Browsing by Author "Luedtke, Robert R."
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Item A guanidine dietary supplement influences pH sensitivity and NSAID activity in acid-sensing ion channels(2015-12-01) Agharkar, Amruta S.; Gonzales, Eric B.; Singh, Meharvan; Luedtke, Robert R.The acid-sensing ion channels (ASICs) are proton sensitive, sodium channels that belong to the epithelial sodium channel/ Degenerin family of ligand-gated ion channels. Activation of the ASIC1a subtype in the central nervous system increases neurodegeneration after ischemic stroke while ASIC3 subtype in the peripheral nervous system is involved in perception of pain. They are emerging targets for ischemic stroke, pain and inflammation. However, we lack selective ligands to target ASICs. In order to gain a better understanding of the channel and to develop selective ligands we must first determine how ASICs are modulated by synthetic as well as endogenous guanidine compounds. This study investigates whether a guanidine dietary supplement, creatine, modulates ASICs. Creatine has been shown to protect from ischemia and benefits patients suffering from muscular dystrophy, osteoarthritis, and fibromyalgia. Furthermore, pain medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit ASICs. Since supplements and NSAIDs are available over-the-counter, the significant amount of the population would consume them simultaneously.However, the interactions of combination of creatine and NSAIDs on ASICs still remain elusive. Here we sought to determine if creatine would modulate ASIC1a and ASIC3 proton sensitivity and if the combination of creatine and NSAIDs would inhibit ASIC3. Our results indicate that, creatine reduces human ASIC1a (hASIC1a) steady-state desensitization and increases their recovery from desensitization. Creatine also slows down the open-state desensitization of hASIC1a. The efficacy of hASIC1a is increased by creatine at higher concentrations. This indicates that, creatine increases channel's reactivation from desensitization by stabilizing the closed conformation of hASIC1a. Creatine's effect on rat ASIC3 (rASIC3) was calcium dependent. Creatine reduced proton sensitivity of rASIC3 in the nominal calcium environment. As previously reported, NSAIDs inhibited steady-state current of rASIC3 which is involved in pain perception. However, creatine reduced NSAIDs efficacy on rASIC3. To summarize, the creatine's effect depends on the desensitized state of hASIC1a and creatine increases the availability of channels for opening. While in rASIC3, creatine reduces proton sensitivity in nominal Ca2+ and antagonizes NSAIDs inhibitory effect. Thus, the use of creatine should be monitored in diseased states and when it is consumed along with NSAIDs.Item Assessment of Sex Differences Following Repeated Mild Head Injuries(2022-05) Duggal, Aakaash; Schreihofer, Derek A.; Sumien, Nathalie; Luedtke, Robert R.There is limited information about sex differences in mRHI, despite some studies suggesting females participating in contact sports experience more head injuries than males. This study will test the hypothesis that mRHI will lead to more severe neurological deficits in female mice than in male mice. C57BL/6 female mice were assigned to sham and mRHI groups (n=30/group). Lightly anesthetized mice received 25 mild head injuries, once a day (M-F) over 5 weeks using a weight drop model that included a free fall with rotational injury. Acutely, mRHI female mice performed worse than sham injured mice on the balance beam (F (1,28) =4.309, P=0.0472) whereas there was no difference in males. 5 weeks and 15 weeks after injury mice underwent a 3-week series of behavioral tests. Both male and female mice in the mRHI groups performed significantly (T-test P< 0.01) worse on the Rotarod than uninjured controls. Only males in MWM showed significant impairment on memory for 5-week and significant impairment on spatial learning and memory for 15-week (Probe T-test P< 0.05). Only 15-week male mice showed deficits in elevated plus maze (EPM) (T-test P< 0.05). Acutely, female mice showed balance deficits that were not apparent in males. Fifteen weeks after mRHI, males no longer displayed deficits in the rotarod, but female mice continued to have a decrease in performance compared to controls (T-test, P<0.05). Unlike the males, female mice did not display any significant deficits in the MWM and EPM.Item Design and Synthesis of Conformationally Flexible Scaffold as Bitopic Ligands for Potent D(3)-Selective Antagonists(MDPI, 2023-01-09) Kim, Ho Young; Lee, Ji Youn; Hsieh, Chia-Ju; Taylor, Michelle; Luedtke, Robert R.; Mach, Robert H.Previous studies have confirmed that the binding of D(3) receptor antagonists is competitively inhibited by endogenous dopamine despite excellent binding affinity for D(3) receptors. This result urges the development of an alternative scaffold that is capable of competing with dopamine for binding to the D(3) receptor. Herein, an SAR study was conducted on metoclopramide that incorporated a flexible scaffold for interaction with the secondary binding site of the D(3) receptor. The alteration of benzamide substituents and secondary binding fragments with aryl carboxamides resulted in excellent D(3) receptor affinities (Ki = 0.8-13.2 nM) with subtype selectivity to the D(2) receptor ranging from 22- to 180-fold. The beta-arrestin recruitment assay