Publications by authors named "Leggy A Arnold"

73 Publications

Assessment of Phenylboronic Acid Nitrogen Mustards as Potent and Selective Drug Candidates for Triple-Negative Breast Cancer.

ACS Pharmacol Transl Sci 2021 Apr 7;4(2):687-702. Epub 2021 Mar 7.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, 3210 N. Cramer Street, Milwaukee, Wisconsin 53211, United States.

Triple-negative breast cancer (TNBC) has limited treatment options and the worst prognosis among all types of breast cancer. We describe two prodrugs, namely, CWB-20145 () and its methyl analogue FAN-NM-CH () that reduced the size of TNBC-derived tumors. The DNA cross-linking of nitrogen mustard prodrugs and was superior to that of chlorambucil and melphalan once activated in the presence of HO. The cellular toxicity of and was demonstrated in seven human cancer cell lines. The TNBC cell line MDA-MB-468 was particularly sensitive toward and . Compound was 10 times more cytotoxic than chlorambucil and 16 times more active than melphalan. An evaluation of the gene expression demonstrated an upregulation of the tumor suppressor genes and supporting a transcriptional mechanism of a reduced tumor growth. Pharmacokinetic studies with showed a rapid conversion of the prodrug. The introduction of a methyl group generated with an increased half-life. An in vivo toxicity study in mice demonstrated that both prodrugs were less toxic than chlorambucil. Compounds and reduced tumor growth with an inhibition rate of more than 90% in athymic nude mice xenografted with MDA-MB-468 cells. Together, the in vivo investigations demonstrated that treatment with and suppressed tumor growth without affecting normal tissues in mice. These phenylboronic acid nitrogen mustard prodrugs represent promising drug candidates for the treatment of TNBC. However, the mechanisms underlying their superior in vivo activity and selectivity as well as the correlation between HO level and in vivo efficacy are not yet fully understood.
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http://dx.doi.org/10.1021/acsptsci.0c00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033613PMC
April 2021

Nebulized MIDD0301 Reduces Airway Hyperresponsiveness in Moderate and Severe Murine Asthma Models.

ACS Pharmacol Transl Sci 2020 Dec 2;3(6):1381-1390. Epub 2020 Dec 2.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States.

We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABAR) modulator. The therapeutic efficacy of nebulized MIDD0301 in reducing airway resistance was investigated in spontaneous breathing mice using a whole-body plethysmograph and in unconscious mice using a forced oscillation technique. Prophylactic nebulized MIDD0301 reduced subsequent methacholine-induced bronchoconstriction in ovalbumin and house dust mite allergic asthma models and in normal mice. Nebulized MIDD0301 exhibited comparable or better therapeutic potency compared to nebulized albuterol and oral montelukast. Prophylactic nebulized MIDD0301 was also effective in reducing bronchoconstriction, comparable to nebulized albuterol or fluticasone, in a steroid resistant asthma mouse model induced by intratracheal installation of lipopolysaccharide and interferon-gamma. Oral dexamethasone was ineffective in this model. Nebulized MIDD0301 was also effective in reversing bronchospasm when dosed after methacholine challenge comparable to albuterol. Pharmacokinetic studies showed that about 0.06% of nebulized MIDD0301 entered the mouse lung when using a whole body plethysmograph and therapeutic levels were sustained in the lung for at least 25 min. Consistent with previous reports on orally dosed MIDD0301, high doses of nebulized MIDD0301 resulted in minimal brain exposure and thus no observable adverse sensorimotor or respiratory depression effects occurred. In addition, no adverse cardiovascular effects were observed following 100 mg/kg i.p. dosing. These results further demonstrate that charged imidazodiazepine MIDD0301 can selectively target lung GABAR without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation.
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http://dx.doi.org/10.1021/acsptsci.0c00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737320PMC
December 2020

Improved scale-up synthesis and purification of clinical asthma candidate MIDD0301.

Org Process Res Dev 2020 Aug 29;24(8):1467-1476. Epub 2020 Jul 29.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States.

