Anthony W Opipari

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Name: Glick, Gary

Organization: University of Michigan , USA

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Polymer

Chemicals
References

Manipulating the Bioenergetics of Alloreactive T Cells Causes Their Selective Apoptosis and Arrests Graft-Versus-Host Disease

Co-reporter:Erin Gatza;Anthony W. Opipari;Daniel R. Wahl;Thomas B. Sundberg;Pavan Reddy;Chen Liu;Gary D. Glick;James L. M. Ferrara

Science Translational Medicine 2011 Volume 3(Issue 67) pp:67ra8

Publication Date(Web):26 Jan 2011

DOI:10.1126/scitranslmed.3001975

Bioenergetic properties differentiate alloreactive T cells from other proliferating cells and can be exploited to arrest GVHD in mice.

NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F1F0-ATPase

Co-reporter:Andrew C. Stelzer;Richard W. Frazee;Chad Van Huis;Joanne Cleary;Anthony W. Opipari Jr.;Gary D. Glick;Hashim M. Al-Hashimi

Biopolymers 2010 Volume 93( Issue 1) pp:85-92

Publication Date(Web):

DOI:10.1002/bip.21306

Abstract

Bz-423 is an inhibitor of the mitochondrial F₁F₀-ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water-soluble Bz-423 analog (5-(3-(aminomethyl)phenyl)-7-chloro- 1-methyl-3-(naphthalen-2-ylmethyl)-1H-benzo][e][1,4]diazepin-2(3H)-one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross-relaxation experiments. Titration experiments with constructs representing residues 1–120 or 1–145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP-F₁ interface resulting in disrupted communication between the peripheral stalk and the F₁-domain of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 29: 85–92, 2010.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Mechanistic basis for differential inhibition of the F1Fo-ATPase by aurovertin

Co-reporter:Kathryn M. Johnson;Lara Swenson;Anthony W. Opipari Jr.;Rolf Reuter;Nawid Zarrabi;Carol A. Fierke;Michael Börsch;Gary D. Glick

Biopolymers 2009 Volume 91( Issue 10) pp:830-840

Publication Date(Web):

DOI:10.1002/bip.21262

Abstract

The mitochondrial F₁F_o-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F₁F_o-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F₁ domain and inhibits F₁F_o-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F₁F_o-ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K_i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F₁F_o-ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F₁F_o-ATPase catalyzes ATP synthesis and hydrolysis. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 830–840, 2009.

Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor

Co-reporter:Joanne Cleary, Kathryn M. Johnson, Anthony W. Opipari Jr., Gary D. Glick

Bioorganic & Medicinal Chemistry Letters 2007 Volume 17(Issue 6) pp:1667-1670

Publication Date(Web):15 March 2007

DOI:10.1016/j.bmcl.2006.12.102

Although PK11195 binds to the peripheral benzodiazepine receptor with nanomolar affinity, significant data exist which suggest that it has another cellular target distinct from the PBR. Here we demonstrate that PK11195 inhibits F1F0-ATPase activity in an OSCP-dependent manner, similar to the pro-apoptotic benzodiazepine Bz-423. Importantly, our data indicate that cellular responses observed with micromolar concentrations of PK11195, which are commonly attributed to modulation of the PBR, are likely a direct result of mitochondrial F1F0-ATPase inhibition.PBR-ligand PK-11195 inhibits F1F0-ATPase activity in an OSCP-depender manner, similar to the pro-apoptotic benzodiazepine Bz-423.

