High-Throughput Screening of Enzyme Inhibitors: Simultaneous Determination of Tight-Binding Inhibition Constants and Enzyme Concentration

Kuzmič, Petr; Elrod, Kyle; Cregar, Lynne; Sideris, Steve; Rai, Roopa; Janc, James W.

doi:10.1006/abio.2000.4685

Cited by 56 publications

(33 citation statements)

References 8 publications

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“…where 0 is the steady-state rate of supercoiled plasmid DNA cleavage in the absence of inhibitor, [E] is the active enzyme concentration, [I] is the concentration of the inhibitor, and is the steady-state rate in the presence of inhibitor (30,31).…”

Section: Methodsmentioning

confidence: 99%

The Nuclease A-Inhibitor Complex Is Characterized by a Novel Metal Ion Bridge

Ghosh

Meiß

Pingoud

et al. 2007

Journal of Biological Chemistry

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Nonspecific, extracellular nucleases have received enhanced attention recently as a consequence of the critical role that these enzymes can play in infectivity by overcoming the host neutrophil defense system. The activity of the cyanobacterial nuclease NucA, a member of the ␤␤␣ Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-Å resolution crystal structure of the NucA-NuiA complex, showing that NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr-135 NuiA hydroxyl oxygen is directly coordinated with the catalytic Mg 2؉ of the nuclease active site, and Glu-24 NuiA also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp-75 and Trp-76 form a second interaction site, contributing to the strength and specificity of the interaction. The crystallographically defined interface is shown to be consistent with results of studies using site-directed NuiA mutants. This mode of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase colicins E7/E9 and from other nuclease-inhibitor complexes that have been studied. The structure of this complex provides valuable insights for the development of inhibitors for related nonspecific nucleases that share the DRGH active site motif such as the Streptococcus pneumoniae nuclease EndA, which mediates infectivity of this pathogen, and mitochondrial EndoG, which is involved in recombination and apoptosis.Nonspecific nucleases are involved in a broad range of functions that include extra-and intracellular digestion, programmed cell death, defense, replication, recombination, and repair (1-3). They also have proven useful for determining nucleic acid structures, mapping mutations, studying the interaction of DNA and RNA with various ligands (4), and sequencing of RNA (5). Most recently, an important role for these nucleases in microbial infectivity has been demonstrated, based on their ability to digest the DNA component of host neutrophil extracellular traps (6 -8). Consequently, these nucleases are now recognized as significant drug targets, and information related to the inhibition of these enzymes is of potential use for inhibitor development. As a result of their ability to degrade nucleic acids nonspecifically, they also represent an endogenous toxic challenge. Therefore, regulation of their activity is critical for the cells that produce them.The ␤␤␣ Me superfamily of nucleases (9) comprises nonspecific, structure-specific, and sequence-specific enzymes that share a structurally conserved active site scaffold and utilize a divalent metal ion. They can be grouped according to sequence motifs into three families: His-Cys box nucleases (e.g. I-PpoI (10)), HNH nucleases (e.g. colicins E7 and E9 (11, 12) and I-HmuI (13)), and DRGH nucleases (e.g. the extracellular nuclease from Serratia marcescens (14), the DNA entry nuclease EndA from Streptococcus pneumoniae (15), the Syncephalast...

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Section: Methodsmentioning

confidence: 99%

The Nuclease A-Inhibitor Complex Is Characterized by a Novel Metal Ion Bridge

Ghosh

Meiß

Pingoud

et al. 2007

Journal of Biological Chemistry

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“…The inhibition dissociation constants (K i ) for inhibitors were determined according to the methods described (41,44). In brief, the HPLC assay was performed in the presence or absence of inhibitors for 2 h at room temperature using ADAM33cat (80 nM) and KL-1 peptide substrate (100 M).…”

Section: Methodsmentioning

confidence: 99%

Catalytic Activity of Human ADAM33

Zou¹,

Zhu²,

Li³

et al. 2004

Journal of Biological Chemistry

104

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“…The increase in fluorescence with time was used as the measure of the reaction rate. Inhibition constants K i (app) were obtained using the program BatchKi (12).…”

Section: Cell Linesmentioning

confidence: 99%

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

Buggy¹,

Cao²,

Bass³

et al. 2006

Molecular Cancer Therapeutics

121

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CRA-024781 is a novel, broad spectrum hydroxamic acidbased inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K i of 0.007 Mmol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of A-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.

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High-Throughput Screening of Enzyme Inhibitors: Simultaneous Determination of Tight-Binding Inhibition Constants and Enzyme Concentration

Cited by 56 publications

References 8 publications

The Nuclease A-Inhibitor Complex Is Characterized by a Novel Metal Ion Bridge

The Nuclease A-Inhibitor Complex Is Characterized by a Novel Metal Ion Bridge

Catalytic Activity of Human ADAM33

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

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