We report an improved and scalable synthesis of MIDD0301, a positive GABA receptor modulator that is under development as oral and inhaled treatments for asthma. In contrast to other benzodiazepines in clinical use, MIDD0301 is a chiral compound that has limited brain absorption. The starting material to generate MIDD0301 is 2-amino-5-bromo-2'-fluorobenzophenone, which has a non-basic nitrogen due to electron withdrawing substituents in the and positions, reducing its reactivity towards activated carboxylic acids. Investigations of peptide coupling reagents on multigram scale resulted in moderate yields due to incomplete conversions. Secondly, basic conditions used for the formation of the seven-membered 1,4-diazepine ring resulted in racemization of the chiral center. We found that neutral conditions comparable to the pKa of the primary amine were sufficient to support the formation of the intramolecular imine but did not enable the simultaneous removal of the protecting group. Both difficulties were overcome with the application of the -carboxyanhydride of D-alanine. Activated in the presence of acid, this compound reacted with non-basic 2-amino-5-bromo-2'-fluorobenzophenone and formed the 1,4-diazepine upon neutralization with triethylamine. Carefully designed workup procedures and divergent solubility of the synthetic intermediates in solvents and solvent combinations were utilized to eliminate the need for column chromatography. To improve compatibility with large scale reactors, temperature-controlled slow addition of reagents generated the imidazodiazepine at -20 °C. All intermediates were isolated with a purity of >97% and impurities were identified and quantified. After the final hydrolysis step, MIDD0301 was isolated in a 44% overall yield and purity of 98.9% after recrystallization. The enantiomeric excess was greater than 99.0%.
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http://dx.doi.org/10.1021/acs.oprd.0c00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497793PMC
August 2020

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABA Receptor.

Molecules 2020 Aug 25;25(17). Epub 2020 Aug 25.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA.

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAR) of the αβγ subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.
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http://dx.doi.org/10.3390/molecules25173864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503500PMC
August 2020

Targeting Nitric Oxide Production in Microglia with Novel Imidazodiazepines for Nonsedative Pain Treatment.

ACS Chem Neurosci 2020 07 18;11(13):2019-2030. Epub 2020 Jun 18.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States.

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC of 260 and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABAR) α subunit, which can assemble with β and γ/δ subunits to form functional GABARs. Mouse microglia contained α, α, and α, in addition to β, γ, and δ, mRNA, enabling a more diverse array of GABARs than human microglia. Benzodiazepines are well-established modulators of GABAR activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABAR antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABARs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the μ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of nonsedative drug candidates for inflammatory pain.
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http://dx.doi.org/10.1021/acschemneuro.0c00324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380323PMC
July 2020

The Effects of pH on the Structure and Bioavailability of Imidazobenzodiazepine-3-Carboxylate MIDD0301.

Mol Pharm 2020 04 26;17(4):1182-1192. Epub 2020 Feb 26.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States.

We describe the effects of pH on the structure and bioavailability of MIDD0301, an oral lead compound for asthma. MIDD0301 interacts with peripheral GABA receptors to reduce lung inflammation and airway smooth muscle constriction. The structure of MIDD0301 combines basic imidazole and carboxylic acid function in the same diazepine scaffold, resulting in high solubility at neutral pH. Furthermore, we demonstrated that MIDD0301 can interconvert between a seven-membered ring structure at neutral pH and an acyclic compound at or below pH 3. Both structures have two stable conformers in solution that can be observed by H NMR at room temperature. Kinetic analysis showed opening and closing of the seven-membered ring of MIDD0301 at gastric and intestinal pH, occurring with different rate constants. However, in vivo studies showed that the interconversion kinetics are fast enough to yield similar MIDD0301 blood and lung concentrations for neutral and acidic formulations. Importantly, acidic and neutral formulations of MIDD0301 exhibit high lung distribution with low concentrations in brain. These findings demonstrate that MIDD0301 interconverts between stable structures at neutral and acidic pH without changes in bioavailability, further supporting its formulation as an oral asthma medication.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b01210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216793PMC
April 2020

Design, synthesis and characterization of novel gamma‑aminobutyric acid type A receptor ligands.

ARKIVOC 2020 2;2020(Pt 7):242-256. Epub 2020 Dec 2.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, USA.