Effect of somatic mutation on DNA binding properties of anti-DNA autoantibodies

Co-reporter:Melissa J. Bobeck;Joanne Cleary;Jenny A. Beckingham;P. Christine Ackroyd;Gary D. Glick

Biopolymers 2007 Volume 85(Issue 5-6) pp:

Publication Date(Web):24 JAN 2007

DOI:10.1002/bip.20691

Autoantibodies that bind DNA are a hallmark of systemic lupus erythematosus. A subset of autoantibody•DNA complexes localize to kidney tissue and lead to damage and even death. 11F8, 9F11, and 15B10 are clonally related anti-DNA autoantibodies isolated from an autoimmune mouse. 11F8 binds ssDNA in a sequence-specific manner and causes tissue damage, while 9F11 and 15B10 bind ssDNA non-specifically and are benign. Among these antibodies, DNA binding properties are mediated by five amino acid differences in primary sequence. Thermodynamic and kinetic parameters associated with recognition of structurally different DNA sequences were determined for each antibody to provide insight toward recognition strategies, and to explore a link between binding properties and disease pathogenesis. A model of 11F8 bound to its high affinity consensus sequence provides a foundation for understanding the differences in thermodynamic and kinetic parameters between the three mAbs. Our data suggest that 11F8 utilizes the proposed ssDNA recognition motif including ^Y32V_L, a hydrogen bonding residue at ⁹¹V_L, and an aromatic residue at the tip of the third heavy chain complementarity determining region. Interestingly, a somatic mutation to arginine at ³¹V_H in 11F8 may afford additional binding site contacts including ^R31V_H, ^R96V_H, and ^R98V_H that could determine specificity. © 2007 Wiley Periodicals, Inc. Biopolymers 85: 471–480, 2007.

Role of conformational dynamics in sequence-specific autoantibody•ssDNA recognition

Co-reporter:Melissa J. Bobeck;Gary D. Glick

Biopolymers 2007 Volume 85(Issue 5-6) pp:

Publication Date(Web):24 JAN 2007

DOI:10.1002/bip.20692

11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at ³¹V_H to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that ^R31V_H contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8•ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8•ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at ³¹V_H. © 2007 Wiley Periodicals, Inc. Biopolymers 85: 481–489, 2007.

Identification of cytotoxic, T-cell-selective 1,4-benzodiazepine-2,5-diones

Co-reporter:Tasha M. Francis, Thomas B. Sundberg, Joanne Cleary, Todd Groendyke, Anthony W. Opipari Jr., Gary D. Glick

Bioorganic & Medicinal Chemistry Letters 2006 Volume 16(Issue 9) pp:2423-2427

Publication Date(Web):1 May 2006

DOI:10.1016/j.bmcl.2006.01.113

A family of 1,4-benzodiazepine-2,5-diones (BZDs) has been synthesized and evaluated against transformed B- and T-cells for lymphotoxic members. A large aromatic group on the C3 position is critical for cytotoxicity. When the C3 moiety contains an electron-rich heterocycle, the resulting BZDs have sub-micromolar potency and are selective for T-cells. Cell death is consistent with apoptosis and does not result from inhibition of the mitochondrial FoF1-ATPase, which is the molecular target of recently reported cytotoxic 1,4-benzodiazepines. Collectively, these studies begin to characterize some of the structural elements required for the activity of a novel family of T-cell-selective lymphotoxic agents.A 1,4-benzodiazepine-2,5-dione (BZD) library was evaluated for lymphotoxic members. When the C3 substituent contains an electron-rich heterocycle, the resulting BZDs have sub-micromolar potency and are selective for T-cells.

Structure activity studies of a novel cytotoxic benzodiazepine

Co-reporter:Anthony Boitano, Cory D Emal, Francesco Leonetti, Neal B Blatt, Thomas A Dineen, Jonathan A Ellman, William R Roush, Anthony W Opipari, Gary D Glick

Bioorganic & Medicinal Chemistry Letters 2003 Volume 13(Issue 19) pp:3327-3330

Publication Date(Web):16 October 2003

DOI:10.1016/S0960-894X(03)00683-8

Analogues of Bz-423, a pro-apoptotic 1,4-benzodiazepine with potent activity in animal models of systemic lupus erythematosus and rheumatoid arthritis, have been designed, synthesized, and evaluated in cell-culture assays. The results of these experiments have defined the structural elements of this new cytotoxic agent required for activity.Analogues of Bz-423, a pro-apoptotic 1,4-benzodiazepine active in animal models of lupus and rheumatoid arthritis, have been designed, synthesized, and evaluated in cell culture assays. These experiments have defined the structural elements of this new cytotoxic agent required for activity.

