Chemistry Commons^™

Glutathione Species And Metabolomic Prints In Subjects With Liver Disease As Biological Markers For The Detection Of Hepatocellular Carcinoma, Juan R. Sanabria, Rajan S. Kombu, Guo Fang Zhang, Yana Sandlers, Jizhou Ai, Rafael A. Ibarra, Rime Abbas, Kush Goyal, Henri Brunengraber

Chemistry Faculty Publications

© 2016 The Authors Background The incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC). Methods The present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. …

Oncogenic Pik3ca Mutations Reprogram Glutamine Metabolism In Colorectal Cancer, Yujun Hao, Yardena Samuels, Qingling Li, Dawid Krokowski, Bo-Jhih Guan, Chao Wang, Zhicheng Jin, Bohan Dong, Bo Cao, Xiujing Feng, Min Xiang, Claire Xu, Stephen Fink, Neal J. Meropol, Yan Xu

Chemistry Faculty Publications

Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to alpha-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110 alpha upregulates GPT2 gene expression through an AKT-independent, PDK1-RSK2-ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not …

Novel Protein Disulfide Isomerase Inhibitor With Anticancer Activity In Multiple Myeloma, Sergei Vatolin, James G. Phillips, Babal K. Jha, Shravya Govindgari, Jennifer Hu, Dale Grabowski, Yvonne Parker, Daniel J. Lindner, Fei Zhong, Clark W. Distelhorst, Mitchell R. Smith, Claudiu Cotta, Yan Xu, Sujatha Chilakala, Rebecca R. Kuang, Samantha Tall, Frederic J. Reu

Chemistry Faculty Publications

Multiple myeloma cells secrete more disulfide bond–rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell–based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow–sparing compound, exhibited a submicromolar IC₅₀ in 10 of 10 multiple myeloma cell lines. An active biotinylated analog …

Early Growth And Development Impairments In Patients With Ganglioside Gm3 Synthase Deficiency, H. Wang, A. Wang, Dan Wang, A. Bright, V. Sency, Aimin Zhou, B. Xin

Chemistry Faculty Publications

Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GSD) causes a complete absence of GM3 and all downstream biosynthetic derivatives. The individuals affected by this disorder manifest severe irritability, intractable seizures and profound intellectual disability. However, we have found that most newborns seem symptom-free for a period of time after birth. In order to further understand the onset of the disease, we investigated the early growth and development of patients with this condition through this study. We compared 37 affected individuals with their normal siblings and revealed that all children with GSD …

Metabolomics Reveals New Mechanisms For Pathogenesis In Barth Syndrome And Introduces Novel Roles For Cardiolipin In Cellular Function, Yana Sandlers, Kelly Mercier, Wimal Pathmasiri, Jim Carlson, Susan Mcritchie, Susan Sumner, Hilary J. Vernon

Chemistry Faculty Publications

Barth Syndrome is the only known Mendelian disorder of cardiolipin remodeling, with characteristic clinical features of cardiomyopathy, skeletal myopathy, and neutropenia. While the primary biochemical defects of reduced mature cardiolipin and increased monolysocardiolipin are well-described, much of the downstream biochemical dysregulation has not been uncovered, and biomarkers are limited. In order to further expand upon the knowledge of the biochemical abnormalities in Barth Syndrome, we analyzed metabolite profiles in plasma from a cohort of individuals with Barth Syndrome compared to age-matched controls via ¹H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry. A clear distinction between metabolite profiles of …

A Systematic Investigation Of Structure/Function Requirements For The Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop Of High-Density Lipoprotein, Xiaodong Gu, Zhiping Wu, Ying Huang, Matthew A. Wagner, Camelia Baleanu Gogonea, Ryan A. Mehl, Jennifer A. Buffa, Anthony J. Didonato, Leah B. Hazen, Paul L. Fox, Valentin Gogonea, John S. Parks, Joseph A. Didonato, Stanley L. Hazen

Chemistry Faculty Publications

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu159–Leu170) in nascent HDL, the so-called “solar flare” (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861–868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we …

The Dual Regulatory Role Of Amino Acids Leu480 And Gln481 Of Prothrombin, Joesph R. Wiencek, Jamila Hirbawi, Vivien C. Yee, Michael Kalafatis

Chemistry Faculty Publications

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser⁴⁷⁸–Val^{479 …}

Identification Of Critical Paraoxonase 1 Residues Involved In High Density Lipoprotein Interaction, Xiaodong Gu, Ying Huang, Bruce S. Levison, Gary Gerstenecker, Anthony J. Didonato, Leah B. Hazen, Joonsue Lee, Valentin Gogonea, Joseph A. Didonato, Stanley L. Hazen