revealed that 21c with 4-(pyridine-4-yl)benzamide can compete well against dopamine with the highest potency (IC(50) = 1.3 nM). Computational studies demonstrated that the high potency of 21c and its analogs was the result of interactions with the secondary binding site of the D(3) receptor. These compounds also displayed minimal effects for other GPCRs except moderate affinity for 5-HT(3) receptors and TSPO. The results of this study revealed that a new class of selective D(3) receptor antagonists should be useful in behavioral pharmacology studies and as lead compounds for PET radiotracer development.Item Design and Synthesis of D(3)R Bitopic Ligands with Flexible Secondary Binding Fragments: Radioligand Binding and Computational Chemistry Studies(MDPI, 2024-01-11) Tian, Gui-Long; Hsieh, Chia-Ju; Taylor, Michelle; Lee, Ji Youn; Luedtke, Robert R.; Mach, Robert H.A series of bitopic ligands based on Fallypride with a flexible secondary binding fragment (SBF) were prepared with the goal of preparing a D(3)R-selective compound. The effect of the flexible linker ((R,S)-trans-2a-d), SBFs ((R,S)-trans-2h-j), and the chirality of orthosteric binding fragments (OBFs) ((S,R)-trans-d, (S,R)-trans-i, (S,S)-trans-d, (S,S)-trans-i, (R,R)-trans-d, and (R,R)-trans-i) were evaluated in in vitro binding assays. Computational chemistry studies revealed that the interaction of the fragment binding to the SBF increased the distance between the pyrrolidine nitrogen and ASP110(3.32) of the D(3)R, thereby reducing the D(3)R affinity to a suboptimal level.Item Development of a mouse model to study the long-term effects of chemotherapy on brain function(2023) Trinh, Oanh; Vann, Philip; Davis, Delaney; Luedtke, Robert R.; Basha, Riyaz; Singh, Meharvan; Sumien, NathaliePurpose: While remaining an effective life-saving intervention for cancer patients, chemotherapy has been associated with many neurotoxic side effects, including chemotherapy-related cognitive impairments (CRCI). Chemotherapy exposure leads to a decline in learning, memory, processing speed, attention, and executive functions, which may persist for more than 20 years post-treatment, impairing the quality of daily lives of survivors. Childhood cancer survivors are particularly vulnerable to chemotherapy and have been impacted in their educational achievements, employment, social relationships, and even life expectancy. Most common childhood cancers are often treated with the folate-inhibitor methotrexate (MTX). Our study aimed at establishing a tumor-free mouse model of MTX-induced brain impairments. We hypothesized that early exposure to MTX would induce impairment in cognition, as well as motor and affective functions. Methods: Male and female C57BL6/J postnatal day 15 pups received intraperitoneal injections of saline or MTX (2 mg/kg) once a day for 3 days. Pups were weaned on PND21, and subsets were behaviorally characterized at 1 or 7 months after MTX exposure (n=6-8 for 1.5 months old, and n=11-13 for 8 months old) for motor, affective and cognitive functions using a comprehensive behavioral test battery. Results: At 1.5 months, coordination and motor learning was significantly impaired in males and improved in females. All other measures did not reveal any other significant effects, however trends of impaired motor and cognitive functions could be discerned. At 8 months, there were no effects of MTX on motor, affective and some cognitive functions. However, MTX exposure led to an impairment on spatial learning and memory and increased swimming speed. Conclusions: Studies at 1.5 months will need to be repeated to increase power and ascertain conclusions on brain functions. Early exposure to MTX treatment led to long-term impairments in both male and female mice and could be used as a model to test interventions to limit CRCI.Item Effect of Sigma-1 Receptor Activation on Renal Injury and Hypertension in Female Mice with Lupus(2023) Dinh, Viet; Chaudhari, Sarika; Young-Stubbs, Cassandra M.; Shimoura, Caroline; Tucker, Selina; Essajee, Salman; Warne, Cooper; Luedtke, Robert R.; Mathis, Keisa W.Systemic lupus erythematosus (SLE) is a female-dominant autoimmune disease with prominent renal injury and hypertension, contributing to its morbidity and mortality. Novel therapies to reduce these detrimental outcomes could be beneficial to SLE patients. The sigma-1 receptor (S1R) is a cytoprotective ligand-regulated chaperone protein that decreases protein aggregation, cellular stress, and cell death, thus preventing tissue injury. S1R activation with pharmacological ligands enhances cytoprotection in autoimmune diseases like multiple sclerosis and Huntington’s disease; however, the efficacy of S1R agonists in SLE is unknown. We hypothesize that S1R activation via the agonist LS-1-127 will reduce renal injury and halt the progression of hypertension in SLE mice. Female SLE (NZBWF1) and control (NZW) mice were weighed and urine collected via metabolic cages weekly starting at 30 weeks of age. Albuminuria was measured via dipsticks. At 33 weeks of age, SLE and control mice were treated with LS-1-127 (10 mg/kg IP) or equal volume of vehicle (10% DMSO; IP) three times a week for two weeks. At 35 weeks, mean arterial pressure (MAP) was measured in conscious mice using indwelling carotid catheters for two consecutive days and then mice were euthanized. Wire myography was used to assess potassium