Browsing by Author "Hurley, Thomas D."
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Item The 5th International Lafora Epilepsy Workshop: Basic science elucidating therapeutic options and preparing for therapies in the clinic(Elsevier, 2020-02) Gentry, Matthew S.; Afawi, Zaid; Armstrong, Dustin D.; Delgado-Escueta, Antonio; Goldberg, Y. Paul; Grossman, Tamar R.; Guinovart, Joan J.; Harris, Frank; Hurley, Thomas D.; Michelucci, Roberto; Minassian, Berge A.; Sanz, Pascual; Worby, Carolyn A.; Serratosa, Jose M.; Biochemistry and Molecular Biology, School of MedicineLafora disease (LD) is both a fatal childhood epilepsy and a glycogen storage disease caused by recessive mutations in either the Epilepsy progressive myoclonus 2A (EPM2A) or EPM2B genes. Hallmarks of LD are aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) that are a disease driver. The 5th International Lafora Epilepsy Workshop was recently held in Alcala de Henares, Spain. The workshop brought together nearly 100 clinicians, academic and industry scientists, trainees, National Institutes of Health (NIH) representation, and friends and family members of patients with LD. The workshop covered aspects of LD ranging from defining basic scientific mechanisms to elucidating a LD therapy or cure and a recently launched LD natural history study.Item ALDH1 Bio-activates Nifuroxazide to Eradicate ALDHHigh Melanoma-Initiating Cells(Elsevier, 2018-12-20) Sarvi, Sana; Crispin, Richard; Lu, Yuting; Zeng, Lifan; Hurley, Thomas D.; Houston, Douglas R.; von Kriegsheim, Alex; Chen, Che-Hong; Mochly-Rosen, Daria; Ranzani, Marco; Mathers, Marie E.; Xu, Xiaowei; Xu, Wei; Adams, David J.; Carragher, Neil O.; Fujita, Mayumi; Schuchter, Lynn; Unciti-Broceta, Asier; Brunton, Valerie G.; Patton, E. Elizabeth; Biochemistry and Molecular Biology, School of Medicine5-Nitrofurans are antibiotic pro-drugs that have potential as cancer therapeutics. Here, we show that 5-nitrofurans can be bio-activated by aldehyde dehydrogenase (ALDH) 1A1/1A3 enzymes that are highly expressed in a subpopulation of cancer-initiating (stem) cells. We discover that the 5-nitrofuran, nifuroxazide, is selective for bio-activation by ALDH1 isoforms over ALDH2, whereby it both oxidizes ALDH1 and is converted to cytotoxic metabolites in a two-hit pro-drug mechanism. We show that ALDH1High melanoma cells are sensitive to nifuroxazide, while ALDH1A3 loss-of-function mutations confer drug resistance. In tumors, nifuroxazide targets ALDH1High melanoma subpopulations with the subsequent loss of melanoma-initiating cell potential. BRAF and MEK inhibitor therapy increases ALDH1 expression in patient melanomas, and effectively combines with nifuroxazide in melanoma cell models. The selective eradication of ALDH1High cells by nifuroxazide-ALDH1 activation goes beyond current strategies based on inhibiting ALDH1 and provides a rational basis for the nifuroxazide mechanism of action in cancer.Item Autoregulatory and structural control of CaMKII substrate specificity(2016-09) Johnson, Derrick Ethan; Hudmon, Andy; Hurley, Thomas D.; Hoang, Quyen Q.; Gallagher, PatriciaCalcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). CaMKII plays a strategic, multifunctional role in coupling the universal second messenger calcium with diverse cellular processes including metabolism, cell cycle control, and synaptic plasticity. CaMKII exhibits broad substrate specificity, targeting numerous substrates with diverse phosphorylation motifs. Binding of the calcium sensor CaM to the autoregulatory domain (ARD) of CaMKII functions to couple kinase activation with calcium signaling. Important sites of autophosphorylation, namely T287 and T306/7 (δ isoform numbering), reside within the ARD and control either CaM dependence or ability to bind to CaMKII respectively, thus determining various activation states of the kinase. Because autophosphorylation is critical to the function of CaMKII in vivo, we sought to determine the relationship between the activation state of the kinase and substrate selectivity. We show that the ARD of activated CaMKII tunes substrate selectivity by competing for substrate binding to the catalytic domain, thus functioning as a selectivity filter. Specifically, in the absence of T287 autophosphorylation, substrate phosphorylation is limited to high-affinity, consensus substrates. T287 autophosphorylation restores maximal kinase activation and broad substrate selectivity by disengaging ARD filtering. The unique multimeric architecture of CaMKII is an ideal sensor which encodes calcium-spike frequency into graded levels of subunit activation/autophosphorylation within the holoenzyme. We find that differential activation states of the holoenzyme produce distinct substrate phosphorylation profiles. Maximal holoenzyme activation/autophosphorylation leads to further broadening of substrate specificity beyond the effect of autophosphorylation alone, which is consistent with multivalent avidity. Thus, the ability of calcium-spike frequency to regulate T287 autophosphorylation and holoenzyme activation permits cellular activity to dictate switch-like behavior in substrate selectivity that is required for diverse cellular responses by CaMKII.Item Bidirectional regulation of YAP and ALDH1A1(2015-08-10) Martien, Matthew F.; Wells, Clark D.; Hurley, Thomas D.; Quilliam, Lawrence A.Breast cancer is the second leading cause of cancer death for women in the United States. Approximately, 1 in 5 women will recur with cancer within 10 years of completing treatment and recent publications have suggested that breast cancer stem cells confer resistance to therapy. These reports highlight aldehyde dehydrogenase 1A1 (ALDH1A1) and Yes-associated protein (YAP) as a biomarker and key mediator of the stem cell phenotype respectively. To further understand how YAP and ALDH1A1 facilitate chemoresistance, this study investigated how ALDH1A1 specific inhibition affected YAP activity and growth of