Publications by authors named "Werner J Geldenhuys"

119 Publications

Development of an a priori computational approach for brain uptake of compounds in an insect model system.

Bioorg Med Chem Lett 2021 May 10;40:127930. Epub 2021 Mar 10.

Emerging Pathogens Institute, Entomology and Nematology Department, University of Florida, Gainesville, FL, USA.

Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure-property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r > 0.5, with the descriptors log(P*MW) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines.
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http://dx.doi.org/10.1016/j.bmcl.2021.127930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096670PMC
May 2021

The Role of ERO1α in Modulating Cancer Progression and Immune Escape.

J Cancer Immunol (Wilmington) 2020 ;2(3):103-115

WVU Cancer Institute, Morgantown, WV 26506, USA.

Endoplasmic reticulum oxidoreductin-1 alpha (ERO1α) was originally shown to be an endoplasmic reticulum (ER) resident protein undergoing oxidative cycles in concert with protein disulfide isomerase (PDI) to promote proper protein folding and to maintain homeostasis within the ER. ERO1α belongs to the flavoprotein family containing a flavin adenine dinucleotide utilized in transferring of electrons during oxidation-reduction cycles. This family is used to maintain redox potentials and protein homeostasis within the ER. ERO1α's location and function has since been shown to exist beyond the ER. Originally thought to exist solely in the ER, it has since been found to exist in the golgi apparatus, as well as in exosomes purified from patient samples. Besides aiding in protein folding of transmembrane and secretory proteins in conjunction with PDI, ERO1α is also known for formation of disulfide bridges. Public databases, such as the Cancer Genome Atlas (TCGA) and The Protein Atlas, reveal ERO1α as a poor prognostic marker in multiple disease settings. Recent evidence indicates that ERO1α expression in tumor cells is a critical determinant of metastasis. However, the impact of increased ERO1α expression in tumor cells extends into the tumor microenvironment. Secretory proteins requiring ERO1α expression for proper folding have been implicated as being involved in immune escape through promotion of upregulation of programmed death ligand-1 (PD-L1) and stimulation of polymorphonuclear myeloid derived suppressor cells (PMN-MDSC's) via secretion of granulocytic colony stimulating factor (G-CSF). Hereby, ERO1α plays a pivotal role in cancer progression and potentially immune escape; making ERO1α an emerging attractive putative target for the treatment of cancer.
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http://dx.doi.org/10.33696/cancerimmunol.2.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894644PMC
January 2020

Small-Molecule HSP27 Inhibitor Abolishes Androgen Receptors in Glioblastoma.

J Med Chem 2021 02 1;64(3):1570-1583. Epub 2021 Feb 1.

Department of Chemistry, Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115, United States.

Androgen receptor (AR) contributes to the progression of glioblastoma (GBM), and antiandrogen agents have the potential to be used for the treatment of GBM. However, AR mutation commonly happens in GBM, which makes the antiandrogen agents less effective. Heat shock 27 kDa protein (HSP27) is a well-documented chaperone protein to stabilize ARs. Inhibition of HSP27 results in AR degradation regardless of the mutation status of ARs, which makes HSP27 a good target to abolish ARs in GBM. Compound I is a HSP27 inhibitor that significantly induces AR degradation in GBM cells the proteasomal pathway, and it selectively inhibits AR-overexpressed GBM cell growth with IC values around 5 nM. The compound also significantly inhibits GBM xenograft at 20 mg/kg and does not cause toxicity to mice up to 80 mg/kg. These results suggest that targeting HSP27 to induce AR degradation in GBM is a promising and novel treatment.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01537DOI Listing
February 2021

Novel Small-Molecule Inhibitor for the Oncogenic Tyrosine Phosphatase SHP2 with Anti-Breast Cancer Cell Effects.

ACS Omega 2020 Oct 25;5(39):25113-25124. Epub 2020 Sep 25.

Department of Biochemistry, West Virginia University, Morgantown, West Virginia 26506, United States.