chloride (KCl)-induced contraction and acetylcholine (ACh)-induced relaxation in excised aorta. Markers of renal injury – urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and creatinine – as well as plasma double-stranded (ds)DNA autoantibodies were measured by ELISA. Albuminuria was present in 44.4% (4 of 9) of SLE mice and no controls. LS-1-127 did not improve albuminuria in SLE mice (50%; 3 of 6). NGAL:creatinine ratio (ng/mg) was higher in SLE mice compared to controls (327.3 ± 119.8 vs 63.2 ± 4.3 ng/mg; n=9–12; P=0.0007). LS-1-127 did not significantly alter NGAL:creatinine ratio in SLE mice (484.3 ± 209.0; n=6) or controls (71.7 ± 5.2; n=10). KIM1:creatinine ratio (ng/mg) did not differ between groups. dsDNA autoantibodies were higher in SLE mice compared to controls (6.9e5 ± 1.1e5 vs. 1.4e5 ± 3.1e4 U/mL; n=9–10; P<0.0001). LS-1-127 did not significantly alter dsDNA autoantibodies in SLE mice (7.1e5 ± 1.2e5; n=6) or controls (1.5e5 ± 4.0e4; n=10). MAP was higher in SLE mice compared to controls (146 ± 4 vs. 123 ± 3 mmHg; n=9–10; P <0.0001). LS-1-127 did not significantly alter MAP in SLE mice (150 ± 8; n=6) or controls (124 ± 2; n=10). KCl-induced aortic contraction was similar in SLE and controls (21 ± 7 vs. 25 ± 4 mM, n=3–4). Sensitivity to KCl after LS-1-127 treatment was 11 ± 3 and 21 ± 2 mM in SLE and controls (n=2–4). ACh-induced aortic relaxation did not differ between groups. In conclusion, two weeks of S1R activation with LS-1-127 did not significantly alter markers of renal injury, autoimmunity, blood pressure, or vascular reactivity in female SLE mice with advanced disease. Further inquiry into the effect of LS-1-127 on the expression of renal proinflammatory cytokines will be conducted. S1R activation at different stages of SLE disease progression also warrants future investigation.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item Evaluation of Substituted N-Phenylpiperazine Analogs as D3 vs. D2 Dopamine Receptor Subtype Selective Ligands(MDPI, 2021-05-26) Lee, Boeun; Taylor, Michelle; Griffin, Suzy A.; McInnis, Tamara; Sumien, Nathalie; Mach, Robert H.; Luedtke, Robert R.N-phenylpiperazine analogs can bind selectively to the D3 versus the D2 dopamine receptor subtype despite the fact that these two D2-like dopamine receptor subtypes exhibit substantial amino acid sequence homology. The binding for a number of these receptor subtype selective compounds was found to be consistent with their ability to bind at the D3 dopamine receptor subtype in a bitopic manner. In this study, a series of the 3-thiophenephenyl and 4-thiazolylphenyl fluoride substituted N-phenylpiperazine analogs were evaluated. Compound 6a was found to bind at the human D3 receptor with nanomolar affinity with substantial D3 vs. D2 binding selectivity (approximately 500-fold). Compound 6a was also tested for activity in two in-vivo assays: (1) a hallucinogenic-dependent head twitch response inhibition assay using DBA/2J mice and (2) an L-dopa-dependent abnormal involuntary movement (AIM) inhibition assay using unilateral 6-hydroxydopamine lesioned (hemiparkinsonian) rats. Compound 6a was found to be active in both assays. This compound could lead to a better understanding of how a bitopic D3 dopamine receptor selective ligand might lead to the development of pharmacotherapeutics for the treatment of levodopa-induced dyskinesia (LID) in patients with Parkinson's disease.Item Graded Mild Head Injury as a Model for Sports Injury(2017-03-14) Metzger, Daniel; Oppong-Gyebi, Anthony; Vann, Philip; Sumien, Nathalie; Luedtke, Robert R.; Schreihofer, Derek; Sun, FenPurpose: To develop a graded model of mild head injury that produces graded behavioral deficits in the mouse. This model will be used to test neuroprotective effects of novel compounds. This study was designed to determine the severity of injury required to cause different behavioral deficits in motor function and cognition. Methods: Young adult male C56/B6J mice were anesthetized daily with isoflurane (20 sec) and 15 sec later were subjected to a weight drop head injury using a tethered steel bar (43 grams) dropped through an acrylic tube from a height of 28 inches. Mice were placed prone on a scored aluminum foil stage 2 cm below the end of the tube. The blow was directed to a 5 mm midline area of the head rostral to the aural canals. The blow causes a break in the scored aluminum foil and allows the mouse to flip 180 degrees and land supine on a foam cushion. This model was chosen to model a hit to the head followed by rotational acceleration indicative of closed head injuries occurring in contact sports. Five groups of mice were randomized to receive 0, 5, 10, 15, 20, or 25 blows, 1 per day M-F. Five days after the final hit, mice then underwent cognitive and behavioral testing consisting of an accelerating Rotorod, Morris water maze, and active avoidance T-maze. Following testing brains will be examined for cell death and inflammation. Results: A total of 30 mice (5 per group) were used for this study. Body weight did not differ among the groups over the course of the study, however waking time after anesthesia was increased in all groups subjected to injury compared to mice anesthetized and not injured. Coordinated movement on an accelerating Rotorod revealed a linear trend for decreased performance with increasing number of head impacts suggesting that a graded approach