-selective estrogen therapy by the DHED prodrug approach for the first time and provide compelling support for further investigation into the full potential of DHED for an efficacious and safe ocular neurotherapy.Item The Antagonist pGlu-betaGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor(MDPI, 2021-09-05) De La Cruz, Daniel L.; Prokai, Laszlo; Prokai-Tatrai, KatalinAfter we identified pGlu-betaGlu-Pro-NH2 as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH's binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-betaGlu-Pro-NH2. The allosteric binding of this TRH antagonist is more robust than TRH's binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.