The oncogenic property of the Src homology phosphotyrosine phosphatase 2 (SHP2) is well-known, but developing specific inhibitors has been very difficult. Based on our previous reports that showed the importance of acidic residues surrounding SHP2 substrate phosphotyrosines for specific recognition, we have rationally designed and chemically synthesized a small-molecule SHP2 inhibitor named 4,4'-(4'-carboxy)-4-nonyloxy-[1,1'-biphenyl]-3,5-diyl)dibutanoic acid (CNBDA). Molecular modeling predicted that CNBDA packs well into the SHP2 active site and makes extended interactions primarily with positively charged and polar amino acids surrounding the active site. PTPase assays showed that CNBDA inhibits SHP2 with an IC of 5 μM. However, the IC of CNBDA toward SHP1, the close structural homologue of SHP2, was 125 μM, suggesting an approximately 25-fold effectiveness against SHP2 than SHP1. Because SHP2 is known for its positive role in breast cancer (BC) cell biology, we tested the effect of SHP2 inhibition with CNBDA in HER2-positive BC cells. Treatment with CNBDA suppressed cell proliferation in 2D culture, anchorage-independent growth in soft agar, and mammosphere (tumorisphere) formation in suspension cultures in a concentration-dependent manner. Furthermore, CNBDA inhibited EGF-induced signaling and expression of HER2 by inhibiting the PTPase activity of SHP2 in BC cells. These findings suggest that CNBDA is a promising anti-SHP2 lead compound with anti-BC cell effects.
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http://dx.doi.org/10.1021/acsomega.0c02746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542598PMC
October 2020

Synthesis of Illudinine from Dimedone and Identification of Activity as a Monoamine Oxidase Inhibitor.

J Org Chem 2020 11 27;85(21):13429-13437. Epub 2020 Aug 27.

The fungal metabolite illudinine is prepared in seven steps and ca. 55% overall yield from dimedone using an "open and shut" (ring-opening and ring-closing) strategy. Tandem ring-opening fragmentation and olefination of dimedone establishes alkyne and vinylarene functionality linked by a neopentylene tether. Oxidative cycloisomerization then provides the illudinine framework. The key innovation in this second-generation synthesis of illudinine is the use of the nitrile functional group, rather than an ester, as the functional precursor to the carboxylic acid of illudinine. The small, linear nitrile (C≡N) is associated with improved selectivity, π-conjugation, and reactivity at multiple points in the synthetic sequence relative to the carboxylic acid ester. Preliminary assays indicate that illudinine and several related synthetic analogues are monoamine oxidase inhibitors, which is the first reported indication of biological activity associated with this natural product. Illudinine was found to inhibit monoamine oxidase B (MAO-B) with an IC of 18 ± 7.1 μM in preliminary assays.
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http://dx.doi.org/10.1021/acs.joc.0c01301DOI Listing
November 2020

Bioenergetic restoration and neuroprotection after therapeutic targeting of mitoNEET: New mechanism of pioglitazone following traumatic brain injury.

Exp Neurol 2020 05 10;327:113243. Epub 2020 Feb 10.

Spinal Cord & Brain Injury Research Center, University of Kentucky, Lexington, KY 40536, USA; Department of Neuroscience, University of Kentucky, Lexington, KY 40536, USA; Lexington VA Healthcare System, Lexington, KY 40502, USA. Electronic address:

Mitochondrial dysfunction is a pivotal event in many neurodegenerative disease states including traumatic brain injury (TBI) and spinal cord injury (SCI). One possible mechanism driving mitochondrial dysfunction is glutamate excitotoxicity leading to Ca-overload in neuronal or glial mitochondria. Therapies that reduce calcium overload and enhance bioenergetics have been shown to improve neurological outcomes. Pioglitazone, an FDA approved compound, has shown neuroprotective properties following TBI and SCI, but the underlying mechanism(s) are unknown. We hypothesized that the interaction between pioglitazone and a novel mitochondrial protein called mitoNEET was the basis for neuroprotection following CNS injury. We discovered that mitoNEET is an important mediator of Ca-mediated mitochondrial dysfunction and show that binding mitoNEET with pioglitazone can prevent Ca-induced dysfunction. By utilizing wild-type (WT) and mitoNEET null mice, we show that pioglitazone mitigates mitochondrial dysfunction and provides neuroprotection in WT mice, though produces no restorative effects in mitoNEET null mice. We also show that NL-1, a novel mitoNEET ligand, is neuroprotective following TBI in both mice and rats. These results support the crucial role of mitoNEET for mitochondrial bioenergetics, its importance in the neuropathological sequelae of TBI and the necessity of mitoNEET for pioglitazone-mediated neuroprotection. Since mitochondrial dysfunction is a pathobiological complication seen in other diseases such as diabetes, motor neuron disease and cancer, targeting mitoNEET may provide a novel mitoceutical target and therapeutic intervention for diseases that expand beyond TBI.
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http://dx.doi.org/10.1016/j.expneurol.2020.113243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089834PMC
May 2020

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2).

J Biol Chem 2020 03 4;295(11):3563-3575. Epub 2020 Feb 4.

Department of Biochemistry, School of Medicine West Virginia University, Morgantown, West Virginia 26506; WVU Cancer Institute, School of Medicine, West Virginia University, Morgantown, West Virginia 26506. Electronic address:

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.
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http://dx.doi.org/10.1074/jbc.RA119.011422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076199PMC
March 2020

Nanoparticle formulation and in vitro efficacy testing of the mitoNEET ligand NL-1 for drug delivery in a brain endothelial model of ischemic reperfusion-injury.

