Pharmaceutical Sciences

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/32090

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    Microfluidic Chip for the evaluation of therapeutics and carrier drugs in breast cancer tissue
    (2023) Rincon, Julio
    Introduction: Breast cancer is the most common cancer in women where 1 in 8 will develop over the course of her lifetime. However, some treatments react differently depending on race and ethnicity. Unfortunately, there are no preclinical models capable of studying pharmacoethnicity differences of drugs. Hence, racial differences are found late during clinical trials or after market adoption. The absence of a model that studies pharmacoethnicity differences results in negative outcomes for the affected race. The objective of this project is to design and evaluate a microfluidic in-vitro platform to test therapeutics and carrier drugs, such as Doxil liposomes, in breast tumor tissue and compare properties based on the tissue’s race and other attributes. The microfluidic device is capable of delivering a dual channel fluid system with the intention of emulating blood and interstitial fluid in tissue. The chip is unique from other systems by directly using human tissue and designing a microfluidic chamber specific to a patient’s tumor allowing it to be used for personalized medicine. Methods: To build a microfluidic platform, a 15 µm tumor section is mounted to a positively charged slide. Then capillary channels are etched at 120 µm diameter with an estimated precision ≤ 10 µm, channel spacing is set to 350 and 500 µm. Channels are etched in glass slides using a 5W UV Laser Marking Machine with 70 mm f-theta lens, at a pulse rate of ≤ 15 ns. Current chip model requires etching two main capillary glass slides. Each slide is then covered by an intermediate thin layer per etched channel side. Afterwards, the slides are assembled with UV resin Loctite 349 and cured under UV light for 20 minutes. Finally, a third slide is used as the interface between the chip and the chromatography tubing. After assembly, the blood and interstitial fluid channels are loaded with deionized water and are flow and pressure tested. As a proof of concept, an acridine orange solution was used with a preliminary chip. Dye penetration was then measured using an inverted microscope. Results: A total of 8 microfluidic chips have been successfully built, and an additional 19 chips are under development. The initial microfluidic chip prototype demonstrated tumor tissue was stained by an acridine orange solution by a total of up to 75 µm past the capillary channels. The preliminary chips exhibited chamber issues where interstitial fluid merged with the blood channel. This has now been successfully corrected in the current chip model. A total of 6 microfluidic chips will be used with a carrier drug, such as Doxil liposomes. Conclusions: After method revisions, where are now capable of developing fully working microfluidic chips. Our preliminary studies demonstrate the capabilities of using our chips to test drug penetration in tissue. Our current objective is to measure drug penetration in breast cancer tumors based on race. Future studies will include additional method development to enable live tissue use and to measure additional drug properties.
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    Impact of pH modulation and NaDC micellular behavior on Amphotericin B solubility
    (2023) Mans, Jaylen C.
    Purpose: Amphotericin B (AmB) has very poor water solubility (< 1 ug/mL), resulting poor oral absorption. Thus, AmB is only commercially produced in intravenous (IV) formulations. One AmB IV formulation, Fungizone® (FZ), uses sodium deoxycholate (NaDC), a surfactant, and sodium phosphate, a pH-modifying agent, to dissolve AmB. As per instructions, the powder is dissolved in water and then diluted lower than NaDC critical micelles concentration with 5% dextrose. To understand why a low concentration NaDC could dissolve AmB, we investigated the mechanism of AmB dissolution in FZ preparation. Methods: AmB solubility in aqueous buffer solutions:Aqueous pH buffers at pH 1.2, 4.5, 6.8, 7.8, and 11 were prepared. AmB powder was mixed with pH buffers at room temperature (RT) or 37C for 24 h and processed for AmB concentration measurement via HPLC. Impact of pH on NaDC micelle formation:NaDC was mixed into pH buffer solutions at 4 mg/mL concentration and separately dissolved into pH 11 buffer at 0.08, 0.2, 0.4, 4, 10, and 20 mg/mL concentrations. The solutions were measured by Dynamic Light Scattering (DLS) to evaluate micelle formation. NaDC micelle formation in the presence of AmB at pH 11:Micelle formation in 4 mg/mL NaDC-Buffer solution was measured by DLS. Next, NaDC was mixed in pH 11 buffer, DI water, and 