Antinociceptive ligand HZ-166 is a GABA α2/α3 receptor subtype-selective potentiator. It has been shown to exhibit anxiolytic-like effects in rodent and rhesus monkeys, as well as reduced sedative/ataxic liabilities. In order to improve the metabolic stability of HZ-166, the ethyl ester moiety was bioisosterically replaced with 2,4-disubstituted oxazoles and oxazolines. The new analogs of HZ-166 were synthesized, characterized, and evalutated for their biological activity and docked in the human full-length heteromeric α1β3γ2L GABA receptor subtype CyroEM structure (6HUO). Importantly no sedation nor ataxia was observed on the rotorod for LKG-I-70 () or KPP-III-51 () at 100 and 120 mg/kg, respectively. These was also no loss of righting response for either ligand.
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http://dx.doi.org/10.24820/ark.5550190.p011.398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909486PMC
December 2020

Synthesis and biological evaluation of calcioic acid.

Steroids 2020 02 6;154:108536. Epub 2019 Nov 6.

Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery (MIDD), University of Wisconsin, Milwaukee, WI 53211, USA. Electronic address:

Herein, we describe the synthesis of calcioic acid following a recently developed synthetic strategy for calcitroic acid. Several improvements to reaction conditions were made, which resulted in higher yields. The improved workup and isolation procedures are described. Furthermore, we investigated the interaction between the vitamin D receptor (VDR) and calcioic acid. Calcioic acid was able to bind VDR with a binding constant of 71 µM. In cells, calcioic acid reduced the transcription of VDR target gene CYP24A1 in the presence 1α,25-dihydroxyvitamin D (1,25(OH)D) but did not induce the transcription of CYP24A1. Therefore, calcioic acid is a very weak VDR antagonist. With the generation of gram quantities, further studies are expected to reveal if calcioic acid is solely a water-soluble metabolite of vitamin D or if it mediates other biological functions through biomolecules other than VDR.
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http://dx.doi.org/10.1016/j.steroids.2019.108536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980330PMC
February 2020

An anthrone-based Kv7.2/7.3 channel blocker with improved properties for the investigation of psychiatric and neurodegenerative disorders.

Bioorg Med Chem Lett 2019 12 14;29(23):126681. Epub 2019 Sep 14.

Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA. Electronic address:

A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC = 0.16 μM in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.
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http://dx.doi.org/10.1016/j.bmcl.2019.126681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858848PMC
December 2019

The parmodulin NRD-21 is an allosteric inhibitor of PAR1 Gq signaling with improved anti-inflammatory activity and stability.

Bioorg Med Chem 2019 09 29;27(17):3788-3796. Epub 2019 Jun 29.

Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA. Electronic address:

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.
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http://dx.doi.org/10.1016/j.bmc.2019.06.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706283PMC
September 2019

Novel Benzodiazepine-Like Ligands with Various Anxiolytic, Antidepressant, or Pro-Cognitive Profiles.

Mol Neuropsychiatry 2019 Apr 23;5(2):84-97. Epub 2019 Jan 23.

Campbell Family Mental Health Research Institute of CAMH, Toronto, Ontario, Canada.

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.
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http://dx.doi.org/10.1159/000496086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528097PMC
April 2019

MIDD0301 - A first-in-class anti-inflammatory asthma drug targets GABA receptors without causing systemic immune suppression.

Basic Clin Pharmacol Toxicol 2019 Jul 21;125(1):75-84. Epub 2019 Feb 21.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin.