Int J Pharm 2020 Mar 29;578:119090. Epub 2020 Jan 29.

Department of Pharmaceutical Sciences, West Virginia University School of Pharmacy, Morgantown, WV 26506, United States; Department of Neuroscience, West Virginia University, School of Medicine, Morgantown, WV 26506, United States. Electronic address:

Ischemic reperfusion injury after a stroke is a leading cause of mortality and disability due to neuronal loss and tissue damage. Mitochondrial dysfunction plays a major role in the reperfusion-injury sequelae, and offers an attractive drug target. Mitochondrial derived reactive oxygen species (ROS) and resultant apoptotic cascade are among the primary mechanisms of neuronal death following ischemia and reperfusion injury. Here we optimized a nanoparticle formulation for the mitoNEET ligand NL-1, to target mitochondrial dysfunction post ischemic reperfusion (IR) injury. NL-1, a hydrophobic drug, was formulated using PLGA polymers with a particle size and entrapment efficiency of 123.9 ± 17.1 nm and 59.7 ± 10.1%, respectively. The formulation was characterized for physical state of NL-1, in vitro release, uptake and nanoparticle localization. A near complete uptake of nanoparticles was found to occur by three hours, with the process being energy-dependent and occurring via caveolar mediated endocytosis. The fluorescent nanoparticles were found to localize in the cytoplasm of the endothelial cells. An in vitro oxygen glucose deprivation (OGD) model to mimic IR was employed for in vitro efficacy testing in murine brain vascular endothelium cells (bEND.3 cells). Efficacy studies showed that both NL-1 and the nanoparticles loaded with NL-1 had a protective activity against peroxide generation, and displayed improved cellular viability, as seen via reduction in cellular apoptosis. Finally, PLGA nanoparticles were found to have a non-toxic profile in vitro, and were found to be safe for intravenous administration. This study lays the preliminary work for potential use of mitoNEET as a target and NL-1 as a therapeutic for the treatment of cerebral ischemia and reperfusion injury.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067674PMC
March 2020

Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

Pharm Res 2020 Jan 27;37(3):43. Epub 2020 Jan 27.

Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia, USA.

Purpose: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia.

Methods: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness.

Results: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay.

Conclusion: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.
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http://dx.doi.org/10.1007/s11095-020-2767-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021357PMC
January 2020

Systemic inhibition of tissue-nonspecific alkaline phosphatase alters the brain-immune axis in experimental sepsis.

Sci Rep 2019 12 11;9(1):18788. Epub 2019 Dec 11.

Department of Microbiology, Immunology, and Cell Biology, School of Medicine, Center for Basic and Translational Stroke Research, Rockefeller Neuroscience Institute, West Virginia University, Morgantown, WV, USA.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme present in many cells and tissues, including the central nervous system. Yet its functions at the brain-immune axis remain unclear. The goal of this study was to use a novel small molecular inhibitor of TNAP, SBI-425, to interrogate the function of TNAP in neuroimmune disorders. Following intraperitoneal (IP) administration of SBI-425, mass spectrometry analysis revealed that the SBI-425 does not cross the blood-brain barrier (BBB) in healthy mice. To elucidate the role of TNAP at the brain-immune axis, mice were subjected to experimental sepsis and received either vehicle or SBI-425 (25 mg/kg, IP) daily for 7 days. While SBI-425 administration did not affect clinical severity outcomes, we found that SBI-425 administration suppressed CD4 + Foxp3+ CD25- and CD8 + Foxp3+ CD25- splenocyte T-cell populations compared to controls. Further evaluation of SBI-425's effects in the brain revealed that TNAP activity was suppressed in the brain parenchyma of SBI-425-treated mice compared to controls. When primary brain endothelial cells were treated with a proinflammatory stimulus the addition of SBI-425 treatment potentiated the loss of barrier function in BBB endothelial cells. To further demonstrate a protective role for TNAP at endothelial barriers within this axis, transgenic mice with a conditional overexpression of TNAP were subjected to experimental sepsis and found to have increased survival and decreased clinical severity scores compared to controls. Taken together, these results demonstrate a novel role for TNAP activity in shaping the dynamic interactions within the brain-immune axis.
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http://dx.doi.org/10.1038/s41598-019-55154-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906465PMC
December 2019

4-Hydroxynonenal and 4-Oxononenal Differentially Bind to the Redox Sensor MitoNEET.

Chem Res Toxicol 2019 06 29;32(6):977-981. Epub 2019 May 29.

Department of Chemistry , Ball State University , Muncie , Indiana 47304 , United States.