is possible with this model. Time to fall was significantly shorter than controls at 15 and 25 hits. Water maze and T-maze tests are ongoing. Conclusions: These data suggest that a graded injury regimen can lead to graded behavioral responses in the young male mouse and will provide a useful model for testing the effectiveness of neuroprotective compounds that have the potential to be used as prophylactic agents for those involved in contact sports.Item In vitro characterization of [(125)I]HY-3-24, a selective ligand for the dopamine D3 receptor(Frontiers Media S.A., 2024-04-24) Lee, Ji Youn; Kim, Ho Young; Martorano, Paul; Riad, Aladdin; Taylor, Michelle; Luedtke, Robert R.; Mach, Robert H.INTRODUCTION: Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [(125)I]HY-3-24. METHODS: In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. RESULTS: HY-3-24 showed high potency at D3R (K(i) = 0.67 +/- 0.11 nM, IC(50) = 1.5 +/- 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R K(i) = 86.7 +/- 11.9 nM and D4R K(i) > 1,000). The K(d) (0.34 +/- 0.22 nM) and B(max) (38.91 +/- 2.39 fmol/mg) values of [(125)I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [(125)I]HY-3-24. Autoradiography results demonstrated that [(125)I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. CONCLUSION: These results suggest that [(125)I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [(125)I]HY-3-24 can be used as the specific D3R radioligand.Item Increased resolution screening of the pharmacogenetic gene CYP2D6 with microarray technology(2019-08) Davis, Carey P.; Budowle, Bruce; Luedtke, Robert R.; Hodge, Lisa M.; Phillips, Nicole R.; Woerner, August E.Autopsy is a primary methodology used for assessing cause and/or manner of death in medicolegal investigations. Some autopsies, however, do not resolve the cause of death unambiguously or there is no evident pathology to determine the cause of death. In addition, in some cases toxicology screens are negative or difficult to interpret because it is challenging to determine if a high concentration of a drug in the body derived from one large dose or has built up over time. Determining the genetic constitution of victims at specific target genes may clarify the cause of some of these unexplained deaths or at least indicate susceptibility to triggering effects. Cytochrome P450 (CYP450) is a super family of enzymes that detoxify foreign chemicals and are involved in the metabolism of drugs. One gene in this family that encodes CYP450 enzymes, CYP2D6, accounts for the metabolism of 25% of all drugs currently on the market. By examining the variability in the CYP2D6 gene, a SNP panel was developed and used to aid in personalized medicine with a long-term outcome of reducing risk in patients who partake in drug therapy. However, paralogs of the CYP2D6 gene can interfere with obtaining accurate typing results. The hypothesis of this dissertation is that it is possible to develop a targeted panel for clinically relevant variants in the CYP2D6 gene using array-based technology that can provide accurate and reliable genotyping results. The goal was first to demonstrate that high throughput sequencing, also known as next generation or massively parallel sequencing, could reliably sequence a complex target using a model system, i.e. the hypervariable regions of the human mitochondrial genome. Then, using this advanced sequencing capability define the baseline genetic variation of the CYP2D6 gene in a selected population and identify those genetic markers associated with metabolism capacity that would be verified against a database of actionable variants that cause reaction to drug exposure. The entire CYP2D6 gene was sequenced to identify SNPs at a population level. These SNPs were compared against a known database of clinically relevant samples with known metabolic responses of the same ethnic background to verify actionable variants. Once these variants were identified, a PCR assay workflow leveraging microarray technology was developed to quickly and efficiently screen individuals of interest by overcoming paralog interference. This assay can be used prior to administering drugs or post mortem to gain information about potential adverse drug reactions.Item Interaction of Ligands for PET with the Dopamine D3 Receptor: In Silico and In Vitro Methods(MDPI, 2021-04-02) Hsieh, Chia-JI; Riad, Aladdin; Lee, Ji Youn; Sahlholm, Kristoffer; Xu, Kuiying; Luedtke, Robert R.; Mach, Robert H.[(18)F]Fallypride and [(18)F]Fluortriopride (FTP) are two different PET radiotracers that bind with sub-nanomolar affinity to the dopamine D3 receptor (D3R). In spite of their similar D3 affinities, the two PET ligands display very different properties for labeling the D3R in vivo: [(18)F]Fallypride is capable of binding to D3R under "baseline" conditions, whereas [(18)F]FTP requires the depletion of synaptic dopamine in order to image the receptor in vivo. These data suggest that [(18)F]Fallypride is able to compete with synaptic dopamine for binding to the D3R, whereas [(18)F]FTP is not. The goal of this study was to conduct a series of docking and molecular dynamic simulation studies to identify differences in the ability of each molecule to interact with the D3R that could explain these differences with respect to competition with synaptic dopamine. Competition studies measuring the ability of each ligand to compete with