Publications -- Laszlo Prokai

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31879

This collection is limited to articles published under the terms of a creative commons license or other open access publishing agreement since 2016. It is not intended as a complete list of the author's works.

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    The impact of 17beta-estradiol on the estrogen-deficient female brain: from mechanisms to therapy with hot flushes as target symptoms
    (Frontiers Media S.A., 2024-01-23) Prokai-Tatrai, Katalin; Prokai, Laszlo
    Sex steroids are essential for whole body development and functions. Among these steroids, 17beta-estradiol (E2) has been known as the principal female" hormone. However, E2's actions are not restricted to reproduction, as it plays a myriad of important roles throughout the body including the brain. In fact, this hormone also has profound effects on the female brain throughout the life span. The brain receives this gonadal hormone from the circulation, and local formation of E2 from testosterone via aromatase has been shown. Therefore, the brain appears to be not only a target but also a producer of this steroid. The beneficial broad actions of the hormone in the brain are the end result of well-orchestrated delayed genomic and rapid non-genomic responses. A drastic and steady decline in circulating E2 in a female occurs naturally over an extended period of time starting with the perimenopausal transition, as ovarian functions are gradually declining until the complete cessation of the menstrual cycle. The waning of endogenous E2 in the blood leads to an estrogen-deficient brain. This adversely impacts neural and behavioral functions and may lead to a constellation of maladies such as vasomotor symptoms with varying severity among women and, also, over time within an individual. Vasomotor symptoms triggered apparently by estrogen deficiency are related to abnormal changes in the hypothalamus particularly involving its preoptic and anterior areas. However, conventional hormone therapies to "re-estrogenize" the brain carry risks due to multiple confounding factors including unwanted hormonal exposure of the periphery. In this review, we focus on hot flushes as the archetypic manifestation of estrogen deprivation in the brain. Beyond our current mechanistic understanding of the symptoms, we highlight the arduous process and various obstacles of developing effective and safe therapies for hot flushes using E2. We discuss our preclinical efforts to constrain E2's beneficial actions to the brain by the DHED prodrug our laboratory developed to treat maladies associated with the hypoestrogenic brain."
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    Effect of estradiol replacement on hippocampal concentrations of estrogens in aged rhesus macaques maintained on an obesogenic diet
    (Elsevier B.V., 2023-09-25) Prokai, Laszlo; Nguyen, Vien; Urbanski, Henryk F.
    Replacement involving estrogens has proven efficacy at treating a wide range of disorders that develop with menopause or after surgical removal of the ovaries. Here, we tested whether an estradiol (E2) replacement paradigm that recapitulates physiological E2 levels in the circulation also recapitulates physiological E2 levels within the hippocampus. E2 was delivered continuously to old ovariectomized (OVX) rhesus macaques, maintained on a high-fat, high-sugar Western-style diet (WSD) for approximately 30 months, via subcutaneous implants; this resulted in physiological concentrations of both estrone (E1) and E2 in the circulation (determined by LC-MS/MS). Surprisingly, however, hippocampal concentrations of E2 were markedly (P < 0.01) higher than in ovary-intact animals maintained on a regular chow diet. The data suggest that E2 replacement paradigms that appear to recapitulate physiological E2 concentrations in the circulation may produce hyper-physiological E2 levels within some brain areas, especially when individuals are maintained on a WSD.
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    Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma
    (MDPI, 2023-08-26) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.
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    Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications
    (MDPI, 2021-02-08) Prokai, Laszlo; Rahlouni, Fatima; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin
    The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17beta-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.
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    Brain-Selective Estrogen Therapy Prevents Androgen Deprivation-Associated Hot Flushes in a Rat Model
    (MDPI, 2020-06-10) Merchenthaler, Istvan; Lane, Malcolm; Stennett, Christina; Zhan, Min; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Hot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17beta-estradiol, E2) are produced from testosterone. Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10beta, 17beta-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.
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    Comparative Proteomics Analysis Reveals Unique Early Signaling Response of Saccharomyces cerevisiae to Oxidants with Different Mechanism of Action