MitoNEET is a CDGSH iron-sulfur protein that has been a target for drug development for diseases such as type-2 diabetes, cancer, and Parkinson's disease. Functions proposed for mitoNEET are as a redox sensor and regulator of free iron in the mitochondria. We have investigated the reactivity of mitoNEET toward the reactive electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) that are produced from the oxidation of polyunsaturated fatty acid during oxidative stress. Proteomic, electrophoretic, and spectroscopic analysis has shown that HNE and ONE react in a sequence selective manner that was unexpected considering the structure similarity of these two reactive electrophiles.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556333PMC
June 2019

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

J Pharmacol Exp Ther 2019 07 22;370(1):25-34. Epub 2019 Apr 22.

Department of Pharmaceutical Sciences, School of Pharmacy (W.J.G.), Department of Microbiology, Immunology and Cell Biology, School of Medicine (R.R.N., K.H.M., L.F.G.), Robert C. Byrd Health Sciences Center (W.J.G., R.R.N., D.P., K.H.M., L.F.G.), and WVU Cancer Institute (W.J.G., K.H.M., L.F.G.), West Virginia University, Morgantown, West Virginia

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase () and mitoNEET () than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.
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http://dx.doi.org/10.1124/jpet.118.255984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538890PMC
July 2019

Targeting the Blood-Brain Barrier to Prevent Sepsis-Associated Cognitive Impairment.

J Cent Nerv Syst Dis 2019 9;11:1179573519840652. Epub 2019 Apr 9.

Graduate Programs in Neuroscience, Department of Neuroscience, School of Medicine, Health Sciences Center, West Virginia University, Morgantown, WV, USA.

Sepsis is a systemic inflammatory disease resulting from an infection. This disorder affects 750 000 people annually in the United States and has a 62% rehospitalization rate. Septic symptoms range from typical flu-like symptoms (eg, headache, fever) to a multifactorial syndrome known as sepsis-associated encephalopathy (SAE). Patients with SAE exhibit an acute altered mental status and often have higher mortality and morbidity. In addition, many sepsis survivors are also burdened with long-term cognitive impairment. The mechanisms through which sepsis initiates SAE and promotes long-term cognitive impairment in septic survivors are poorly understood. Due to its unique role as an interface between the brain and the periphery, numerous studies support a regulatory role for the blood-brain barrier (BBB) in the progression of acute and chronic brain dysfunction. In this review, we discuss the current body of literature which supports the BBB as a nexus which integrates signals from the brain and the periphery in sepsis. We highlight key insights on the mechanisms that contribute to the BBB's role in sepsis which include neuroinflammation, increased barrier permeability, immune cell infiltration, mitochondrial dysfunction, and a potential barrier role for tissue non-specific alkaline phosphatase (TNAP). Finally, we address current drug treatments (eg, antimicrobials and intravenous immunoglobulins) for sepsis and their potential outcomes on brain function. A comprehensive understanding of these mechanisms may enable clinicians to target specific aspects of BBB function as a therapeutic tool to limit long-term cognitive impairment in sepsis survivors.
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http://dx.doi.org/10.1177/1179573519840652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456845PMC
April 2019

Inhibition of the FAD containing ER oxidoreductin 1 (Ero1) protein by EN-460 as a strategy for treatment of multiple myeloma.

Bioorg Med Chem 2019 04 10;27(8):1479-1488. Epub 2019 Feb 10.

Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morganton, WV 26506, United States; Cancer Center, West Virginia University, Morgantown, WV 26506, United States. Electronic address:

Multiple myeloma (MM) cells demonstrate high basal endoplasmic reticulum (ER) stress and are typically exquisitely sensitive to agents such as proteasome inhibitors that activate the unfolded protein response. The flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum oxidoreductin enzyme (Ero1L) catalyzes de-novo disulfide bridge formation of ER resident proteins and contributes to proper protein folding. Here we show that increased Ero1L expression is prognostic of poor outcomes for MM patients relapsing on therapy. We propose that targeting protein folding via inhibition of Ero1L may represent a novel therapeutic strategy for the treatment of MM. In this report we show that treatment of MM cells with EN-460, a known inhibitor of ERO1L, was sufficient to inhibit cell proliferation and induce apoptosis. Furthermore, we show that cell death correlated in part with induction of ER stress. We also show that EN460 inhibited the enzyme activity of Ero1L, with an IC50 of 22.13 μM, consistent with previous reports. However, EN-460 was also found to inhibit other FAD-containing enzymes including MAO-A (IC = 7.91 μM), MAO-B (IC = 30.59 μM) and LSD1 (IC = 4.16 μM), suggesting overlap in inhibitor activity and the potential need to develop more specific inhibitors to enable pharmacological validation of ERO1L as a target for the treatment of MM. We additionally prepared and characterized azide-tagged derivatives of EN-460 as possible functional probe compounds (e.g., for photo-affinity labeling) for future target-engagement studies and further development of structure-activity relationships.
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http://dx.doi.org/10.1016/j.bmc.2019.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554731PMC
April 2019

Extracellular Vesicles Secreted in Response to Cytokine Exposure Increase Mitochondrial Oxygen Consumption in Recipient Cells.