dopamine in the beta-arrestin assay were also conducted. The results of the in silico studies indicate that FTP has a weaker interaction with the orthosteric binding site of the D3R versus that of Fallypride. The results of the in silico studies were also consistent with the IC50 values of each compound in the dopamine beta-arrestin competition assays. The results of this study indicate that in silico methods may be able to predict the ability of a small molecule to compete with synaptic dopamine for binding to the D3R.Item Ligand with Two Modes of Interaction with the Dopamine D2 Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism(ACS Publications, 2020-09-15) Agren, Richard; Zeberg, Hugo; Stepniewski, Tomasz Maciej; Free, R. Benjamin; Reilly, Sean W.; Luedtke, Robert R.; Arhem, Peter; Ciruela, Francisco; Sibley, David R.; Mach, Robert H.; Selent, Jana; Nilsson, Johanna; Sahlholm, KristofferA solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 muM DA (>25 and >90% of control, respectively), whereas recovery was less prominent ( approximately 20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 muM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.Item Low-dose methotrexate exposure induced long-term cognitive deficits in mice(2022) Trinh, Oanh; Sumien, Nathalie; Vann, Philip; Davis, Delaney; Luedtke, Robert R.; Basha, Riyaz; Singh, MeharvanPurpose: Chemotherapy-related cognitive impairment (CRCI) remains a mysterious morbidity that threatens the quality of life of up to 70% cancer survivors in the United States. Longitudinal studies of CRCI highlighted deficits in memory, learning, attention, motor, and executive functions for up to 20 years after the completion of chemotherapy paradigm. These deficits, especially if happened during childhood, can negatively impact educational achievement, employment, self-independence, and life expectancy of approximately 500,000 adult survivors currently living in the U.S. Given that acute lymphoblastic leukemia (ALL) is the most common diagnosis of childhood cancers worldwide, the folate-inhibitor methotrexate (MTX) has been at the backbone of ALL-treatment with a substantial risk of neurotoxicity. The purpose of this study was to establish a tumor-free mouse model representative of MTX-induced CRCI in childhood ALL survivors and study the long-term effects of chemotherapy treatment on brain function. We hypothesized that MTX administration at a very young age will induce long-term cognitive impairments. Methods: At post-natal day 15, male and female C57BL6/J pups received intraperitoneal injections of either saline (n=12) or MTX (2 mg/kg; n=12) once a day for 3 days. The pups were weaned at post-natal day 21 and allowed to age. At 8-month-old, animals underwent behavioral tests to assess motor, affective and cognitive functions. Results: MTX administration impaired cognitive flexibility in males and impaired spatial learning and memory in females, indicating potential sex- and test-dependent behavioral outcomes. MTX increased performance in coordinated-running test, and increased swimming speed. Anxiety-like behaviors were not affected by the treatment. Conclusions: These preliminary results suggested that low dose MTX-treatment induced sex-dependent cognitive deficits while affective and motor functions were not negatively affected. This study will be repeated, and behaviors will be assessed at other time points to establish complete neurobehavioral profiles of mice affected by MTX chemotherapy.Item Pharmacogenetics of Select Genes in the Opiate Metabolism and Response Pathways(2018-08) Wendt, Frank R.; Budowle, Bruce; Phillips, Nicole R.; LaRue, Bobby L.; Luedtke, Robert R.; Clark, Abbot F.Pharmacogenetics and pharmacogenomics aim to elucidate the underlying genetic variation contributing to adverse drug reactions, differential enzyme activity, and resulting appropriate drug dosage on the individual and population levels. Studies with this goal in mind typically rely on targeted genotyping of select single nucleotide polymorphisms (SNPs) and/or insertion/deletion (INDEL) polymorphisms within a gene that have demonstrated significant association with the rate of drug absorption, distribution, excretion, and/or metabolism. This approach may enable association and characterization of clinically relevant polymorphisms with a phenotype of interest and may provide guidance regarding appropriate prescription medication practices for medical professionals. Additionally, these data, namely those of the cytochrome p450, family 2, subfamily D, polypeptide 6 gene (CYP2D6), have contributed to identifying cause and/or manner of death in some death investigations which initially were negative medico-legal autopsies. Though invaluable to medical genetics, the chemistry of targeted genotyping approaches, including genome-wide association studies and SNP-targeted massively parallel sequencing, inherently lack the capability to discover novel or rare polymorphisms that may be enriched in pharmacogenetically-valuable cohorts (i.e., individuals who have experienced idiosyncratic responses to codeine/morphine). Relatively recently, the pharmacogenetics community has utilized comprehensive (i.e., full-gene) and/or combinatorial (i.e., multi-gene) genetic studies using multiple genes whose protein products are involved in a drug metabolism/response pathway. The multi-gene approach is demonstrably