5% dextrose solution at 0.08, 0.2, 0.4, 4, 10, and 20 mg/mL concentrations, and measured via DLS. Additionally, AmB was mixed into NaDC media solutions at 0.1 and 5 mg/ml and measured via DLS. Enhancement of NaDC and pH 11 on AmB solubility:NaDC was dissolved in pH buffer at 4, 10, and 20 mg/mL. AmB powder then was added into the NaDC solution and mixed at RT. At 1, 12, and 24 h, samples were collected prepared for HPLC analysis. Fungizone® Preparation and Characterization:Lab FZ and commercial FZ solutions were prepared. The lab FZ solution was prepared by mixing ingredients at RT until the solution was visibly clear. Commercial FZ powder was reconstituted with DI water. FZ solutions were diluted 1:50 with filtered 5% dextrose. Following dilution, the commercial FZ was characterized via pH measurements, DLS analysis, and HPLC measurements. Results: AmB is not stable at extreme pH with heat; therefore, solubility studies were performed at RT. AmB (pKa 5.5 and 10) had the highest solubility at pH 11 (216 ug/mL). NaDC has different micellular behavior in different pH conditions, generating lower CMCs at pH 7.8 and 11. Further, pH 11 was able to increase aqueous AmB solubility in the presence of NaDC. Conclusion: Both pH 11 and NaDC can increase AmB aqueous solubility; however, pH 11 combined with NaDC can synergistically enhance AmB solubility significantly. Additionally, pH 11 facilitated stable AmB-NaDC micelles formation below NaDC critical micelle concentration in water. Thus, the initial reconstitution of FZ in water generated a high pH facilitated AmB dissolution via AmB-NaDC micelles formation. Once the micelles formed, they were stable for further dilution with 5% dextrose below the CMC of NaDC.
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    Pharmacokinetic and Physicochemical Evaluation of Novel Drug Candidates for Retinitis Pigmentosa
    (2023) Garrett, Meredith; Curry, Stacy; Feris, Sherri; Martin, Stephen; Clark, Abbot; Kastellorizios, Michail
    Purpose: Retinitis pigmentosa is a set of inherited ocular diseases that affect nearly 3 million people worldwide. The condition is inherited and causes the progressive deterioration of the retina. Retinitis pigmentosa begins with the loss of rod photoreceptors which cause night blindness and a decrease in peripheral vision. After significant loss of rod cells, cone cells also begin to die, decreasing central vision until complete blindness. More than 150 genetic mutations in 80 different genes have thus far been identified to contribute to progression pathways of the condition. Despite ongoing stem cell and gene therapy investigations, thus far there are no curative options. Most existing treatments focus on slowing the progression of retinal deterioration by reducing oxidative stress on the retina. Unfortunately, these treatments only achieve limited success and cannot halt progression. Recently, the sigma 2 receptor (σ2r) was identified to be endoplasmic reticulum membrane protein 97 (TMEM97). This protein (σ2r/TMEM97) has been shown to have neuroprotective effects on retinal cells and is thus of interest as a potential drug target for retinitis pigmentosa. Here we synthesized and tested a series of six compounds which have previously been found to modulate σ2r/TMEM97. To determine which of these compounds is a suitable drug candidate, each underwent in vivo and in vitro testing with the goal of selecting the best candidate for further clinical development. Methods: We tested the compounds in a rat model to determine retinal uptake following intravitreal injection. Each drug was dissolved in dimethyl sulfoxide (DMSO) and injected into the eye. At set time points, animals were sacrificed, and retinas were isolated from harvested eyes. The retina was separated and homogenized using sonication. A small portion was removed and underwent protein precipitation to purify the sample. The samples were then analyzed via liquid chromatography mass spectrometry (LCMS) to find the drug concentration remaining at each timepoint. In addition to obtaining a pharmacokinetic profile, the compounds were physiochemically characterized for chemical stability, solubility, in vitro drug release from vitreous humor, thermal analysis, and surface tension. Conclusion: Our goal is to select those drug candidates with the highest chance of clinical success. The pharmacokinetic profiles as well as physicochemical characteristics and stability of the compounds obtained in this study revealed important differences between the compounds that were used in selecting which to advance to in vivo efficacy testing. Ongoing studies include completion of physicochemical characterization and in vivo efficacy in a retinitis pigmentosa rat model that will be used to identify top candidates for further development.
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    Molecular Docking Studies for Designing and Identification of Novel Bitopic Ligands for Sigma-1 Receptor
    (2023) Ponce, Aiyana; Kumari, Pratibha; Liu, Jin
    Purpose: Our goal is to identify more specific ligands for the Sigma-1 Receptor (S1R) by designing and docking various bitopic ligands then ranking them based on their interactions. The S1R is an intracellular, multifunctional receptor that is a target in many pathologies. S1R is found throughout the body, within the membranes of the nucleus, ER, and mitochondria, and the CNS is the primary site of activity. Bitopic ligands are those that combine the high affinity by binding to orthosteric sites and high selectivity by binding to allosteric sites on the same receptor. They are used to gain insights on molecular functioning of the receptor. Methods: A set of 3 linkers were tested with a known allosteric binding compound and the one with highest affinity was selected to continue in the design process. Nine bitopic ligands were designed using known allosteric compounds, the selected linker, and a high affinity orthosteric compound. Ligands were minimized with Avogadro then PDBQT files for minimized ligands and S1R open-state confirmation were prepared with AutoDock Tools. All newly designed ligands were docked on S1R using Vina to determine their binding affinities. Interactions were visualized using Pymol and quantified using Protein Ligand Interaction Profiler. Results: After testing 3 linkers from the literature, we identified 1 that gave highest affinity with our allosteric compound and selected to use it in the design of new bitopic ligands. Our results shows that all ligands docked in a bend conformation in the S1R. Specifically, bitopic ligands having benzazepine derivatives showed greater affinity to S1R. The number of hydrophobic bonds, hydrogen bonds, pi bonds, and salt bridges were identified for each interaction between the ligand and S1R. Conclusions: The 1-2 compounds with the highest affinity and favorable interactions are candidates to be used for future drug design models.
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    Assessing tumor biopsy decellularization using contact angle method
    (2023) Spano, Giacomo; Guyot, Savannah; Banda, Brenda Natalia
    Purpose: More than 30% of new female cancer cases each year are breast cancer, according to the National Cancer Institute, whose risk increases progressively with advancing age. To date, off-target toxicity remains a main drawback of anticancer therapy. Several drug delivery systems are developed to bypass this problem, and among these the nanoparticle-based delivery platforms have proved extremely promising. A successful example of this approach is liposome formulation which today is used to encapsulate drugs such as doxorubicin (DOXIL) and paclitaxel. A critical role in the development of anticancer drugs is played by preclinical testing, but animal testing is very expensive, time-consuming, and limited due to their different physiology. Using patient tissue biopsies may help identify promising drugs to advance to clinical trials, saving time and money. Extracellular matrix (ECM) in this context is well known to present an impact on anticancer drug efficacy by acting as a barrier between drug molecules and targeting cancer cells, but little has been done so far to quantify and characterize the behavior of drugs and formulations on it. ECM hydrophobicity, in particular, can affect drug penetration through tissue and tissue membranes changing drug absorption. We hypothesize that contact angle can be used as part of the pre-clinical screening of promising anti-cancer drug candidates by assessing ECM hydrophobicity and ECM-drug interactions. Here, we optimized a tissue decellularization process to test ECM hydrophobicity and interaction with liposomes using contact angle measurement. Methods: An in-house modified optical goniometer instrument was used to measure the contact angle on glass slides and de-identified, commercially available breast cancer tissue sections (US Biomax Inc). Samples were deparaffinized by consecutive washing cycles in xylene, ethanol, and water. The slides were dried in the incubator for 60 minutes at 60 °C to ensure adequate tissue adherence. Tissues were then decellularized by up to three freeze-thaw cycles in water 0.1% w/v SDS (Sodium dodecyl sulfate). Contact angle measurements obtained at every step of the deparaffinization and decellularization process were used to determine the optimum tissue processing method. The contact angle of liposomal suspensions was then collected on glass and tissue sections that were processed accordingly. Results: The optimum decellularization process for cancer biopsy sections was two freeze-thaw cycles using pure water; SDS was found to cause some tissue detachment from the glass slide. After each freezing cycle, contact angle was trending upwards. This indicates that tissue decellularization results in progressively more hydrophobicity, likely due to the removal of water-soluble proteins and polysaccharides. This effect was more pronounced in tumor tissue compared to healthy tissue from the same patient. Contact angles obtained using liposomal suspensions were lower than water contact angles and followed similar patterns. Conclusions: -Contact angle measurements can be used to quantify and optimize tissue decellularization. - Decellularized biopsies are slightly more hydrophobic, an effect more pronounced in tumor tissue compared to healthy. - Liposomes reduce contact angle due to their inherent surfactant-like properties. - Contact angle may be used to distinguish how anti-cancer liposomes interact with tumor and healthy tissue.