We report a 28-day repeat dose immunotoxicity evaluation of investigational drug MIDD0301, a novel oral asthma drug candidate that targets gamma amino butyric acid type A receptors (GABA R) in the lung. The study design employed oral administration of mice twice daily throughout the study period with 100 mg/kg MIDD0301 mixed in peanut butter. Compound dosing did not reveal signs of general toxicity as determined by animal weight, organ weight or haematology. Peanut butter plus test drug (in addition to ad libitum standard rodent chow) did not affect weight gain in the adult mice, in contrast to weight loss in 5 mg/kg prednisone-treated mice. Spleen and thymus weights were unchanged in MIDD0301-treated mice, but prednisone significantly reduced the weight of those organs over the 28-day dosing. Similarly, no differences in spleen or thymus histology were observed following MIDD0301 treatment, but prednisone treatment induced morphological changes in the spleen. The number of small intestine Peyer's patches was not affected by MIDD0301 treatment, an important factor for orally administered drugs. Circulating lymphocyte, monocyte and granulocyte numbers were unchanged in the MIDD0301-treated animals, whereas differential lymphocyte numbers were reduced in prednisone-treated animals. MIDD0301 treatment did not alter IgG antibody responses to dinitrophenyl following dinitrophenyl-keyhole limpet haemocyanin immunization, indicating that systemic humoral immune function was not affected. Taken together, these studies show that repeated daily administration of MIDD0301 is safe and not associated with adverse immunotoxicological effects in mice.
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http://dx.doi.org/10.1111/bcpt.13206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565495PMC
July 2019

Design and Evaluation of Heterobivalent PAR1-PAR2 Ligands as Antagonists of Calcium Mobilization.

ACS Med Chem Lett 2019 Jan 3;10(1):121-126. Epub 2018 Dec 3.

Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, United States.

A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331166PMC
January 2019

Alternative binding sites at the vitamin D receptor and their ligands.

Mol Cell Endocrinol 2019 04 14;485:1-8. Epub 2019 Jan 14.

Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery (MIDD), University of Wisconsin, Milwaukee, WI, 53211, USA. Electronic address:

In recent decades, the majority of ligands developed for the vitamin D receptor (VDR) bind at its deeply buried genomic ligand binding pocket. Theses ligands can be categorized into agonists and partial agonists/antagonists. A limited number of ligands, most of them peptides, bind the VDR‒coactivator binding site that is formed in the presence of an agonist and inhibit coactivator recruitment, and therefore transcription. Another solvent exposed VDR‒ligand binding pocket was identified for lithocholic acid, improving the overall stability of the VDR complex. Additional proposed interactions with VDR are discussed herein that include the alternative VDR‒ligand binding pocket that may mediate both non-genomic cellular responses and binding function 3 that was identified for the androgen receptor. Many VDR ligands increase blood calcium levels at therapeutic concentrations in vivo, thus the identification of alternative VDR‒ligand binding pockets might be crucial to develop non-calcemic and potent ligands for VDR to treat cancer and inflammatory disease.
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http://dx.doi.org/10.1016/j.mce.2019.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444937PMC
April 2019

A novel GABA receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.

Am J Physiol Lung Cell Mol Physiol 2019 02 29;316(2):L385-L390. Epub 2018 Nov 29.

Department of Anesthesiology, Vagelos College of Physicians and Surgeons, Columbia University , New York, New York.

Airway smooth muscle (ASM) cells express GABA A receptors (GABARs), and previous reports have demonstrated that GABAR activators relax ASM. However, given the activity of GABARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.
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http://dx.doi.org/10.1152/ajplung.00356.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397346PMC
February 2019

Discovery and Optimization of Novel Hydrogen Peroxide Activated Aromatic Nitrogen Mustard Derivatives as Highly Potent Anticancer Agents.

J Med Chem 2018 10 10;61(20):9132-9145. Epub 2018 Oct 10.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery , University of Wisconsin, Milwaukee , 3210 North Cramer Street , Milwaukee , Wisconsin 53211 , United States.

We describe several new aromatic nitrogen mustards with various aromatic substituents and boronic esters that can be activated with HO to efficiently cross-link DNA. In vitro studies demonstrated the anticancer potential of these compounds at lower concentrations than those of other clinically used chemotherapeutics, such as melphalan and chlorambucil. In particular, compound 10, bearing an amino acid ester chain, is selectively cytotoxic toward breast cancer and leukemia cells that have inherently high levels of reactive oxygen species. Importantly, 10 was 10-14-fold more efficacious than melphalan and chlorambucil for triple-negative breast-cancer (TNBC) cells. Similarly, 10 is more toxic toward primary chronic-lymphocytic-leukemia cells than either chlorambucil or the lead compound, 9. The introduction of an amino acid side chain improved the solubility and permeability of 10. Furthermore, 10 inhibited the growth of TNBC tumors in xenografted mice without obvious signs of general toxicity, making this compound an ideal drug candidate for clinical development.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00559DOI Listing
October 2018

A high-throughput screening assay for pyruvate carboxylase.

