Monitoring Compound Integrity With Cytochrome P450 Assays and qHTS

The authors describe how room temperature storage of a 1120-member compound library prepared in either DMSO or in a hydrated-DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z′ factors of 0.71 and 0.62, with 17% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes, respectively. The authors tested the library using quantitative high-throughput screening to generate potency values for every library member, which was measured at 7 time intervals spanning 37 weeks. They calculated the minimum significant ratio (MSR) from these potency values at each time interval and found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical quality control results. Based on this study, the authors recommend that DMSO samples be stored in 1536-well plates for <4 months at room temperature. Furthermore, the study illustrates the degree and time scale of apparent compound potency changes due to sample storage. (Journal of Biomolecular Screening 2009:538-546)

[1]  D. J. Tomlinson,et al.  Nuclear spin relaxation and self-diffusion in the binary system, dimethylsulphoxide (DMSO)+ water , 1971 .

[2]  Brian J. Eastwood,et al.  Methods for the Design and Analysis of Replicate‐Experiment Studies to Establish Assay Reproducibility and the Equivalence of Two Potency Assays , 2010 .

[3]  James A Chan,et al.  Compound library management. , 2002, Methods in molecular biology.

[4]  Caroline Engeloch,et al.  Stability of Screening Compounds in Wet DMSO , 2008, Journal of biomolecular screening.

[5]  J. Cowie,et al.  ASSOCIATION IN THE BINARY LIQUID SYSTEM DIMETHYL SULPHOXIDE – WATER , 1961 .

[6]  T. Riss,et al.  Bioluminescent assays for ADMET. , 2008, Expert opinion on drug metabolism & toxicology.

[7]  Sandra L. Nelson,et al.  The Effect of Freeze/Thaw Cycles on the Stability of Compounds in DMSO , 2003, Journal of biomolecular screening.

[8]  David J. Liu,et al.  Luminogenic cytochrome P450 assays , 2006, Expert opinion on drug metabolism & toxicology.

[9]  John S Lazo,et al.  Building a Pharmacological Lexicon: Small Molecule Discovery in Academia , 2007, Molecular Pharmacology.

[10]  Stevan Djuric,et al.  Studies of the relative stability of TFA adducts vs non-TFA analogues for combinatorial chemistry library members in DMSO in a repository compound collection. , 2003, Journal of combinatorial chemistry.

[11]  O. Trubetskoy,et al.  Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes , 2005, Journal of biomolecular screening.

[12]  Eva J Gordon.  Small-molecule screening: it takes a village... , 2007, ACS chemical biology.

[13]  Sam Michael,et al.  A robotic platform for quantitative high-throughput screening. , 2008, Assay and drug development technologies.

[14]  Sandra L. Nelson,et al.  The Effect of Room-Temperature Storage on the Stability of Compounds in DMSO , 2003, Journal of biomolecular screening.

[15]  R. J. Fort,et al.  Viscosities of binary liquid mixtures , 1966 .

[16]  Christopher P Austin,et al.  Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening. , 2009, Assay and drug development technologies.

[17]  Sam Michael,et al.  Compound Management for Quantitative High-Throughput Screening , 2008, JALA.

[18]  R. Ellson,et al.  In situ DMSO hydration measurements of HTS compound libraries. , 2005, Combinatorial chemistry & high throughput screening.

[19]  William P. Janzen,et al.  High Throughput Screening: Methods and Protocols , 2002 .

[20]  Hua Tang,et al.  Studies on Repository Compound Stability in DMSO under Various Conditions , 2003, Journal of biomolecular screening.

[21]  J Richard Archer,et al.  History, evolution, and trends in compound management for high throughput screening. , 2004, Assay and drug development technologies.

[22]  U Schopfer,et al.  Screening Library Evolution through Automation of Solution Preparation , 2007, Journal of biomolecular screening.

[23]  Investigation of 3 Industry-Wide Applied Storage Conditions for Compound Libraries , 2007, Journal of biomolecular screening.

[24]  J. Bloomfield,et al.  Mechanisms of Nucleophilic Displacement in Aqueous Dimethyl Sulfoxide Solutions , 1961 .

[25]  R. N. Havemeyer.  Freezing point curve of dimethyl sulfoxide--water solutions. , 1966, Journal of pharmaceutical sciences.

[26]  I. Kariv,et al.  Development of a Miniaturized 384-Well High Throughput Screen for the Detection of Substrates of Cytochrome P450 2D6 and 3A4 Metabolism , 2001, Journal of biomolecular screening.

[27]  Dongyu Sun,et al.  Quality Assessment and Analysis of Biogen Idec Compound Library , 2006, Journal of biomolecular screening.

[28]  P. Cleveland,et al.  Nanoliter dispensing for uHTS using pin tools. , 2005, Assay and drug development technologies.

[29]  Adam Yasgar,et al.  Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Kim E. Garbison,et al.  The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies , 2006, Journal of biomolecular screening.

[31]  D. Rasmussen,et al.  Phase Diagram for the System Water–Dimethylsulphoxide , 1968, Nature.

[32]  Kurt Scudder,et al.  High throughput sonication: evaluation for compound solubilization. , 2005, Combinatorial chemistry & high throughput screening.

[33]  Christopher P Austin,et al.  High-throughput screening assays for the identification of chemical probes. , 2007, Nature chemical biology.