Chemistry Faculty Publications

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815–3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to …

Orally Active And Selective Tubulin Inhibitors As Anti-Trypanosome Agents, Vishal Nanavaty, Rati Lama, Ranjodh Sandhu, Bo Zhong, Daniel Kulman, Viharika Bobba, Anran Zhao, Bibo Li, Bin Su

Chemistry Faculty Publications

Objectives: There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis. Methods: We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host …

Structural Insights Into High Density Lipoprotein: Old Models And New Facts, Valentin Gogonea

Chemistry Faculty Publications

The physiological link between circulating high density lipoprotein (HDL) levels and cardiovascular disease is well-documented, albeit its intricacies are not well-understood. An improved appreciation of HDL function and overall role in vascular health and disease requires at its foundation a better understanding of the lipoprotein's molecular structure, its formation, and its process of maturation through interactions with various plasma enzymes and cell receptors that intervene along the pathway of reverse cholesterol transport. This review focuses on summarizing recent developments in the field of lipid free apoA-I and HDL structure, with emphasis on new insights revealed by newly published nascent and …

Modified Electrodes With Grafted Dna And Oligonucleotides For Detection And Quantification Of Peroxynitrate, Heba Azmy Salim

ETD Archive

Peroxynitrite (ONOO-) is a strong oxidizing and nitrating agent, and its formation has been correlated with many pathological conditions. It is generated in-vivo through the diffusion-controlled reaction between nitric oxide and superoxide. Peroxynitrite has been linked to nitration of protein and DNA as well as to DNA strand breaks. Accumulation of mutations and/or other kinds of DNA damage represent a carcinogenic risk. The accurate measurement of peroxynitrite concentration has been a challenge since this analyte is very unstable and reacts with many cellular targets. Development of analytical techniques capable of rapid and sensitive detection of this fast-reacting and damaging agent …

Profiling Cell Surface Sialylation And Desialylation Dynamics Of Immune Cells, Dan Wang

ETD Archive

Sialic acids (SAs) are a diverse family of naturally occurring 2-keto-3-deoxy-nononic acids that are involved in a wide range of biological processes, including early fetal development, cellular recognition, and utilization by microbes. While it is clear that cell surface SAs are highly involved in the immune system, the sialylation status of individual immune cells and functions are still unknown. In this study, I combined the newly developed LC-MS/MS methods with flow cytometry and confocal microscopy to systematically study the sialylation and desialylation dynamics of macrophages at different conditions.

First, I developed an accurate LC-MS/MS method to quantify free SA in …

Development And Validation Of Uplc/Ms/Ms Methods For Quantification Of Gangliosides In The Clinical Study Of Ganglioside Gm3 Synthase Deficiency, Qianyang Huang

ETD Archive

Gangliosides are a large subclass of glycosphingolipids that structurally characterized by the addition of mono- or poly-sialyated carbohydrate moieties onto the ceramide scaffold. They are presented at significant abundance in the central nervous system of vertebrates. Their interactions with trans-membrane receptors, especially receptor tyrosine kinase, are believed to play critical roles in modulating signal transduction events during cell proliferation, differentiation, migration, and adhesion. Furthermore, the disruption and dysfunction on their metabolism have been found to be associated with various neurodegenerative disorders, such as Alzheimer disease, Parkinson’s disease, and GM3 synthase deficiency. GM3 synthase deficiency (GSD) is a newly identified neurological …

Study Of Electron Transfer Through The Reductase Domain Of Neuronal Nitric Oxide Synthase And Development Of Bacterial Nitric Oxide Synthase Inhibitors, Yue Dai

ETD Archive

Crystal structure of neuronal Nitric Oxide Synthase reductase (nNOSr) implies that large-scale domain motion is essential for electron transfer. However, the details are not well understood. To address this, we generated a functioning “Cys-lite” version of nNOSr and then replaced the nNOSr Glu816 and Arg1229 residues with Cys in the FMN and FAD domains (CL5SS) in order to allow cross-domain disulfide bond formation under pH 9 or to cross-linking using bis-maleimides. Cross-linked CL5SS exhibited a =95% decrease in cytochrome c reductase activity and reduction of the disulfide bond restored the activities. The results demonstrate that a conformational equilibrium involving FMN …

The Role Of Rnase L In Type I Diabetes And Development Of Quantitative Lc-Ms/Ms Methods For The Pharmacological Studies Of Anti-Cancer Drug Candidates, Chun Zeng