Front Cell Neurosci 2019 14;13:51. Epub 2019 Feb 14.

Department of Physiology and Pharmacology, West Virginia University School of Medicine, Morgantown, WV, United States.

Extracellular vesicles (EVs) are small, membrane-bound nanoparticles released from most, if not all cells, and can carry functionally active cargo (proteins, nucleic acids) which can be taken up by neighboring cells and mediate physiologically relevant effects. In this capacity, EVs are being regarded as novel cell-to-cell communicators, which may play important roles in the progression of neurodegenerative diseases, like Alzheimer's disease (AD). Aside from the canonical physical hallmarks of this disease [amyloid β (Aβ) plaques, neurofibrillary tangles, and widespread cell death], AD is characterized by chronic neuroinflammation and mitochondrial dysfunction. In the current study, we sought to better understand the role of tumor necrosis factor-alpha (TNF-α), known to be involved in inflammation, in mediating alterations in mitochondrial function and EV secretion. Using an immortalized hippocampal cell line, we observed significant reductions in several parameters of mitochondrial oxygen consumption after a 24-h exposure period to TNF-α. In addition, after TNF-α exposure we also observed significant upregulation of two microRNAs (miRNAs; miR-34a and miR-146a) associated with mitochondrial dysfunction in secreted EVs. Despite this, when naïve cells are exposed to EVs isolated from TNF-α treated cells, mitochondrial respiration, proton leak, and reactive oxygen species (ROS) production are all significantly increased. Collectively these data indicate that a potent proinflammatory cytokine, TNF-α, induces significant mitochondrial dysfunction in a neuronal cell type, in part the secretion of EVs, which significantly alter mitochondrial activity in recipient cells.
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http://dx.doi.org/10.3389/fncel.2019.00051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383587PMC
February 2019

Binding of thiazolidinediones to the endoplasmic reticulum protein nutrient-deprivation autophagy factor-1.

Bioorg Med Chem Lett 2019 04 1;29(7):901-904. Epub 2019 Feb 1.

Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, WV 26506, United States. Electronic address:

Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]-containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.
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http://dx.doi.org/10.1016/j.bmcl.2019.01.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542280PMC
April 2019

Crystal structure of the mitochondrial protein mitoNEET bound to a benze-sulfonide ligand.

Commun Chem 2019 3;2. Epub 2019 Jul 3.

Department of Biochemistry, School of Medicine West Virginia University, Morgantown, WV 26506, USA.

MitoNEET (gene ) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
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http://dx.doi.org/10.1038/s42004-019-0172-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205193PMC
July 2019

An Effective Approach to Teaching Pharmacogenomics in the First Year of Pharmacy Curriculum.

Am J Pharm Educ 2018 10;82(8):6345

School of Pharmacy, West Virginia University, Morgantown, West Virginia.

To develop an effective method in teaching pharmacogenomics as a part of a new course, Biopharmaceutics and Pharmacogenomics. Teaching effectiveness was measured by quizzes, retrospective pre- and post-surveys, team activities, and journal reflections. Four team activities were included in the course: genomic disease, patient case, genetic counselor and a debate about personalized medicine. Outcomes and course impact were evaluated at the end of the course. The evaluation methods included the assessment of knowledge, students' perceptions regarding the utility of team activities, the impact of the course on students' confidence to discuss pharmacogenomics with health care providers or patients, and long-term knowledge retention, measured in the following P2 semester. Seventy-six students were enrolled in the course. Multiple assessments during the course demonstrated that students' knowledge of pharmacogenomics improved. The team activities had a positive impact on student learning, and the course improved their confidence level to discuss pharmacogenomics with another health care provider or a patient. While 86% of the students considered themselves "unconfident," "somewhat unconfident" or "neither confident nor unconfident" at the beginning of the course, 91% reported being "confident" or "somewhat confident" by the end of the course. This increase in confidence was statistically significant. Furthermore, students showed knowledge retention six months after taking the course. Implementation of a new course in pharmacogenomics was effective and well received by the students. It also prepared students for system-based therapeutics courses later in the curriculum.
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http://dx.doi.org/10.5688/ajpe6345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221532PMC
October 2018

Combination of cabazitaxel and plicamycin induces cell death in drug resistant B-cell acute lymphoblastic leukemia.

Leuk Res 2018 09 6;72:59-66. Epub 2018 Aug 6.

Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV, United States; Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, United States; WVU Cancer Institute, West Virginia University, Morgantown, WV, United States. Electronic address:

Bone marrow microenvironment mediated downregulation of BCL6 is critical for maintaining cell quiescence and modulating therapeutic response in B-cell acute lymphoblastic leukemia (ALL). In the present study, we have performed a high throughput cell death assay using BCL6 knockdown REH ALL cell line to screen a library of FDA-approved oncology drugs. In the process, we have identified a microtubule inhibitor, cabazitaxel (CAB), and a RNA synthesis inhibitor, plicamycin (PLI) as potential anti-leukemic agents. CAB and PLI inhibited cell proliferation in not only the BCL6 knockdown REH cell line, but also six other ALL cell lines. Furthermore, combination of CAB and PLI had a synergistic effect in inhibiting proliferation in a cytarabine-resistant (REH/Ara-C) ALL cell line. Use of nanoparticles for delivery of CAB and PLI demonstrated that the combination was very effective when tested in a co-culture model that mimics the in vivo bone marrow microenvironment that typically supports ALL cell survival and migration into protective niches. Furthermore, exposure to PLI inhibited SOX2 transcription and exposure to CAB inhibited not only Mcl-1 expression but also chemotaxis in ALL cells. Taken together, our study demonstrates the utility of concomitantly targeting different critical regulatory pathways to induce cell death in drug resistant ALL cells.
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http://dx.doi.org/10.1016/j.leukres.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414069PMC
September 2018

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Bioorg Med Chem Lett 2018 06 23;28(10):1937-1942. Epub 2018 Mar 23.

Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV 26506, USA; West Virginia University Cancer Institute, West Virginia University, Morgantown, WV 26506, USA. Electronic address:

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508634PMC
June 2018

Disulfiram-based disulfides as narrow-spectrum antibacterial agents.

Bioorg Med Chem Lett 2018 05 10;28(8):1298-1302. Epub 2018 Mar 10.

Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV, USA; Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA. Electronic address:

Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893419PMC
May 2018

Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.

J Biol Chem 2018 03 29;293(11):4134-4148. Epub 2018 Jan 29.

From the Departments of Biochemistry and

CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism. Dynamic regulation of CoA in different tissues and organs supports metabolic flexibility. Two mammalian Nudix hydrolases, Nudt19 and Nudt7, degrade CoA Nudt19 and Nudt7 possess conserved Nudix and CoA signature sequences and specifically hydrolyze the diphosphate bond of free CoA and acyl-CoAs to form 3',5'-ADP and 4'-(acyl)phosphopantetheine. Limited information is available on these enzymes, but the relatively high abundance of and mRNA in the kidney and liver, respectively, suggests that they play specific roles in the regulation of CoA levels in these organs. Here, we analyzed mice and found that deletion of elevates kidney CoA levels in mice fed , indicating that Nudt19 contributes to the regulation of CoA Unlike what was observed for the regulation of Nudt7 in the liver, Nudt19 transcript and protein levels in the kidney did not differ between fed and fasted states. Instead, we identified chenodeoxycholic acid as a specific Nudt19 inhibitor that competed with CoA for Nudt19 binding but did not bind to Nudt7. Exchange of the Nudix and CoA signature motifs between the two isoforms dramatically decreased their Furthermore, substitutions of conserved residues within these motifs identified amino acids playing different roles in CoA binding and hydrolysis in Nudt19 and Nudt7. Our results reveal that the kidney and liver each possesses a distinct peroxisomal CoA diphosphohydrolase.
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http://dx.doi.org/10.1074/jbc.RA117.001358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857999PMC
March 2018

LPA signaling is regulated through the primary cilium: a novel target in glioblastoma.

Oncogene 2018 03 11;37(11):1457-1471. Epub 2018 Jan 11.

WVU Cancer Institute, West Virginia University School of Medicine, Morgantown, WV, USA.

The primary cilium is a ubiquitous organelle presented on most human cells. It is a crucial signaling hub for multiple pathways including growth factor and G-protein coupled receptors. Loss of primary cilia, observed in various cancers, has been shown to affect cell proliferation. Primary cilia formation is drastically decreased in glioblastoma (GBM), however, the role of cilia in normal astrocyte or glioblastoma proliferation has not been explored. Here, we report that loss of primary cilia in human astrocytes stimulates growth rate in a lysophosphatidic acid (LPA)-dependent manner. We show that lysophosphatidic acid receptor 1 (LPAR1) is accumulated in primary cilia. LPAR1 signaling through Gα12/Gαq was previously reported to be responsible for cancer cell proliferation. We found that in ciliated cells, Gα12 and Gαq are excluded from the cilium, creating a barrier against unlimited proliferation, one of the hallmarks of cancer. Upon loss of primary cilia, LPAR1 redistributes to the plasma membrane with a concomitant increase in LPAR1 association with Gα12 and Gαq. Inhibition of LPA signaling with the small molecule compound Ki16425 in deciliated highly proliferative astrocytes or glioblastoma patient-derived cells/xenografts drastically suppresses their growth both in vitro and in vivo. Moreover, Ki16425 brain delivery via PEG-PLGA nanoparticles inhibited tumor progression in an intracranial glioblastoma PDX model. Overall, our findings establish a novel mechanism by which primary cilium restricts proliferation and indicate that loss of primary cilia is sufficient to increase mitogenic signaling, and is important for the maintenance of a highly proliferative phenotype. Clinical application of LPA inhibitors may prove beneficial to restrict glioblastoma growth and ensure local control of disease.
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http://dx.doi.org/10.1038/s41388-017-0049-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854509PMC
March 2018