basal-like breast cancer cells, which are enriched in cancer stem cells. Intriguingly, attenuation of growth by ALDH1A1 inhibition was observed when cells were plated on a reconstituted basement membrane. Further, the inhibition of cell growth correlated with cytosolic retention of YAP and a reduction in YAP signaling. In a complementary analysis, the overexpression of YAP correlated with an increased level of ALDH1A1 transcript. Results from this study indicate a novel mechanism by which basal-like breast cancer cells utilize YAP to maintain the stem cell phenotype and also suggest ALDH1A1 as a potential therapeutic target for breast cancer therapy.Item Brain glycogen serves as a critical glucosamine cache required for protein glycosylation(Elsevier, 2021) Sun, Ramon C.; Young, Lyndsay E.A.; Bruntz, Ronald C.; Markussen, Kia H.; Zhou, Zhengqiu; Conroy, Lindsey R.; Hawkinson, Tara R.; Clarke, Harrison A.; Stanback, Alexandra E.; Macedo, Jessica K.A.; Emanuelle, Shane; Brewer, M. Kathryn; Rondon, Alberto L.; Mestas, Annette; Sanders, William C.; Mahalingan, Krishna K.; Tang, Buyun; Chikwana, Vimbai M.; Segvich, Dyann M.; Contreras, Christopher J.; Allenger, Elizabeth J.; Brainson, Christine F.; Johnson, Lance A.; Taylor, Richard E.; Armstrong, Dustin D.; Shaffer, Robert; Waechter, Charles J.; Vander Kooi, Craig W.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.; Drake, Richard R.; Gentry, Matthew S.; Biochemistry and Molecular Biology, School of MedicineGlycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.Item Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors.(ACS, 2015-02-26) Morgan, Cynthia A.; Hurley, Thomas D.; Department of Biochemistry & Molecular Biology, IU School of MedicineAldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson?s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit submicromolar inhibition constants but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.Item Development of 2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one inhibitors of Aldehyde Dehydrogenase 1A (ALDH1A) as potential adjuncts to ovarian cancer chemotherapy(Elsevier, 2021-02) Huddle, Brandt C.; Grimley, Edward; Chtcherbinine, Mikhail; Buchman, Cameron D.; Takahashi, Cyrus; Debnath, Bikash; McGonigal, Stacy C.; Mao, Shuai; Li, Siwei; Felton, Jeremy; Pan, Shu; Wen, Bo; Sun, Duxin; Neamati, Nouri; Buckanovich, Ronald J.; Hurley, Thomas D.; Larsen, Scott D.; Biochemistry and Molecular Biology, School of MedicineThere is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.Item Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1(Elsevier, 2015-06-05) Morgan, Cynthia A.; Hurley, Thomas D.; Department of Biochemistry & Molecular Biology, IU School of MedicineThe human aldehyde dehydrogenase (ALDH) superfamily consists of at least 19 enzymes that metabolize endogenous and exogenous aldehydes. Currently, there are no commercially available inhibitors that target ALDH1A1 but have little to no effect on the structurally and functionally similar ALDH2. Here we present the first human ALDH1A1 structure, as the apo-enzyme and in complex with its cofactor NADH to a resolution of 1.75 and 2.1Å, respectfully. Structural comparisons of the cofactor binding sites in ALDH1A1 with other closely related ALDH enzymes illustrate a high degree of similarity. In order to minimize discovery of compounds that inhibit both isoenzymes by interfering with their conserved cofactor binding sites, this study reports the use of an in vitro, NAD(+)-independent, esterase-based high-throughput screen (HTS) of 64,000 compounds to discover novel, selective inhibitors of ALDH1A1. We describe 256 hits that alter the esterase activity of ALDH1A1. The effects on aldehyde oxidation of 67 compounds were further analyzed, with 30 selectively inhibiting ALDH1A1 compared to ALDH2 and ALDH3A1. One compound inhibited ALDH1A1 and ALDH2, while another inhibited ALDH1A1, ALDH2, and the more distantly related ALDH3A1. The results presented here indicate that this in vitro enzyme activity screening protocol successfully identified ALDH1A1 inhibitors with a high degree of isoenzyme selectivity. The compounds identified via this screen plus the screening methodology itself represent a starting point for the development of highly potent and selective inhibitors of ALDH1A1 that may be utilized to better understand the role of this enzyme in both normal and disease states.Item Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones(American Chemical Society, 2014-02-13) Kimble-Hill, Ann C.; Parajuli, Bibek; Chen, Che-Hong; Mochly-Rosen, Daria; Hurley, Thomas D.; Department of Biochemistry & Molecular Biology, IU School of MedicineAldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme-selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active-site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure–activity relationships for each ALDH isoenzyme.Item Development of selective inhibitors for human aldehyde dehydrogenase 3A1 (ALDH3A1) for the enhancement of cyclophosphamide cytotoxicity(Wiley, 2014-03) Parajuli, Bibek; Georgiadis, Taxiarchis M.; Fishel, Melissa L.; Hurley, Thomas D.; Biochemistry & Molecular Biology, School of MedicineAldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemoresistance, by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues, such as mafosfamide (MF), ifosfamide (IFM), and 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 could permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1-selective inhibitor, CB29, previously identified in a high-throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as ALDH3A1 non-expressing lung fibroblast (CCD-13Lu) cells, is unaffected by treatment with CB29 and its analogues alone. However, sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumor cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small-molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which might be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines.