scholarly journals Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived from High-Throughput Screening

2016 ◽  
Vol 21 (10) ◽  
pp. 1075-1089 ◽  
Author(s):  
Douglas S. Auld ◽  
Marta Jimenez ◽  
Kimberley Yue ◽  
Scott Busby ◽  
Yu-Chi Chen ◽  
...  

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.

2019 ◽  
Vol 60 (5) ◽  
pp. 1082-1097 ◽  
Author(s):  
Panneerselvam Krishnamurthy ◽  
Yukiko Fujisawa ◽  
Yuya Takahashi ◽  
Hanako Abe ◽  
Kentaro Yamane ◽  
...  

2017 ◽  
Vol 23 (4) ◽  
pp. 375-383 ◽  
Author(s):  
Lisa M. Ogawa ◽  
Neil T. Burford ◽  
Yu-Hsien Liao ◽  
Caitlin E. Scott ◽  
Ashley M. Hine ◽  
...  

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.


2004 ◽  
Vol 383 (3) ◽  
pp. 551-559 ◽  
Author(s):  
Sheraz GUL ◽  
Richard BROWN ◽  
Earl MAY ◽  
Marie MAZZULLA ◽  
Martin G. SMYTH ◽  
...  

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.


2017 ◽  
Vol 280 ◽  
pp. S263-S264
Author(s):  
Caroline Gomes ◽  
Barbara Birk ◽  
Eric Fabian ◽  
Julian Doersam ◽  
Catharina Wilhelmina van Dongen ◽  
...  

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