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    Prediction of Protospacer Adjacent Motif (PAM) dependencies in CRISPR-Cas9 systems and design of novel Cas9 with broad PAM compatibility
    (2023) Pham, Tyler; Madugula, Sita Sirisha; Liu, Jin
    Purpose: CRISPR-Cas9 gene editing capabilities has experienced many limiting factors and biological constraints preventing its rapid adoption and mass utilization. Of interest is the Cas9 protein’s ability and necessity to recognize unique Protospacer Adjacent Motif (PAM) DNA sequences prior to its gene editing functionality. This recognition constraint has limited the scope of targetable regions of DNA, and thus prevents access to extensive sections of DNA by the Cas9 protein. Given the multitude of Cas9 species, it has become a challenge to fully comprehend the wide variations in this relationship. Machine learning (ML) applications have increasingly been developed to discern obscure patterns and relationships to aid in the analysis and design of the next generation of proteins. In this project, we hypothesize that the relationship between PAM-I domain sequences and its corresponding PAM DNA sequence can be computationally understood to predict new PAM DNA sequences and novel PAM-interacting (PAM-I) protein domain sequences. Specifically, our model attempts to directly associate such a relationship with a sequence-based approach between amino acid and DNA. The implementation of computational technologies into understanding biological function can facilitate the advancements in overcoming the innate constraints in the CRISPR-Cas9 system and provide a pipeline toward modern protein engineering. Method: Protein and DNA sequence data were extracted from public database sources. From EMBL-Uniprot queries, Cas9 protein sequences were obtained for various species. From NCBI-GenBank, protein CRISPR sequences were obtained from each protein’s respective genomic DNA matched by accession. Collection of PAM DNA sequences were gathered from predictive alignments utilizing NCBI-BLAST. Construction of our model utilizes a transformer architecture implementing text embedding on the sequence data. Results: The final construction of our database contains a total of 795 unique Cas9 protein sequences, from which their corresponding PAM-I domains were extracted. From their respective genomic DNA, a total of 18,445 CRISPR sequences were found. From which, we have aligned and collected a large set of PAM DNA targets for each protein species. With our collection of Cas9 protein domain sequences and their associated PAM DNA sequences, we have trained and tested a novel ML model to discern and classify the relationship between the two associated sequences. To further expand the relationship, a similar transformer ML model will be developed to methodically generate unique protein domain sequences capable of recognizing PAM DNA sequence targets. Final accuracy results from our prediction and generation models remain in progress and are pending, with the expectation of reaching at least 50% for both models. Conclusion: Given its absence and public availability, a unique database of protein PAM-I domains and their associated PAM DNA sequences has been successfully developed and curated to facilitate the development and testing of our novel ML models. The results and outcomes of this project can create an opportunity to directly integrate into a modern protein engineering pipeline to build and test new libraries of Cas9 proteins.
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    Structural optimization of VU0609159 as an activator of Slack potassium channels
    (2023) Peprah, Paul; Du, Yu; Spitznagel, Brittany; Mishra, Nigam; Qunies, Alshaima'a; Weaver, David; Emmitte, Kyle
    Purpose: Slack (Slo2.2) is a sodium-activated potassium channel widely expressed throughout the brain and encoded by the KCNT1 gene. This channel modulates the firing patterns and general excitability of many types of neurons, contributing to neuronal resting membrane potential, action potential repolarization, and afterhyperpolarization. Increasing evidence suggests that channelopathies that alter Slack activity, triggers cognitive dysfunction, as has been found for Fragile X Syndrome (FXS), the most common cause of intellectual disability (ID) and inherited autism. FXS results from the absence of fragile X mental retardation protein (FMRP) which interacts directly with Slack channels to regulate outward currents termed IKNa in neuronal cells. Prior studies involving animal models of FXS demonstrated that lack of FMRP leads to reduced IKNa currents due to diminished Slack activity, affecting neuronal function. We therefore hypothesize that small molecule activators of Slack have potential utility as therapeutics for neurological disorders associated with Slack hypofunction. Thus, the objective of this study is to discover small molecule activators of Slack potassium channels that may be used as in vitro probes to investigate such a hypothesis. A high-throughput screen using a thallium (Tl+) flux assay identified the hit compound