Anal Biochem 2018 06 12;550:90-98. Epub 2018 Apr 12.

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA. Electronic address:

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.
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http://dx.doi.org/10.1016/j.ab.2018.04.012DOI Listing
June 2018

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABA Receptors in the Lung.

Mol Pharm 2018 05 2;15(5):1766-1777. Epub 2018 Apr 2.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery , University of Wisconsin-Milwaukee , Milwaukee , Wisconsin 53211 , United States.

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric αβγ selective GABA receptor (GABAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by αβγ GABARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4 T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4 T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAR ligands.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b01013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954213PMC
May 2018

Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ-Aminobutyric Acid Type A Receptor (GABAR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability.

J Med Chem 2018 03 6;61(6):2422-2446. Epub 2018 Mar 6.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery , University of Wisconsin-Milwaukee , 3210 N. Cramer St. , Milwaukee , Wisconsin 53211 , United States.

Recent reports indicate that α6β2/3γ2 GABAR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABAR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABAR α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABARs and were functionally silent at diazepam sensitive α1β2/3γ2 GABARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABAR α6β2/3γ2 subtypes.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941172PMC
March 2018

Synthesis of chiral GABA receptor subtype selective ligands as potential agents to treat schizophrenia as well as depression.

ARKIVOC 2018;2018(4):158-182. Epub 2018 Mar 11.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States.

A series of novel imidazobenzodiazepine analogs of the lead chiral ligand SH-053-2'F--CH (), an α2/α3/α5 (Bz)GABA (A)ergic receptor subtype selective ligand, which reversed PCP-induced prepulse inhibition (PPI) of acoustic startle, were synthesized. These chiral ()-CH ligands are targeted for the treatment of schizophrenia and depression. These new ligands were designed by modifying the liable ester functionality in to improve the metabolic stability, cytotoxicity, and activity as compared to . Based on the data to date, the most promising ligands are the -cyclopropyl amide GL-I-55 () and the methyl bioisostere GL-I-65 (). The metabolic stability, cytotoxicity and locomotor effects are described in this report. Based on these results, and are the most promising for further pharmacology.
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http://dx.doi.org/10.24820/ark.5550190.p010.460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413308PMC
March 2018

Novel VDR antagonists based on the GW0742 scaffold.

Bioorg Med Chem Lett 2018 02 19;28(3):351-354. Epub 2017 Dec 19.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discover, University of Wisconsin-Milwaukee, WI 53211, USA. Electronic address:

The vitamin D receptor is a nuclear hormone receptor that regulates cell proliferation, cell differentiation and calcium homeostasis. The receptor is endogenously activated by 1,25-dihydroxyvitamin D, which induces transcription of VDR targets genes regulated by coactivator binding. VDR antagonists and partial agonists have been developed based on the secosteroid scaffold of vitamin D. Only a few non-secosteroid VDR antagonists are known. Herein, we report the rational design of non-secosteroid VDR antagonists using GW0742 as a scaffold. GW0742 is a PPARδ agonist previously identified by our group as a VDR antagonist. Several modifications including the replacement of the thiazole ring with an oxazole ring led to compound 7b, which inhibited VDR-mediated transcription (IC = 660 nM) without activating PPARδ-mediated transcription. However, inhibition of transcription mediated by other nuclear receptors was observed.
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http://dx.doi.org/10.1016/j.bmcl.2017.12.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008168PMC
February 2018

Parallel Chemistry Approach to Identify Novel Nuclear Receptor Ligands Based on the GW0742 Scaffold.

ACS Comb Sci 2017 10 5;19(10):646-656. Epub 2017 Sep 5.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin , Milwaukee, Wisconsin 53211, United States.