    (MDPI, 2020-12-26) Pandey, Prajita; Zaman, Khadiza; Prokai, Laszlo; Shulaev, Vladimir
    The early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H2O2, cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H2O2, 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.
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    Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry
    (MDPI, 2016-01-16) Prokai, Laszlo; Stevens, Stanley M., Jr.
    Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.
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    Treatment with an orally bioavailable prodrug of 17beta-estradiol alleviates hot flushes without hormonal effects in the periphery
    (Springer Nature, 2016-08-01) Merchenthaler, Istvan; Lane, Malcolm; Sabnis, Gauri; Brodie, Angela; Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin
    Estrogen deprivation has a profound effect on the female brain. One of the most obvious examples of this condition is hot flushes. Although estrogens relieve these typical climacteric symptoms, many women do not want to take them owing to unwanted side-effects impacting, for example, the uterus, breast and blood. Therefore, there is a need for developing safer estrogen therapies. We show here that treatment with 10beta,17beta-dihydroxyestra-1,4-dien-3-one (DHED), a novel brain-targeting bioprecursor prodrug of the main human estrogen, 17beta-estradiol, alleviates hot flushes in rat models of thermoregulatory dysfunction of the brain. Oral administration of DHED elicits a significant reduction of tail skin temperature (TST) rise representing hot flushes in the morphine-dependent ovariectomized rat model and results in the restoration of estrogen deprivation-induced loss of diurnal rhythm in TST. These beneficial effects occur without detrimental peripheral hormonal exposure; thus, the treatment avoids potentially harmful stimulation of estrogen-sensitive peripheral organs, including the uterus and the anterior pituitary, or the proliferation of MCF-7a breast cancer cell xenografts. Our promising preclinical assessments warrant further considerations of DHED for the development of a brain-selective 17beta-estradiol therapy to relieve hot flushes without undesirable peripheral side-effects.
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    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, Katalin
    After 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.
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    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, Laszlo
    We 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.
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    A Novel Prodrug Approach for Central Nervous System-Selective Estrogen Therapy
    (MDPI, 2019-11-19) Prokai-Tatrai, Katalin; Prokai, Laszlo
    Beneficial effects of estrogens in the central nervous system (CNS) results from the synergistic combination of their well-orchestrated genomic and non-genomic actions, making them potential broad-spectrum neurotherapeutic agents. However, owing to unwanted peripheral hormonal burdens by any currently known non-invasive drug administrations, the development of estrogens as safe pharmacotherapeutic modalities cannot be realized until they are confined specifically and selectively to the site of action. We have developed small-molecule bioprecursor prodrugs carrying the para-quinol scaffold on the steroidal A-ring that are preferentially metabolized in the CNS to the corresponding estrogens. Here, we give an overview of our discovery of these prodrugs. Selected examples are shown to illustrate that, independently of the route of administrations and duration of treatments, these agents produce high concentration of estrogens only in the CNS without peripheral hormonal liability. 10beta,17beta-Dihydroxyestra-1,4-dien-3-one (DHED) has been the best-studied representative of this novel type of prodrugs for brain and retina health. Specific applications in preclinical animal models of centrally-regulated and estrogen-responsive human diseases, including neurodegeneration, menopausal symptoms, cognitive decline and depression, are discussed to demonstrate the translational potential of our prodrug approach for CNS-selective and gender-independent estrogen therapy with inherent therapeutic safety.
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    [beta-Glu(2)]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain
    (MDPI, 2021-06-09) Prokai-Tatrai, Katalin; Nguyen, Vien; Prokai, Laszlo
    Selective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu(2)]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [beta-Glu(2)]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [beta-Glu(2)]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [beta-Glu(2)]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.
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    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, Laszlo
    Using 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.
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    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, Laszlo
    The 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.