Design and synthesis of small molecule agonists of EphA2 receptor.

Eur J Med Chem 2018 Jan 10;143:1261-1276. Epub 2017 Oct 10.

Rammelkamp Center for Research and Department of Medicine, MetroHealth Campus, Case Western Reserve University School of Medicine, Cleveland, OH, USA; Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA. Electronic address:

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure-activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor.
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http://dx.doi.org/10.1016/j.ejmech.2017.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752149PMC
January 2018

Allicin-inspired pyridyl disulfides as antimicrobial agents for multidrug-resistant Staphylococcus aureus.

Eur J Med Chem 2018 Jan 12;143:1185-1195. Epub 2017 Oct 12.

Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV, USA; Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA. Electronic address:

A chemical library comprised of nineteen synthesized pyridyl disulfides that emulate the chemical reactivity of allicin (garlic) was evaluated for antimicrobial activity against a panel of pathogenic bacteria. Gram-positive species including vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus (VISA, VRSA) demonstrated the highest level of susceptibility toward analogs with S-alkyl chains of 7-9 carbons in length. Further biological studies revealed that the disulfides display synergy with vancomycin against VRSA, cause dispersal of S. aureus biofilms, exhibit low cytotoxicity, and decelerate S. aureus metabolism. In final analysis, pyridyl disulfides represent a novel class of mechanism-based antibacterial agents that have a potential application as antibiotic adjuvants in combination therapy of S. aureus infections with reduced vancomycin susceptibility.
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http://dx.doi.org/10.1016/j.ejmech.2017.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817985PMC
January 2018

MitoNEET (CISD1) Knockout Mice Show Signs of Striatal Mitochondrial Dysfunction and a Parkinson's Disease Phenotype.

ACS Chem Neurosci 2017 12 27;8(12):2759-2765. Epub 2017 Sep 27.

Department of Pharmaceutical Sciences and Center for Neurodegenerative Disease and Aging, Northeast Ohio Medical University, College of Pharmacy , Rootstown, Ohio 44272, United States.

Mitochondrial dysfunction is thought to play a significant role in neurodegeneration observed in Parkinson's disease (PD), yet the mechanisms underlying this pathology remain unclear. Here, we demonstrate that loss of mitoNEET (CISD1), an iron-sulfur containing protein that regulates mitochondrial bioenergetics, results in mitochondrial dysfunction and loss of striatal dopamine and tyrosine hydroxylase. Mitochondria isolated from mice lacking mitoNEET were dysfunctional as revealed by elevated reactive oxygen species (ROS) and reduced capacity to produce ATP. Gait analysis revealed a shortened stride length and decreased rotarod performance in knockout mice, consistent with the loss of striatal dopamine. Together, these data suggest that mitoNEET KO mice exhibit many of the characteristics of early neurodegeneration in PD and may provide a novel drug discovery platform to evaluate compounds for enhancing mitochondrial function in neurodegenerative disorders.
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http://dx.doi.org/10.1021/acschemneuro.7b00287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964979PMC
December 2017

Synthesis and structure-activity relationships of carbohydrazides and 1,3,4-oxadiazole derivatives bearing an imidazolidine moiety against the yellow fever and dengue vector, Aedes aegypti.

Pest Manag Sci 2018 Feb 23;74(2):413-421. Epub 2017 Oct 23.

Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA.

Background: 1,3,4-Oxadiazole and imidazolidine rings are important heterocyclic compounds exhibiting a variety of biological activities. In this study, novel compounds with oxadiazole and imidazolidine rings were synthesized from 3-(methylsulfonyl)-2-oxoimidazolidine-1-carbonyl chloride and screened for insecticidal activities. The proposed structures of the 17 synthesized compounds were confirmed using elemental analysis, infrared (IR), proton nuclear magnetic resonance ( H-NMR), and mass spectroscopy.