We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferator-activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were applied, such as a solid-phase palladium-catalyzed Suzuki coupling. In addition, tetrazole-based compounds were generated as a bioisostere for carboxylic acid-containing ligand GW0742. The new compounds were investigated for their ability to activate PPARδ mediated transcription and their cross-reactivity with the vitamin D receptor (VDR), another member of the nuclear receptor superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742, where ∼65 of the compounds synthesized exhibited partial PPARδ activity (23-98%) with EC values ranging from 0.007-18.2 μM. Some ligands, such as compound 32, were more potent inhibitors of VDR-mediated transcription with significantly reduced PPARδ activity than GW0742, however, none of the ligands were completely selective for VDR inhibition over PPARδ activation of transcription.
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http://dx.doi.org/10.1021/acscombsci.7b00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643073PMC
October 2017

Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABA Receptor Modulators.

Mol Pharm 2017 06 1;14(6):2088-2098. Epub 2017 May 1.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee , Milwaukee, Wisconsin 53201, United States.

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic αβγ GABAR selective compound 1 and acidic αβγ selective GABAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4 T lymphocytes and directly modulated their transmembrane currents by acting on GABARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAR ligands.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497587PMC
June 2017

Hydrogen peroxide activated quinone methide precursors with enhanced DNA cross-linking capability and cytotoxicity towards cancer cells.

Eur J Med Chem 2017 Jun 24;133:197-207. Epub 2017 Mar 24.

Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, 3210 N. Cramer Street, Milwaukee, WI, 53211, USA. Electronic address:

Quinone methide (QM) formation induced by endogenously generated HO is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of HO-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with HO and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves HO-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development.
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http://dx.doi.org/10.1016/j.ejmech.2017.03.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652303PMC
June 2017

Pharmacological and antihyperalgesic properties of the novel α2/3 preferring GABA receptor ligand MP-III-024.

Brain Res Bull 2017 May 4;131:62-69. Epub 2017 Mar 4.

University of Wisconsin-Milwaukee, Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, Milwaukee, WI 53201, USA.

γ-Aminobutyric acid type A (GABA) receptors are located in spinal nociceptive circuits where they modulate the transmission of pain sensory signals from the periphery to higher centers. Benzodiazepine-type drugs bind to GABA receptors containing α1, α2, α3, and α5 subunits (α1GABA, α2GABA, α3GABA and α5GABA receptors, respectively) through which they inhibit the transmission of these signals. In the present study we describe the novel benzodiazepine site positive allosteric modulator modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024). MP-III-024 displayed preference for α2GABA and α3GABA receptors relative to α1GABA and α5GABA receptors as well as an improved metabolic profile relative to subtype-selective positive modulators that are available currently. Administration of MP-III-024 resulted in a dose- and time-dependent reversal of mechanical hyperalgesia. On locomotor activity and schedule-controlled responding, MP-III-024 was ineffective across the doses tested. These data provide further evidence that α2GABA and α3GABA receptors play an important role in the antihyperalgesic effects and may not be involved in some of the undesired effects of benzodiazepine-like drugs. Further, these findings suggest that MP-III-024 is a suitable research tool for investigating the role of α2GABA and α3GABA receptors in the behavioral properties of benzodiazepine-like drugs in mice.
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http://dx.doi.org/10.1016/j.brainresbull.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501353PMC
May 2017

Synthesis and Characterization of a Novel γ-Aminobutyric Acid Type A (GABA) Receptor Ligand That Combines Outstanding Metabolic Stability, Pharmacokinetics, and Anxiolytic Efficacy.

J Med Chem 2016 12 28;59(23):10800-10806. Epub 2016 Nov 28.

Lilly Research Laboratories, Eli Lilly and Company , Indianapolis, Indiana 42685, United States.

1,4-Benzodiazepines are used in the treatment of anxiety disorders but have limited long-term use due to adverse effects. HZ-166 (2) has been shown to have anxiolytic-like effects with reduced sedative/ataxic liabilities. A 1,3-oxazole KRM-II-81 (9) was discovered from a series of six bioisosteres with significantly improved pharmacokinetic and pharmacodynamic properties as compared to 2. Oxazole 9 was further characterized and exhibited improved anxiolytic-like effects in a mouse marble burying assay and a rat Vogel conflict test.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215686PMC
December 2016

Optimization of substituted imidazobenzodiazepines as novel asthma treatments.