Next-generation therapeutics

Co-reporter:Gary D. Glick, David L. Pompliano

Current Opinion in Chemical Biology 2002 Volume 6(Issue 4) pp:415-417

Publication Date(Web):1 August 2002

DOI:10.1016/S1367-5931(02)00354-X

Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding

Co-reporter:Jennifer A. Beckingham, Gary D. Glick

Bioorganic & Medicinal Chemistry 2001 Volume 9(Issue 9) pp:2243-2252

Publication Date(Web):September 2001

DOI:10.1016/S0968-0896(01)00076-1

11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k1 are fast and above the modified Smoluchowski limit for a diffusion limited interaction (105–106 M−1 s−1). The dependency of k1 on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.11F8 is an anti-ssDNA autoantibody involved in the pathogenesis of murine lupus. Since DNA binding is a key first step in the tissue damage caused by 11F8, the mechanism by which it binds DNA was investigated. Recognition of ssDNA occurs via an initial encounter complex that is followed by a second slower step, during which the binding interfaces are optimised. This second step is critical in determining the specificity if 11F8.

Signaling pathways and effector mechanisms pre-programmed cell death

Co-reporter:Neal B. Blatt, Gary D. Glick

Bioorganic & Medicinal Chemistry 2001 Volume 9(Issue 6) pp:1371-1384

Publication Date(Web):June 2001

DOI:10.1016/S0968-0896(01)00041-4

Apoptosis is a complex biochemical process that involves all aspects of the cell from the plasma membrane to the nucleus. Apoptosis stimuli are mediated by many different cellular processes including protein synthesis and degradation, the alteration in protein phosphorylation states, the activation of lipid second messenger systems, and disruption of normal mitochondrial function. Despite this diversity in signal transduction, all apoptotic pathways are believed to converge ultimately with the activation of caspases leading to the characteristic morphological changes of apoptosis. In this review, we discuss what is known about these pathways and its implication for normal cellular function.Apoptosis, or pre-programmed cell death, is a complex biochemical process that involves all aspects of the cell from the plasma membrane to the nucleus. Apoptosis stimuli are mediated by many different cellular processes including protein synthesis and degradation, the alteration in protein phosphorylation states, the activation of second messenger systems, and disruption of normal mitochondrial function. Despite this diversity in signal transduction, all apoptotic pathways are believed to converge ultimately with the activation of caspases leading to the characteristic morphological changes of apoptosis. In this review, we discuss what is known about these pathways and its implication for normal cellular function.

Design, synthesis, and analysis of conformationally constrained nucleic acids

Co-reporter:Gary D. Glick

Biopolymers 1998 Volume 48(Issue 1) pp:

Publication Date(Web):22 JUN 1999

DOI:10.1002/(SICI)1097-0282(1998)48:1<83::AID-BIP8>3.0.CO;2-E

In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids. © 1998 John Wiley & Sons, Inc. Biopoly 48: 83–96, 1998

Inhibiting oxidative phosphorylation restrains autoimmune disease

Co-reporter:Gary D. Glick

Biochimica et Biophysica Acta (BBA) - Bioenergetics (August 2016) Volume 1857(Supplement) pp:e14

Publication Date(Web):August 2016

DOI:10.1016/j.bbabio.2016.04.380

Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax

Co-reporter:Neal B. Blatt, Anthony E. Boitano, Costas A. Lyssiotis, Anthony W. Opipari, Gary D. Glick

Free Radical Biology and Medicine (1 November 2008) Volume 45(Issue 9) pp:1232-1242

Publication Date(Web):1 November 2008

DOI:10.1016/j.freeradbiomed.2008.07.022

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F1F0-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.