more successful in predicting phenotypic expressions and more efficacious for patient outcomes compared to as single-gene approach. While mainly elucidating multigenic profiles of psychiatric drugs and disorders, to date, it is reasonable to consider that more efficacious patient outcomes can be achieved using the pathways responsible for other pathologies or drug metabolism and response pathways. The goal of this dissertation was to develop a comprehensive genetic profiling system using the full gene region of five genes that have demonstrated associations between specific SNPs and opiate metabolism/response. The in silico phases of this dissertation aimed to characterize the genes encoding CYP2D6, uridine diphosphate glucuronosyltransferase family 1 polypeptide B7 (UGT2B7), adenosine triphosphate (ATP) binding cassette subfamily B number 1 (ABCB1; p-glycoprotein; multidrug resistance protein 1), opioid receptor mu 1 (OPRM1; MOR1), and catechol-O-methyltransferase (COMT) on the individual SNP and full-gene haplotype levels. Subsequent empirical evaluation of these genes was performed on a cohort of deceased tramadol-exposed Finns using targeted genotyping and exome-wide analyses. This dissertation research has 1) described previously uncharacterized individual SNPs that are associated with the metabolism of tramadol to its primary metabolite, O-desmethyltramadol; 2) evaluated the utility of full-gene information for predicting metabolizer phenotype; 3) produced a massively parallel sequencing panel to genotype opiate-metabolism genes in a more comprehensive and combinatorial manner than previously attempted; 4) demonstrated the increased predictive capabilities of a multigenic opiate metabolizer phenotyping system; and 5) identified additional genetic targets that may have predictive phenotypic value.Item Selective Activation of D3 Dopamine Receptors Ameliorates DOI-Induced Head Twitching Accompanied by Changes in Corticostriatal Processing(MDPI, 2023-06-10) Estrada-Sanchez, Ana M.; Rangel-Barajas, Claudia; Howe, Andrew G.; Barton, Scott J.; Mach, Robert H.; Luedtke, Robert R.; Rebec, George V.D3 receptors, a key component of the dopamine system, have emerged as a potential target of therapies to improve motor symptoms across neurodegenerative and neuropsychiatric conditions. In the present work, we evaluated the effect of D3 receptor activation on the involuntary head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) at behavioral and electrophysiological levels. Mice received an intraperitoneal injection of either a full D3 agonist, WC 44 [4-(2-fluoroethyl)-N-[4-[4-(2-methoxyphenyl)piperazin 1-yl]butyl]benzamide] or a partial D3 agonist, WW-III-55 [N-(4-(4-(4-methoxyphenyl)piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide] five minutes before the intraperitoneal administration of DOI. Compared to the control group, both D3 agonists delayed the onset of the DOI-induced head-twitch response and reduced the total number and frequency of the head twitches. Moreover, the simultaneous recording of neuronal activity in the motor cortex (M1) and dorsal striatum (DS) indicated that D3 activation led to slight changes in a single unit activity, mainly in DS, and increased its correlated firing in DS or between presumed cortical pyramidal neurons (CPNs) and striatal medium spiny neurons (MSNs). Our results confirm the role of D3 receptor activation in controlling DOI-induced involuntary movements and suggest that this effect involves, at least in part, an increase in correlated corticostriatal activity. A further understanding of the underlying mechanisms may provide a suitable target for treating neuropathologies in which involuntary movements occur.Item Strategies & mechanisms to reduce locomotor impairment in aging & Parkinson's disease(2021-05) Kasanga, Ella A.; Salvatore, Michael; Sumien, Nathalie; Luedtke, Robert R.; Bugnariu, Nicoleta L.; Goulopoulou, StylianiThe maintenance of physical function throughout the lifespan is a hallmark of successful aging. However, vulnerability to motor impairment during aging is evident in a substantial fraction of those reaching their seventh to ninth decade of life. Aging-related Parkinsonism is a major source of aging-related motor impairment and manifests similarly to Parkinson's disease (PD). Such disability is associated with a loss of independent living, frailty and mortality. Aging is a major risk factor for these two conditions and with the expected exponential increase in the aging population, their prevalence will also increase. Thus, there is the need to identify interventions which can attenuate this motor impairment, and elucidate the mechanisms mediating their protective benefits. Both pharmacological and non-pharmacological interventions, including exercise, have been proposed to ameliorate motor impairment in this target population. However, most of these interventions are instituted in preclinical models before motor function decline is evident. Also, in the quest to elucidate the underlying neurobiological mechanisms, most studies investigate the role of striatal dopamine (DA) regulation which is presumed to be paramount for the initiation or maintenance of locomotor activities. However, many studies do not report a corresponding increase in striatal DA regulation despite improved motor function. This dissertation research, therefore, evaluates interventions