VU0609159 (VU159) as a moderately potent Slack activator. Here we report our efforts to develop structure-activity relationships (SAR) in the VU159 series through an iterative, systematic optimization plan using parallel library synthesis. Method: Our approach involved identifying multiple regions of VU159 that could be readily diversified and using short efficient synthetic routes to produce small libraries of analogs. Systematic substitution using a variety of monomers was carried out around the western benzoxazolone ring, the central amide and linker region as well as the eastern aromatic ring. Structure and purity of all analogs were confirmed using spectra obtained from a Bruker Fourier 300HD NMR spectrometer and an Agilent 6230 time-of-flight LC/MS. Cellular activity was then evaluated using a Tl+ flux assay in HEK-293 cells that stably express wild-type (WT) Slack channels. Results: Each region of the VU159 scaffold proved tolerant of modification to some degree. Multiple fused heterocyclic rings proved competent replacements for the benzoxazolone ring with some analogs providing superior potency. Synthesis of alkylated linkers identified preferred enantiomers. Secondary amides were preferred to tertiary amides. Likewise, cyclic linkers were generally less effective than acyclic linkers. Finally, substitution of the eastern aryl ring was allowed with some analogs demonstrating enhanced potency. Conclusion: Our systematic optimization plan has identified multiple slack activator analogs with improved activity relative to VU159. Multiple regions of the scaffolds are amenable to SAR development, which greatly enhances the probability of reaching our goal of highly optimized in vitro probes. Additional modifications, including preparation of analogs that combine optimal features, could provide additional SAR and analogs with optimal potency for use as an in vitro probe.
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    Ligand Identification for the Orthosteric site of Sigma 1 Receptor using Computational Molecular Docking and Virtual Screening Methods
    (2023) Olson, Zachary; Liu, Jin; Kumari, Pratibha
    Purpose: The 𝝈1 Receptor (Sig-1R) is a ligand operated membrane protein resides in the mitochondria-associated-membranes of the endoplasmic reticulum (ER). At the molecular level, Sig-1R has several important roles in cellular homeostasis, including Ca2+ regulation, and helping chaperone the unfolded protein response. This ER stress has been found to be one of the factors leading to cytokine storm and clinical deterioration in patients with a coronavirus infection, leading to an interest in drugs which modulate the response of the Sig-1R for treatment of COVID-19. These receptors are found throughout the CNS as well as the periphery, explaining its wide range of effects throughout the body. At the organ level, studies conducted on the Sig-1R have implicated its involvement in neurodegenerative diseases such as Parkinson’s and Alzheimer’s Disease, cardiac diseases such as heart failure and cardiovascular disease (CVD), and major depressive disorder. This implication in a wide variety of disease states means it has a large potential as a drug target. Our study's purpose is to identify novel potential drug candidates at the orthosteric binding site of Sig-1R with high binding affinity, specificity, and favorable PK parameters using a structure-based drug design approach. Method: Prior work in this lab found a list of the top 1000 orthosteric ligands by docking Sig-1R against libraries containing 9,270 small drug-like molecules using the TACC drug discovery tool. These libraries were extracted from the ZINC database. From this list of 1000 compounds, we selected the top 130 compounds (binding affinity cut-off ≥ -11.0 kcal/mol) and re-docked against the Sig-1R using the efficient docking suite Glide in Maestro. Also, we analyzed the pharmacokinetic/ADME parameters of these compounds using SwissADME, identifying possible candidates to use as our scaffold to try and design a ligand with even stronger binding affinity to the orthosteric site of Sig1R. Furthermore, we docked 130 compounds with the Dopamine Receptor D2 (D2R) to analyze their specificity for the Sig-1R. Results: Using an extra precision molecular docking in Glide, we found that our molecule 106 (-12.88 kcal/mol), molecule 105 (-12.83 kcal/mol), and molecule 100 (-12.29 kcal/mol) all had very high binding affinity for the Sig-1R. Both Molecules 105 and 100 had favorable PK parameters, as both were estimated to be BBB permeable, as well as not breaking any aspects of Lipinski’s Rule of Five. Molecule 100 was also found to have relatively low binding affinity (-7.6 kcal/mol) for the D2R. Conclusion: Using our computational molecular docking methods, we have identified molecule 100 as a ligand with strong affinity and specificity for the Sig1R, as well as favorable PK parameters. This could be a strong candidate to use as a chemical scaffold to develop a ligand with even stronger binding affinity for Sig-1R, which can eventually go on to in-vitro assays to confirm activity.
