bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Organophosphate (OP)‐Induced Structural Changes in Acetylcholinesterase on Kinetics of OP Inhibition and Oxime Reactivation
Authors: Yu Wanlu , Kovalevsky Andrey , Blumenthal Donald K. , Cheng Xiaolin , Gerlits Oksana , Fajer Mikolai , Ho Kwok‐Yiu , Taylor Palmer , Radic Zoran 
Published: June 2021

Organophosphate (OP) pesticides and nerve warfare agents covalently inhibit human acetylcholinesterase (hAChE) compromising cholinergic neurotransmission that can lead to fatal outcome. Effective therapy of OP intoxication requires that both, excess OP is removed from circulation and that activity of OP inhibited tissue hAChE is recovered. For both reactions nucleophilic oxime reactivation can play an essential role. For oxime assisted OP bioscavenging it is also important that the rate of inhibition of bioscavenger AChE is not compromised. Recently determined structures of diethylphosphorylated hAChE (POX‐hAChE) formed by paraoxon (POX) inhibition, both our unpublished and PDB released (5HF5 and 5HF8) show significant structural changes of the acyl pocket, immediately close to the active Ser203 when compared to the non‐inhibited enzyme. Those are likely to compromise nucleophilic oxime attack and rate of reactivation, but could also influence rate of hAChE inhibition by POX. Inhibition of AChE by dimethylphosphate analogue of POX (dmPOX) does not lead to conformational changes in the conjugated AChE (dmPOX‐AChE). In order to investigate effect of conformational changes in POX‐hAChE on rates of POX inhibition and oxime reactivation we studied dependence of rates of those reactions on reaction temperatures compared to dmPOX reactions. No difference was observed in temperature dependences of inhibition by diethyl‐ and dimethylphosphates. However, we observed strong temperature dependence of the oxime 2PAM reactivation, where reactivation of POX‐hAChE at 37 °C appeared an order of magnitude faster than the reaction at 22 °C. Reactivation of dimethylphosphorylated hAChE (dmPOX‐hAChE), as well as both dimetyhyl‐ and diethyl‐ phosphorylated Y337A/F338A hAChE mutant by 2PAM was significantly less temperature dependent, consistent with the absence of compromising conformational changes in dmPOX‐hAChE and in the mutant. This result indicates that formation of distorted acyl pocket loop in POX‐hAChE is not limiting for inhibition reaction but affects rates of oxime reactivation. Ability of POX‐hAChE to change acyl pocket loop conformation at the higher temperature allows for faster oxime reactivation and emphasizes the importance of conformational flexibility of hAChE for the efficient oxime reactivation.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The JAVA Based Computational Tool for Pairwise Comparison of Protein Backbone Folds in Liganded and Apo 3D Structures of the alpha/beta Hydrolase Fold Proteins
Authors: Zheng Zihan , Rohrer Jacqueline , RADIC ZORAN 
Published: June 2021

Cholinesterases, carboxylesterases and lipases are some of prominent catalytically active proteins with specifically similar, globular tertiary structure fold termed “alpha/beta hydrolase fold”. Ligand binding, both reversible and covalent, frequently results in small, yet systematic and potentially functionally important changes in the conformation of their alpha carbon backbones that can be easily overseen by commonly used overlays based on overall minimization of backbone atom RMSD analysis. In particular, backbone conformations of cholinesterases ‐ acetylcholinesterase and butyrylcholinesterase ‐ show only small conformational changes. This is revealed in more than 200 PDB deposited cholinesterase X‐ray structures in complexes with structurally diverse ligands that affect their function.We developed a novel, reference point based principle for overlay‐independent pairwise comparison of liganded and non‐liganded alpha carbon backbone conformations from respective PDB (Protein Data Bank) deposited 3D structure entries and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each alpha carbon and chosen reference point in each pair of compared structures, as well as on differences in the angle between center of mass, reference point and each of alpha carbons in the comparison. Combination of the two comparison criteria reveals subset of backbone alpha carbons in two structures that maintains best their relative positions in the 3D space and that can be used as tethering points for overlay of compared structures. Created overlay has capacity to reveal small but systematic local changes in the backbone conformation frequently masked by the overall RMSD minimization approach.Our systematic analysis of a number of PDB deposited alpha/beta hydrolase fold structures revealed small, yet functionally important conformational changes in their backbone consistent with experimental observations and instrumental in revealing molecular mechanisms in respective catalytic reactions. These findings are important for complete molecular target template characterization in the structure based development of novel antidotes of exposure to organophosphates which covalently inhibit this family of enzymes.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant R21 NS098998 from NINDS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the molecular basis of a phenylketonuria‐causing mutation in phenylalanine hydroxylase
Authors: Khan Crystal A. , Meisburger Steve P. , Ando Nozomi , Fitzpatrick Paul F. 
Published: June 2021