designed to prevent further motor decline in both aging and PD rat models after the onset of motor decline. It is hypothesized that improvement in motor function from the implemented interventions: caloric restriction, treadmill exercise and a pharmacological therapy-ceftriaxone, may not be dependent on only increased striatal, but also nigral, dopaminergic transmission. From several angles of intervention to mitigate motor decline in the models used, it is clear that motor function preservation or recovery can occur if interventions are initiated at a time-point when motor decline is already evident. The body of results show that preservation of motor function is not associated with preservation or restoration of striatal tyrosine hydroxylase expression, the rate-limiting enzyme in the synthesis of DA. Taken together, this dissertation delineates the efficacy of select interventions to attenuate motor decline and identifies key mechanistic targets for possible translation in this vulnerable population.Item The 6' and 7' Residue of the Second Transmembrane Domain of Ligand-Gated Ion Channels Influence Gating and Picrotoxin Sensitivity(2005-07-01) Gonzales, Eric B.; Dillon, Glenn; Luedtke, Robert R.; Martin, MichaelGonzales, Eric B., The 6’ and 7’ residue of the second transmembrane domain of ligand-gated ion channels influence gating and picrotoxin sensitivity. Doctor of Philosophy (Pharmacology and Neuroscience), July 2005, pp213, 4 tables, 33 illustrations, 89 titles. The GABAA and glycine receptor are members of the Cys-loop family of ion channels. These receptors mediate rapid neurotransmission in the nervous system. Picrotoxin (PTX) interacts within the channel near the TM2 2’-6’ position, with the most critical interaction at the 6’ position. The present studied addressed the stoichiometric dependence and molecular requirements of the TM2 6’ position on PTX sensitivity. I hypothesized that there is not a stoichiometric dependence and that residues with a hydroxyl group remain sensitive to PTX. Further, work previously completed in the laboratory demonstrated that the TM2 7’ position influences channel kinetics in the serotonin type-3 receptor. However, similar work has not been performed in other members of the Cys-loop family of receptors. I hypothesize that the TM2 7’ position influences both gating in the glycine α1 receptor similarly to that in the serotonin type-3 receptor. Additionally, the TM2 7’ position could influence the PTX in the glycine α1 receptor. Picrotoxin sensitivity was determined to not be stoichiometrically dependent on the subunit location of the T6’F mutation in α1β2 or α1β2γ2 GABAA receptors, a single T6’F mutation was sufficient to eliminate PTX sensitivity. The α1(T6’F) β2 receptor showed PTX concentration-dependent stimulation. Picrotoxin sensitivity had a rank order of potency in α1β2(mutant) as follows: Serine [greater than] Threonine = Alanine [greater than] Cysteine [greater than] Tryptophan. In several cases, the kinetics of the T6’F mutant receptors exhibited rapid desensitization during prolonged application of agonist. In combinations of subunits with the T6’F mutation, two or fewer mutant receptors appeared to have normal phenotypes. Three or four T6’F mutations exhibited rapid desensitization, and strongly suggests that the stoichiometery of the α1β2 GABAA receptor is two α subunits and three β subunits in the receptor. The Gly α1(T7’L) mutation exhibited enhanced glycine sensitivity with slower gating kinetics than the wild type (approximately 3-fold slower); the T7’A mutation had significantly reduced glycine affinity. The glycine EC50 kinetics of the α1(T7’A) mutant receptor was more complex than the wild type. There appears to be a complex interaction between agonist binding and gating of the channel that is disrupted by the 7’ position mutations. Picrotoxin sensitivity in the mutation were enhanced with either a T7’A or T7’L substitution. The data suggest that the 7’ residue may play an accessory role in shaping the PTX site. Finally, these residues are also critical in gating of the receptor. Residues critical for gating and PTX sensitivity may be coupled in the normal function of the Cys-loop family of receptors.Item THE EFFECT OF LS-1-137, A NOVEL PHENYLACETAMIDE SIGMA 1 RECEPTOR SELECTIVE AGONIST ON SCOPOLAMINE-DEPENDENT COGNITIVE DEFICIT IN C57BL/6J MICE.(2014-03) Malik, Maninder; Rangel-Barajas, Claudia; Griffin, Suzy; Sumien, Nathalie; Singh, Meharvan; Maurice, Tangui; Mach, Robert; Luedtke, Robert R.Cognitive deficits are observed in aged population and in patients with Alzheimer’s Disease, Parkinson’s Disease, traumatic brain injury and stroke. Cognitive deficits often involve alterations in brain signaling. Currently available therapeutic drugs provide only symptomatic relief and generally become ineffective as disease progresses. Therefore, novel therapeutic agents are needed to retard and/or arrest the progressive loss of memory forming cells. Scopolamine-induced memory impairment model provides a relatively rapid and reversible screening paradigm for cognition enhancement drug discovery. In this study, mice were administered scopolamine and were used to evaluate the ability of LS-1-137, a novel drug, to improve the cognitive deficits. Our study results indicate that LS-1-137 may represent a novel therapeutic agent for the treatment of age and disease related