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    Animal model of kidney disease induced by high folic acid
    (2023) Aldeeb, Sara; Yan, Liang-Jun
    Purpose: The kidney is a vital organ that helps the body eliminate waste and toxic substances and return nutrients and vital substances back into the bloodstream. Kidney disease can be categorized into acute kidney injury (AKI) or chronic kidney disease (CKD). This may be caused by numerous risk factors such as ischemia, sepsis, drug toxicity and drug overdose, exposure to heavy metals, and diabetes. However, the exact prognosis from an AKI to CKD is not fully understood. In addition, approximately 37 million individuals in the United States population currently suffer from CKD. Despite the high prevalence of CKD, information is lacking on our understanding of the pathogenesis of AKI and CKD and there are still no available therapeutics that can be used to combat kidney disease effectively. This highlights an urgent need to further study the pathological mechanisms underlying AKI, CKD, and AKI progression to CKD. In this regard, animal models of kidney disease are imperative. Methods: This presentation reviews a widely used animal model of kidney disease, which is induced in mice with folic acid (FA). While a low dose of FA is nutritionally favorable, a high dose of FA is toxic to the kidneys. A high dose of FA is injected intra-peritoneally in the mice. Following a brief description of the procedure for disease induction by FA, major mechanisms of FA-induced kidney injury are then reviewed. This includes observing oxidative stress levels, mitochondrial abnormalities such as impaired bioenergetics and mitophagy, ferroptosis, pyroptosis, and increased expression of fibroblast growth factor 23 (FGF23). This is completed to explore possible pathological mechanisms of kidney disease and thereby the efficacy of a variety of therapeutic approaches may be evaluated. These procedural methods required Institutional Animal Care and Use Committee (IACUC) clearance and proper laboratory training to ensure ethical laboratory practices. The presentation will also highlight an overview of how to obtain IACUC clearance and ethical practice certification. Results: This animal model of inducing high doses of FA can induce both AKI and CKD in mice and therefore can be used to further study AKI to CKD progression. Conclusion: Given that the animal model is reproducible and can recapitulate human kidney disease phenotypes, it should be useful for both studying the pathological mechanisms of kidney disease and identifying effective therapeutic targets to fight kidney disease. This presentation is supported by PDRT at UNTHSC
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    Chronic Intermittent Hypoxia Increases Oxidative Stress and Impairs Spatial Memory in Male and Female Rats
    (2023) Gardner, Jennifer J.; Mabry, Steve; Bradshaw, Jessica L.; Wilson, E. Nicole; Little, Joel; Goulopoulou, Styliani; Cunningham, Rebecca L.
    Obstructive sleep apnea (OSA) is characterized by complex phenotypes and increased long-term risk of neurodegenerative disease. The impact of OSA in women is unknown due to sex differences in clinical presentation contributing to underdiagnosis. Using chronic intermittent hypoxia (CIH) to model OSA in rodents, our previous studies have shown CIH exposure increases oxidative stress and inflammation in male rats. However, the impact of CIH in female rats remains unclear. The objective of this study was to assess sex differences in CIH-mediated oxidative stress and rodent behaviors associated with neurodegenerative disease. Young adult male and female Long Evans and Sprague Dawley rats were exposed to CIH or normoxia for 14-15 days. Spatial memory and fine and gross motor skills were assessed. Plasma oxidative stress was measured and neuronal expression in the dorsal hippocampus was quantified. Female rats exhibited better spatial memory than males with increased neuronal expression in the CA1 region of the hippocampus. In both males and females, CIH impaired spatial memory and increased circulating oxidative stress. Yet, CIH increased CA1 neuronal expression in female rats only. CIH did not impact gross or fine motor skills, regardless of sex. Our preliminary findings indicate CIH increases oxidative stress and impairs spatial memory in males and females, but the impact of CIH on hippocampal neurons and region-specific contributions to spatial memory may be sexually dimorphic.