Phenylketonuria (PKU) is a metabolic disorder in which the conversion of excess dietary phenylalanine to tyrosine is disrupted. Phenylalanine hydroxylase (PheH) is responsible for this hydroxylation reaction and uses tetrahydrobiopterin (BH4) and molecular oxygen as cosubstrates. The prevalence of PKU in the U.S. is 1 in 10,000 newborns and was the leading cause of mental retardation prior to the discovery of treatment by a protein‐restricted diet. R68S PheH has been shown to cause a mild form of PKU but the mechanism of action has been controversial. Studies have reported that mutations to Arg68 cause the enzyme to become fully active and lose sensitivity to phenylalanine as an allosteric activator. Arg68 is located in the regulatory domain of PheH, which is responsible for the activation of the enzyme. The current model for activation states that the regulatory domains of two subunits on the tetrameric PheH exist in a monomer‐dimer equilibrium and the dimer is stabilized by binding of phenylalanine. We characterized R68S PheH and found that the R68S PheH is fully active compared to the wild‐type enzyme. Sedimentation velocity experiments showed the isolated regulatory domain of R68S PheH does experience a concentration‐dependent shift from a monomer to a dimer; however, phenylalanine had no effect on this shift. The mutant protein displayed a fluorescence emission spectrum different from that of the wild‐type protein that is unchanged in the presence of phenylalanine. SAXS experiments showed no transitions when the protein was treated with 1 mM phenylalanine. Limited proteolysis experiments showed R68S PheH was more proteolytically sensitive than wild‐type PheH. The data suggest that R68S PheH exhibits constitutively active behavior due to the destabilization of the regulatory domain, which causes the protein to be more proteolytically sensitive.Support or Funding InformationNIH F31GM116452This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introducing allosteric regulation into homing endonucleases via tryptophan modification
Authors: Danon Sonja , Kaiser Brett K. 
Published: June 2021

Homing endonucleases (HEGs) are DNA cleaving enzymes present in microbial genomes that recognize unusually long DNA target sites (20–22 bp). Given their high specificity for DNA targets, these enzymes may be extremely useful genome editing reagents. Introducing allosteric control into these enzymes would enable them to have a broader applicability in genome editing approaches. Previous studies have shown that tryptophan residues near the active site of enzymes can play an important role in enzyme activity. Mutating these residues to glycine results in loss of enzymatic activity and can be rescued by the addition of indole in solution. However, this approach hasn't been demonstrated with DNA cleaving enzymes. The goal of this study is to test whether this approach can be used to introduce allosteric regulation into HEGs. We generated tryptophan to glycine single point mutations in I‐SmaMI, a homing endonuclease of the LAGLIDADG family, based on the structure of I‐SmaMI bound to DNA. We are expressing these proteins in bacteria and utilizing an in vitro cleavage assay to assess the level of activity of the mutants in the presence and absence of indole. These studies may have important genome editing applications and are likely to provide insight into the mechanism by which HEGs bind and cleave DNA.Support or Funding InformationMurdock Charitable TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A High‐Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase
Authors: Li Shunan , Cheung Keith , Tchaga Grigoriy , Yuan Gordon , Xu Jianping 
Published: June 2021

Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity.The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample.In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of Cdc42 Inhibition as the Mechanism of Action of the Anti‐metastatic Drug MBQ‐167, Using Budding Yeast as a Model System
Authors: Rivera‐Robles Michael John , Medina‐Velázquez Julia , Asencio‐Torres Gabriela , González‐Crespo Sahily , Rymond Brian C. , Rodríguez‐Medina José R. , Dharmawardhane Suranganie 
Published: June 2021

Cdc42 (Cell division control protein 42 homolog), a Rho GTPase, is highly conserved among eukaryotes in its structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and regulate cell polarity and motility. Its role in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae, but was subsequently shown to also establish directional motility in animal cells. Accordingly, in cancer, Cdc42 promotes migration, invasion, and spread of tumor cells. Thus, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer, and designed and characterized the small molecule MBQ‐167 as a potent inhibitor against Cdc42 and the homolog Rac. We recently reported that MBQ‐167 inhibits cancer cell and tumor growth and spread in‐vitro and in‐vivo using a mouse xenograft model of metastatic breast cancer. Since budding yeast express a single Cdc42 gene and do not express Rac, we used this well characterized model of polarization to validate the MBQ‐167 specificity for Cdc42 inhibition. Using a tetracycline regulated transcriptional inactivation (Tet‐Off) system in the promoter region of the yeast CDC42 gene, we knocked‐down Cdc42 supplementing the media with doxycycline (DOX; IC=10μg/μL). Next, we analyzed the growth, budding pattern, and Cdc42 activity of yeast exposed to DOX, MBQ‐167, or in combination. As expected, maximum growth, doubling time and budding polarity of yeast with the Tet‐Off system were reduced when treated with DOX (EC50=10μg/mL). The same effect was seen with MBQ‐167 (ED50=100μM) alone and in combination with DOX. Ultimately, pull‐downs of the active Cdc42‐GTP complex confirmed our hypothesis that MBQ‐167 disrupts cell polarity through impeding Cdc42 activation. Hence, we have validated MBQ‐167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors as potential therapeutics against cancer metastasis.Support or Funding InformationThis work is partially supported by grants from the Susan G Komen for the Cure and the Puerto Rico Science and Technology Research Trust (S.D.), NIH/NIGMS P20GM103475 (JRRM), and NIH/NIGMS‐RISE R25 GM061838 (MJR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A kinetic assay for non‐automated drug screening
Authors: Zem Gregory C. , Chimayan A , Aleksanyan V , Gordon J , Gomez F , Seyedroudbari A , Chang J , Botello T , Tan N , Arefin D , Tobar D , Khachekian A , Gama L , Durodola E , Batty J , Plascencia C , Barillas L , Roverud A , Kreuz S , Sarkisyan L , Lee F , Munoz J , Reque L , Abed V , Kinog L , Oppenheimer SB 
Published: June 2021

A non‐automated kinetic assay has been used in this laboratory to screen reagents that unclump fixed yeast, a model for unclumping human cancer cells, human blood cells and pathogens (FASEB J, 26:62ab; 28:234ab; 29:65ab; 31:539ab). Fixed yeast is the material of choice because its surface properties are similar to live cells without unknowns associated with live cell metabolism (Acta Histochem 104: 99–106). Here the assay is tested to determine if it also can be used to assess cell clumping instead of unclumping, of importance in many drug screening venues, using a calcium induced flocculation model. In addition, the precision of the assay is tested using reagents that do not clump or unclump cells. O.05 ml of Prefer (Anatech Ltd, Battle Creek, MI) fixed yeast (Saccharomyces cerevisae) were added to 1 ml droplets of deionized water on glass microscope slides. The percentages of single yeast and clumps (2 or more cells) were assessed with light microscopes (100X, 200X) before and after addition of 1, 2 or 3 mg of calcium sulfate dihydrate (droplets with no additions served as controls) and at 20 min, 40 min and 60 min with agitation. The results showed that calcium sulfate flocculated the yeast in a concentration and time dependent manner. For example for calcium sulfate, 1, 2 and 3 mg per ml, in 30 trials, with p values generally below 0.05 and tight standard error bars, the percentages of single yeast at 60 min were: 1 mg/ml 40 percent vs 67 percent in controls, 2 mg/ml 27% vs 67 percent in controls, 3 mg/ml 14 percent vs 72 percent in controls. The assay, therefore, assesses the kinetics of cell clumping (flocculation). When 3 reagents, D‐melizitose, D‐melibiose, L‐ascorbic acid were tested at 1, 2 and 3 mg per ml at 20, 40 and 60 min, the assay picked up little or no effect on the yeast. For example in 90 trials with tight error bars each time point showed little to no significant difference between experimentals and controls. The results indicate that the assay kinetically measures cell clumping and is reproducible and precise in generating data. The non‐automated assay is ideal for drug screening by hundreds of investigators at a time especially when expense is a consideration.Support or Funding InformationCSUN Foundation and Corporation Accounts, U.S. Presidential AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Studies of Drug‐Like Inhibitors of P‐glycoprotein using ATPase Assays, Electron Spin Resonance Spectroscopy and Cancer Cell Models
Authors: Chen Gang , Ballou‐Crawford Jett , Aljowni Maha , Lippert Alexander , Wise John G. , Vogel Pia D. 
Published: June 2021