ETD Archive

PROJECT I: The cause of type I diabetes continues to be a focus of investigation. In this study, we found that 2-5A dependent RNase L (RNase L), an IFN-a-inducible enzyme that functions in IFN action against viruses and cell proliferation, played an important role in dsRNA-induced onset of type I diabetes. By using RNase L deficient RIP-B7.1 mice which are more vulnerable to environmental harmful factors such as viral infection, we demonstrated that deficiency of RNase L in mice resulted in a significant delay of diabetes onset induced by polyinosinic:polycytidylic acid (poly I:C), a type of dsRNA, and streptozotocin (STZ). …

Application Of Advanced Analytical Technologies To Drug Development Studies And Cancer Detection, Ramakrishna Reddy Voggu

ETD Archive

In the past decade, bioanalytical method development has become an integral part of the clinical diagnosis, biomarker discovery, and drug discovery and development. The new and emerged bioanalytical techniques allow the quantitative and qualitative analysis of bio molecules with remarkably high sensitivity and specificity. Specifically, these bioanalytical methods based on LC-MS and methylation-specific PCR are well suited for detecting low-abundance metabolites in various biological fluids and DNA in plasma and tissues for biomarker investigation. They offer great clinical promise for early disease diagnosis and therapeutic intervention. This dissertation broadly organized into two parts, part one talks about the application of …

Tmco1 Is An Er Ca2+ Load-Activated Ca2+ Channel, Qiao-Chu Wang, Qiaoxia Zheng, Haiyan Tan, Bing Zhang, Xiaoling Li, Yuxiu Yang, Jie Yu, Yang Liu, Hao Chai, Xi Wang, Zhongshuai Sun, Jiu-Qiang Wang, Shu Zhu, Fengli Wang, Maojun Yang, Caixia Guo, Heng Wang, Qingyin Zheng, Yang Li, Quan Chen, Aimin Zhou, Tie-Shan Tang

Chemistry Faculty Publications

Maintaining homeostasis of Ca2+stores in theendoplasmic reticulum (ER) is crucial for properCa2+signaling and key cellular functions. The Ca2+-release-activated Ca2+(CRAC) channel is respon-sible for Ca2+influx and refilling after store depletion,but how cells cope with excess Ca2+when ER storesare overloaded is unclear. We show that TMCO1 is anER transmembrane protein that actively preventsCa2+stores from overfilling, acting as what we terma ‘‘Ca2+load-activated Ca2+channel’’ or ‘‘CLAC’’channel. TMCO1 undergoes reversible homotetra-merization in response to ER Ca2+overloadingand disassembly upon Ca2+depletion and forms aCa2+-selective ion channel on giant liposomes.TMCO1 knockout mice reproduce the main clinicalfeatures of human cerebrofaciothoracic (CFT)dysplasia spectrum, a developmental disorder linkedto TMCO1 dysfunction, and …

Determination Of Mln0128, An Investigational Antineoplastic Agent, In Human Plasma By Lc–Ms/Ms, Sandeep R. Kunati, Yan Xu

Chemistry Faculty Publications

MLN0128, an mTOR kinase inhibitor, is currently undergoing clinical investigation for treatment of a variety of cancers. To support this work, an LC–MS/MS method has been developed for the determination of MLN0128 in human plasma. A structural analog STK040263 was used as the internal standard. Both MLN0128 and the IS were first extracted from plasma using methyl tert-butyl ether; then separated on a Waters XTerra® MS C18 column using a mobile phase consisting of methanol–acetonitrile–10.0 mm ammonium formate (34:6:60, v/v/v) at a flow rate of 0.300 mL min−1. Quantitation of MLN0128 was done by positive electrospray ionization tandem mass spectrometry …

Targeting Radioresistant Breast Cancer Cells By Single Agent Chk1 Inhibitor Via Enhancing Replication Stress, Yao Zhang, Jinzhi Lai, Zhanwen Du, Jinnan Gao, Shuming Yang, Shashank Gorityala, Xiahui Xiong, Ou Deng, Zhefu Ma, Chunhong Yan, Gonzalo Susana, Yan Xu, Junran Zhang

Chemistry Faculty Publications

Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher …

Sialyltransferase Inhibition And Recent Advances, Libo Wang, Ying Liu, Lijun Wu, Xue-Long Sun

Chemistry Faculty Publications

Sialic acids, existing as terminal sugars of glycoconjugates, play important roles in various physiological and pathological processes, such as cell–cell adhesion, immune defense, tumor cell metastasis, and inflammation. Sialyltransferases (STs) catalyze the transfer of sialic acid residues to non-reducing oligosaccharide chains of proteins and lipids, using cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) as the donor. Elevated sialyltransferase activity leads to overexpression of cell surface sialic acids and contributes to many disease developments, such as cancer and inflammation. Therefore, sialyltransferases are considered as potential drug targets for disease treatment. Inhibitors of sialyltransferases thus are of medicinal interest, especially for the cancer …