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    Redox Profiling of NAD Kinase in Acute Kidney Injury
    (2023) Quach, Emily; Yan, Liang-Jun
    Title: Redox Profiling of NAD Kinase in Acute Kidney Injury Authors: Emily Q. Quach Pharm.D. Candidate 2023, Liang-Jun Yan PhD. Abstract Background: Acute kidney injury (AKI) is a common clinical disorder in hospitalized patients and is characterized by a rapid decline in kidney function reflected by a rapid decrease in glomerular filtration rate and a decrease in urine output or anuria. Besides supportive therapy for AKI, there are no effective treatments. Therefore, further understanding of the underlying pathology of this disorder is needed. Purpose: Oxidative stress has been postulated to be one of the major mechanisms underlying AKI; and NAD kinase (NADK), the sole enzyme making NADP from NAD, is a key player in oxidative stress and redox imbalance. Nevertheless, the role of NADK in AKI remains unknown. In this study, we propose to investigate the redox profile of cytosolic NADK in mice models. Methods: AKI-induced mice model by a single intraperitoneal injection of folic acid (250 mg/kg). Three days after the folic acid injection, mice will be euthanized followed by the collection of blood and the kidneys. Kidney function will be measured by quantitation of blood urea nitrogen, serum uric acid, and creatinine. For NADK profiling, we will analyze NADK protein expression, enzymatic activities, and NADK protein oxidative modifications. NADK protein oxidation will be quantitated by Western blot detection of protein carbonyls labeled with biotin-linked reactive aldehyde probes. Results: The NADK protein oxidation is expected to increase upon AKI induction concurrent with a decreased enzymatic activity of NADK. Additionally, we expect to observe changes in NADK protein expression in AKI. Conclusion: the results of our study will provide insights into the role of NADK in AKI and may also indicate that NADK could be a potential target for future prevention and therapy for acute kidney injury.
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    Identification of New Allosteric Modulators for the mGlu2 Receptor by using a Ligand-based Drug Discovery Approach
    (2023) Nguyen, Trong; Kumari, Pratibha; Mathew, Ezek; Liu, Jin
    Purpose: The human mGlu receptors are G protein-coupled receptors located within the central nervous system. These receptors normally bind to glutamate, which is the primary excitatory neurotransmitter in the body. The receptors can then assist in modulating the transmission of excitatory signals within the brain. These characteristics help to make the mGlu2 receptor a potential, novel target for future drug development, particularly for the treatment of certain neurologic or neuropsychiatric disorders, such as schizophrenia or depression. However, most allosteric ligands bind non-selectively on both mGlu2 and mGlu3 receptors. A pharmacological tool that assists with distinguishing ligands specific to mGlu2 and mGlu3 receptor subtypes will be pivotal to speed-up the drug discovery process. Our purpose in this study is to find novel ligands of potential allosteric modulators for the mGlu2 receptor by using already identified modulators through a ligand-based drug designing approach. Methods: The potential allosteric ligands for the mGlu2 receptor were obtained by performing similarity searches on the online databases, ZINC and Drugbank. The original compounds used as the basis for the similarity searches came from a previously compiled list of Top 39 ZINC mGlu2 ligands (from the Liu Lab). Once the ligands were downloaded, they were converted into the appropriate file formats for molecular docking. Due to time constraints, it was decided that we would only dock the compounds whose original ligands had <10 results obtained from similar searching through ZINC. The selected ligands were then docked using Autodock Vina and visualized using Pymol. The Top 3 ligands were then determined based on their presence within the mGlu2 allosteric binding pocket and their predicted binding affinity for the receptor. Additionally, these ligands were also analyzed using a previously developed machine learning model. Specifically, the machine learning model would predict mGlu2 ligand likeness and binding affinity for each of the obtained ligands. Results: A total of 1507 allosteric ligands were obtained for the mGlu2 receptor through the similarity searches. Machine learning model analysis of the similar ligands deemed that 88.89% of them were more likely to be mGlu2 ligands. Additionally, 83.50% of the ligands were deemed to have a high predicted binding affinity for the mGlu2 receptor. A total of 46 compounds were docked to the mGlu2 receptor using Autodock Vina, and their predicted binding affinities were obtained. The Top 3 similar ligands for the mGlu2 receptor, listed in order, exhibited binding affinities of -12.5 kcal/mol, -12.3 kcal/mol and -11.0 kcal/mol. Conclusion: We were able to identify 1507 potential ligands for the mGlu2 receptor through similarity searches. Through further molecular docking of 46 of the similar ligands, we have determined three specific allosteric ligands for the mGlu2 receptor that are comparable or slightly better to their original counterparts. However, we believe additional research and investigation is required for validation of their potential efficacy. Future studies should involve analysis of the specific protein-ligand interactions that exist between the mGlu2 receptor and the three similar allosteric ligands, followed by comparison with the interactions present in their original counterparts.