Results: None of the compounds showed larvicidal activity at the tested concentrations against first-instar Aedes aegypti larvae. However, nine compounds exhibited promising adulticidal activity, with mortality rates of ≥80% at 5 µg per mosquito. Further dose-response bioassays were undertaken to determine median lethal dose (LD ) values. Compounds 1, 2b, 2c, 2d, 2 g, 3b, 3c, 3 g, and 3 h were effective, with typical LD values of about 5 - 10 µg per mosquito against female Ae. aegypti. Compounds 2c (bearing a nitro group on the aromatic ring; LD = 2.80 ± 0.54 µg per mosquito) and 3 h (double halogen groups at 2,4 position on the phenyl ring; LD = 2.80 ± 0.54 µg per mosquito) were the most promising compounds.

Conclusion: Preliminary mode of action studies failed to show consistent evidence of either neurotoxic or mitochondria-directed effects. Further chemical synthesis within this series may lead to the development of new effective insecticides. © 2017 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.4722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817975PMC
February 2018

Exploring Adenosine Receptor Ligands: Potential Role in the Treatment of Cardiovascular Diseases.

Molecules 2017 Jun 1;22(6). Epub 2017 Jun 1.

Department of Pharmaceutical Sciences, West Virginia University, School of Pharmacy, Morgantown, WV 26506, USA.

Cardiovascular diseases remain the number one diseases affecting patients' morbidity and mortality. The adenosine receptors are G-protein coupled receptors which have been of interest for drugs target for the treatment of multiple diseases ranging from cardiovascular to neurological. Adenosine receptors have been connected to several biological pathways affecting the physiology and pathology of the cardiovascular system. In this review, we will cover the different adenosine receptor ligands that have been identified to interact with adenosine receptors and affect the vascular system. These ligands will be evaluated from clinical as well as medicinal chemistry perspectives with more emphasis on how structural changes in structure translate into ligand potency and efficacy. Adenosine receptors represent a novel therapeutic target for development of treatment options treating a wide variety of diseases, including vascular disease and obesity.
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http://dx.doi.org/10.3390/molecules22060917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568125PMC
June 2017

Pioglitazone treatment following spinal cord injury maintains acute mitochondrial integrity and increases chronic tissue sparing and functional recovery.

Exp Neurol 2017 07 30;293:74-82. Epub 2017 Mar 30.

Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, Lexington, KY 40536-0509, USA; Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY 40536-0509, USA. Electronic address:

Pioglitazone is an FDA-approved PPAR-γ agonist drug used to treat diabetes, and it has demonstrated neuroprotective effects in multiple models of central nervous system (CNS) injury. Acute treatment after spinal cord injury (SCI) in rats is reported to suppress neuroinflammation, rescue injured tissues, and improve locomotor recovery. In the current study, we additionally assessed the protective efficacy of pioglitazone treatment on acute mitochondrial respiration, as well as functional and anatomical recovery after contusion SCI in adult male C57BL/6 mice. Mice received either vehicle or pioglitazone (10mg/kg) at either 15min or 3h after injury (75kdyn at T9) followed by a booster at 24h post-injury. At 25h, mitochondria were isolated from spinal cord segments centered on the injury epicenters and assessed for their respiratory capacity. Results showed significantly compromised mitochondrial respiration 25h following SCI, but pioglitazone treatment that was initiated either at 15min or 3h post-injury significantly maintained mitochondrial respiration rates near sham levels. A second cohort of injured mice received pioglitazone at 15min post injury, then once a day for 5days post-injury to assess locomotor recovery and tissue sparing over 4weeks. Compared to vehicle, pioglitazone treatment resulted in significantly greater recovery of hind-limb function over time, as determined by serial locomotor BMS assessments and both terminal BMS subscores and gridwalk performance. Such improvements correlated with significantly increased grey and white matter tissue sparing, although pioglitazone treatment did not abrogate long-term injury-induced inflammatory microglia/macrophage responses. In sum, pioglitazone significantly increased functional neuroprotection that was associated with remarkable maintenance of acute mitochondrial bioenergetics after traumatic SCI. This sets the stage for dose-response and delayed administration studies to maximize pioglitazone's efficacy for SCI while elucidating the precise role that mitochondria play in governing its neuroprotection; the ultimate goal to develop novel therapeutics that specifically target mitochondrial dysfunction.
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http://dx.doi.org/10.1016/j.expneurol.2017.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473659PMC
July 2017

High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor.

Bioorg Med Chem Lett 2017 05 28;27(9):2029-2037. Epub 2017 Feb 28.

Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506, United States.

In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC=4.078μM) and surprisingly a DAPK1 kinase agonist/activator (EC=39.525μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.
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http://dx.doi.org/10.1016/j.bmcl.2017.02.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435482PMC
May 2017