cognitive deficits. Purpose (a): Cognitive deficits are observed in patients with Alzheimer’s Disease, Parkinson’s Disease, traumatic brain injury and stroke. These deficits often involve alterations in cholinergic signaling. Currently available therapeutic drugs provide only symptomatic relief and generally become ineffective as a neurodegenerative disorder progresses. Therefore, novel therapeutic agents are needed to retard and/or arrest the progressive loss of memory forming cells. Methods (b): A filtration-binding assay was used to characterize the binding properties of a novel sigma compound at D2-like dopamine receptors, muscarinic receptors and at sigma receptors. Co-immunoprecipitation assay was used for the quantification of Sigma 1 receptor-binding immunoglobulin protein (BiP) complex formation. LS-1-137 mediated brain-derived neurotrophic factor (BDNF) release was analyzed using enzyme-linked immunosorbent assay (ELISA). In this study, male C57BL/6J mice injected with scopolamine were used as experimental model to evaluate the in vivo cognitive properties of the test drug. The neuroprotective properties were evaluated using water maze and active avoidance test. Results (c): LS-1-137 binds with high affinity (Ki = 3.2 nM) at sigma 1 receptors and is 80-fold selective for sigma 1 compared to sigma 2 receptor. LS-1-137 binds with low affinity at D2-like (D2, D3 and D4) dopamine and muscarinic receptors. LS-1-137 was found to partially reverse the learning and memory deficits associated with scopolamine administration using a water maze test and an active avoidance task. LS-1-137 treatment modulates sigma 1 receptor- BiP complex formation and also triggers the release of BDNF from rat astrocytes. Conclusions (d): LS-1-137 may represent a novel candidate cognitive enhancer for the treatment of cholinergic muscarinic-dependent cognitive deficits.Item The Influence of CNS stimulants, opioid antagonists and an NMDA antagonist on the reinforcing effect of cocaine using a progressive-ratio schedule(1996-07-01) Li, Donghang; Forster, Michael J.; Martin, Michael; Luedtke, Robert R.It has been hypothesized that there is a common dopaminergic pathway mediating the reward properties of abused drugs, and that dopamine is involved in tolerance to the reinforcing effect of cocaine. The progressive-ratio (PR) schedule can be used to test both potentiation and reduction of the reinforcing effects of cocaine by other factors. Under the PR schedule, an increasing number of responses is required to obtain each subsequent cocaine injection, and failure to complete the required number of responses within 1 h of the previous cocaine injection terminates the session. The number of total reinforcers obtained during a session is defined as “the breaking point” and was used as the primary dependent measure. Fisher F344 male rats acquired the self-administration task under the PR schedule within forty sessions and showed a stable daily acquisition baseline. The breaking point and inter-reinforcer time (ISRT) were positively correlated within each ratio. A motor-incapacitating side effect of a pretreatment can be determined by a change in the relationship between the ISRT and the breaking point. d-Amphetamine pretreatment (0.32-3.2 mg/kg, i.p., 30 min) potentiates the reinforcing effect of cocaine as demonstrated by a higher breaking point of self-administration without changing the ISRT. Morphine pretreatment (0.32-3.2 mg/kg, i.p., 30 min failed to change the breaking point of cocaine self-administration but it did increase the ISRT. These results support an additive reinforcing effect for amphetamines and cocaine, but do not support an additive reinforcing effect of morphine and cocaine. The reinforcing effect of cocaine was reduced by pretreatment with ketamine (0.032-0.32 mg/kg, i.p., 20 min) as indicated by a reduction in the breaking point. In a concurrent experiment, animals were trained to self-administer cocaine under a fixed ratio 2 schedule (FR2). Ketamine pretreatment did not modify the ISRT in FR2 trained animals except at the highest dose (0.32 mg/kg, i.p., 20 min), where significant motor incoordination was observed. Both chronic treatment with cocaine (20 mg/kg/ 8hr x 7 days, iv) or amphetamine (3.2 mg/kg /12 hr x 7 days, i.p.) resulted in a reduction in breaking point at any given dose, providing direct evidence of tolerance and cross-tolerance to the reinforcing effects of cocaine. Chronic treatment with ketamine (0.32 mg/kg/8hr x 7 days, i.v.) failed to modify either the breaking point under a PR schedule of reinforcement or the ISRT under a FR2 schedule of reinforcement. Co-administration of ketamine (0.32 mg/kg/8hr x 7 days, i.v.) with chronic cocaine (20 mg/kg/8hr x 7days, i.v.) failed to prevent tolerance to the reinforcing effect of cocaine as indicated by either the breaking point under a PR schedule of reinforcement of the ISRT under an FR2 schedule of reinforcement. These data indicate that the breaking point in the PR schedule is more sensitive to changes in the dopamine reward system, whereas changes in rate of response are not consistently related to the changes in the dopamine reward system. These data support the use of PR schedule as a better method than FR schedule for determining reward properties of drugs of abuse with fewer complications due to the central nervous system inhibitory effects of some drugs of abuse.