Eur J Med Chem 2017 Jan 24;126:550-560. Epub 2016 Nov 24.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, WI, 53211, United States. Electronic address:

We describe the synthesis of analogs of XHE-III-74, a selective α4β3γ2 GABAR ligand, shown to relax airway smooth muscle ex vivo and reduce airway hyperresponsiveness in a murine asthma model. To improve properties of this compound as an asthma therapeutic, a series of analogs with a deuterated methoxy group in place of methoxy group at C-8 position was evaluated for isotope effects in preclinical assays; including microsomal stability, cytotoxicity, and sensorimotor impairment. The deuterated compounds were equally or more metabolically stable than the corresponding non-deuterated analogs and increased sensorimotor impairment was observed for some deuterated compounds. Thioesters were more cytotoxic in comparison to other carboxylic acid derivatives of this compound series. The most promising compound 16 identified from the in vitro screens also strongly inhibited smooth muscle constriction in ex vivo guinea pig tracheal rings. Smooth muscle relaxation, determined by reduction of airway hyperresponsiveness with a murine ovalbumin sensitized and challenged model, showed that 16 was efficacious at low methacholine concentrations. However, this effect was limited due to suboptimal pharmacokinetics of 16. Based on these findings, further analogs of XHE-III-74 will be investigated to improve in vivo metabolic stability while retaining the efficacy at lung tissues involved in asthma pathology.
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http://dx.doi.org/10.1016/j.ejmech.2016.11.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253108PMC
January 2017

Calcitroic Acid-A Review.

ACS Chem Biol 2016 10 6;11(10):2665-2672. Epub 2016 Sep 6.

Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee , 3210 N. Cramer Street, Milwaukee, Wisconsin 53211, United States.

Calcitroic acid was isolated and characterized almost four decades ago, but little is known about this important vitamin D metabolite. Four reported synthetic strategies to generate calcitroic acid are presented that highlight the scientific progress in the field of chemistry directed to vitamin D analog synthesis. The most recent synthesis described the generation of calcitroic acid with an overall yield of 12.8% in 13 steps. The endogenous formation of calcitroic acid has been demonstrated in perfused rat kidney using 24,25,26,27-tetranor-1,23(OH)D. Although, the majority of vitamin D metabolism is mediated by 24-hydoxylase (CYP24A1), it is not clear why the formation of calcitroic acid was not observed in the presence of recombinant CYP24A1 enzyme. Furthermore, it is not known if enzyme 1α-hydroxylase (CYP27B1) can convert calcioic acid into calcitroic acid. In addition to the lack of research investigating the endogenous formation of calcitroic acid, the physiological role of calcitroic acid remains unknown. Only a few reports mentioned the biological activity of calcitroic acid in connection with the vitamin D receptor (VDR). When administered subcutaneously, calcitroic acid has anthracitic properties and elevates calcium blood levels when administered intravenously. In vitro, calcitroic acid at higher concentrations has been shown to bind VDR and induce gene transcription. However, these studies were not carried out in cells derived from target organs of calcitroic acid such as kidney, liver, and intestine. We can conclude that our current knowledge of calcitroic acid is limited, and more studies are needed to identify its physiological role.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074857PMC
http://dx.doi.org/10.1021/acschembio.6b00569DOI Listing
October 2016

Characterization of GABA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells.

J Pharmacol Toxicol Methods 2016 Nov - Dec;82:109-114. Epub 2016 Aug 18.

Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address:

Introduction: Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABA receptor.

Methods: iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABA receptor subunits were quantified by qRT-PCR.

Results: We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15 to 25MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC of 0.43μM. Positive GABA receptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC values of 0.42μM, 1.56μM, and 0.23μM, respectively. The α2/α3/α5 selective compound HZ-166-induced the highest potentiation (efficacy) of 810% of the current induced by 100nM GABA. Quantification of GABA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain.

Discussion: iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change.
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http://dx.doi.org/10.1016/j.vascn.2016.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116268PMC
May 2017