Multidrug resistance (MDR) in cancer and other diseases is frequently associated with transmembrane efflux proteins, one of which is P‐glycoprotein (P‐gp). Over‐expression of P‐gp in cancer cells increases the efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al., Molecular pharmacology 86, 716–726, 2014), and several of these inhibitors were shown to successfully overcome MDR in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). In this study, we expanded our cell culture screening to evaluate 25 new compounds identified by our in silico screening method and report that over 30% of these compounds re‐sensitized DU145TXR cancer cells to chemotherapy, while not exhibiting significant toxicity to the non‐cancerous human lung fibroblast (HFL‐1) cell line. The best of the identified hit compounds were further investigated with regard to the mechanism of inhibition using ATPase activity assays and electron spin resonance spectroscopy (ESR). Several of the best compounds were found to inhibit ATPase activity. Using a spin‐labeled ATP analog and ESR spectroscopy, we assessed the effects of the identified inhibitors on ATP binding to P‐gp. Variants of one of the original hit compounds, SMU 29, that were computationally predicted to interact more strongly with P‐gp have been synthesized and were analyzed here for their effects on P‐gp ATPase activity and ATP binding characteristics.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring immune cells‐mediated cytolysis through impedance technology allows kinetic analysis of reagent efficacy
Authors: Cerignoli Fabio , Xi Biao , Guenther Garret , Muir Lincoln , Lamarche Brandon , Abassi Yama 
Published: June 2021

In vitro characterization of immunotherapy reagents efficacy and combination synergies is necessary before moving to expensive animal models and clinical studies. Current standard assays like Chromium‐51 release, ATP‐based luminescence cell monitoring or flow cytometry are difficult to implement in a high throughput environment and are based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we validate an impedance‐based platform for monitoring cytotoxic activity of immune cells in the context of cancer immunotherapy assays. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384‐wells cell culture plates, without the use of labeling or cell modification. We present a large set of validation including CD19 Car T models, EpCAM/CD3 BiTE antibodies and checkpoint combination therapies with anti PD‐1/PD‐L1 and CTLA‐4 antibodies. Data comparison with Annexin V staining/Flow Cytometry shows perfect correlation between the drop in impedance signal and % of apoptotic cells. Long time monitoring (> 7 days) in combination with low effector:target ratios reveal the occurrence of resistance mechanisms in target cells and exhaustion in effector cells. Such dynamic response has been observed in animal models and patient clinical trials, but it is difficult to monitor in short time assays. Overall, our results demonstrate the value of such label‐free approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays but that is highly relevant when testing combinations. Thanks to the availability of 384‐wells format and minimal sample handling, the technology is ideally suited for applications in large reagent validation screening or therapeutic protocol validation directly on patient samples.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of novel glucose‐6‐phosphate dehydrogenase activators to correct G6PD deficiency
Authors: HWANG SUNHEE 
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. As no medications are available to treat G6PD deficiency directly, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis of human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.