To evaluate the use of a bioassay system to monitor compound sample integrity we used the Prestwick library that is available in adequate supply and represents a well-characterized set of compounds which is widely used in both pharmaceutical and academic screening laboratories.28
The Prestwick library contains small molecules of which 90% are marketed drugs and 10% are bioactive alkaloids that were selected to give a high degree of both structural and pharmacological diversity. We have analyzed this library for structural diversity which suggests that the Prestwick collection is very diverse, although more limited in scope because of its size compared to larger compound collections such as those found in PubChem.29
Activity of CYP isozyme assay
A large amount of activity was observed for each isozyme in the compound library regardless of storage conditions. Initially, the CYP 2D6 isozyme revealed inhibition by 32% of the library (357 compounds), while the CYP 1A2 isozyme was inhibited by 17% of the library (187 compounds). We found 52% of the CYP 1A2 isozyme active set was shared on average with the active set of the CYP 2D6 isozyme and 36% of the CYP 2D6 active set was shared with the CYP 1A2 active set at any time point of the experiment. The average potency of the compounds was found to be near 1 μM for both isozymes (average pIC50 = 5.8 ±0.6). Overall, both assays provided a large robust dataset to evaluate potency values across the time course of the experiment.
CYP assays show high performance
We evaluated the stability of the CYP isozyme assays themselves by calculating the MSR of control compounds at each time point. For the set of plates used to assay the library stored in DMSO, titrations of the CYP 1A2 control inhibitor furafylline yielded MSR values that varied from 1.7 to 2.9 over the 37 weeks of the experiment, with pIC50s that varied from 6.4 to 5.4. Comparable stability was observed in the plates used to assay the library stored in the hydrated-DMSO conditions, with control compound titrations yielding MSR values that varied between 1.2 and 2.8. The CYP 2D6 isozyme assay also showed good stability although several anomalous plates caused the control inhibitor titrations of quinidine during the sixth time point measurement to inflate the MSR value to 17.7. However, the CYP 2D6 control inhibitor MSR values at other points of the experiment in both hydrated-DMSO and DMSO working sets ranged between 1.3 and 1.9. The CYP 2D6 isozyme assay also had lower signal-to-background overall and showed a Z-factor of 0.62, compared to the CYP 1A2 assay which showed an Z-factor of 0.71. Overall, the average control baseline MSR value (excluding the one inflated point) for both assays was approximately 2.0.
As a further test of reproducibility of the assays, we compared CYP actives determined from an unused (e.g. newly received from compound management) preparation of the library in both CYP assays. The potencies of CYP 1A2 library actives determined from the freshly prepared library copy showed an MSR = 1.9. Similarly, the CYP 2D6 assay showed an MSR = 1.6 using the fresh library copy. Therefore, both CYP isozyme assays showed high performance throughout the time course of the experiment.
Time dependent performance of the CYP assays
We measured the MSR, MR and AUC for each time point for the libraries prepared in either DMSO or using a hydrated-DMSO mixture. The hydrated-DMSO mixture contained 67/33% v/v DMSO/water that yields a mole fraction of 0.63 in water, which was chosen due to its special properties of near maximal viscosity30–32
and proximity to the DMSO-hydrate/DMSO eutectic,18, 33
where compounds are expected to deviate most from ideal solubility.10, 34, 35
The overall trends in compound library performance as measured by the CYP 1A2 isozyme assayed against the DMSO working copy is shown in . The reproducibility of IC50 values decreased as the experiment progressed, and consequently, the number of outliers increased. For example, in week 1, five outlying compounds were initially observed () while at the conclusion of the experiment thirty-six outliers were observed (), out of 51 distinct outliers that appeared during the experiment. In general, compounds decreased in efficacy over time in concert with the decreases in potency (). Of the 126 compounds that had apparent CRCs in the initial as well as both replicates in the final time point assays, 103 showed a decrease in efficacy and 91 showed a decrease in potency, with 74 demonstrating a simultaneous decrease in potency and efficacy. Also indicative of the overall trend, 109 compounds showed a decrease in Hill AUC. The decrease in IC50 reproducibility is also reflected by the compound library MSR values, which show a progressive increase from 2.0 in week 1 to 4.5 in week 37 ().
CYP 1A2 assay over 37 weeks against the working-DMSO library copy
Examining both library conditions in the two CYP isozyme assays showed a similar trend (). In all cases, the IC50 value reproducibility decreased over time, as indicated by increases in MSR values (). Additionally, in general for both assays, compound potency decreased over time, as tracked by decreases in the MRs (). The MRs represent the initial IC50 value divided by the IC50 value at the time point of interest for a compound, averaged over all compounds, such that MR<1 indicates less potency for the compound copy. After the initial assay, lower MR values were recorded, with an average of 0.84 over 37 weeks. Although the overall trend in MR values is downward, from measurement to measurement the values did increase at some time points.
Time-dependent performance of the two CYP assays against the DMSO and hydrated–DMSO working libraries
When we compared the potency differences between the two storage conditions, we observed that the changes occurred similarly in the two working copies. We compared the DMSO and hydrated-DMSO working copies to each other by taking the difference in log potencies between the two datasets at each time point for each CYP isozyme assay. For CYP 1A2, the DMSO to hydrated average MR was 0.92 +/− 0.20, and for CYP 2D6, the average MR was 0.93 +/− 0.12 suggesting similar changes in potency under either storage condition.
The hydrated-DMSO working library showed only slightly greater variance over time than the DMSO working copy. It is possible that the DMSO working copy achieved a water content of at least 10% within the first day of preparation, or equilibrated to hydrated conditions,36
as this can occur within a day in 1,536-well plates filled to a starting volume of 2 to 5 uL with 100% DMSO,37, 38
which would serve to minimize the differences between the two working copies. We found that in the CYP 1A2 assay, MSR values increased from 2.0 to 4.5 for the DMSO samples, while the hydrated-DMSO samples increased from 2.5 to 5.9. In the CYP 2D6 assay, MSR values increased from 2.5 to 4.7 for the DMSO working samples, with a maximum MSR of 5.3 (with 2.5% to 13.5% of active compounds identified as outliers), while the hydrated-DMSO working samples MSR values increased from 2.8 to 6.6. However, both storage conditions showed acceptable MSR values (library MSRs ≤ 3-fold) up to the 100 day time-point, after which IC50
variability became unacceptable as did potencies decreases relative to the starting point. As well, we note that in either condition a larger number of compounds became inactive after the first 100 days: 96±3% of the original actives were fit to high quality CRCs on average for the first 100 days while for the last two time points this average dropped to 79±7%.
Summary of Analytical QC results
The QC result of the library as received from the supplier prepared in DMSO (, 77% found) was comparable to the DMSO stored copy (, 81% found) and the two stored copies showed a similar percentage of compounds found at the end of the experiment (). Differences between the two working conditions as observed by LC-MS were small. There was little difference in the numbers of compounds found in both the DMSO and hydrated-DMSO working copies: 763 and 786, respectively, at the conclusion of the experiment (approximately 70% found in either copy, ). The number of compounds uncharacterized by LC-MS under hydrated conditions, n=212, and those with an apparent increase in purity, n=51, were comparable to the DMSO-working copy. However, the number of compounds showing >95% purity was higher in the hydrated-DMSO working (52%) copy than in the DMSO-working copy (41%). In contrast, the both working libraries conditions showed a reduction in compound QC, compared to the DMSO stored copies.
Analytical QC results for DMSO and hydrated-DMSO conditions
In a recent publication by Engeloch et al.37
duplicate copies of a 2mM, 1,404 member compound library were stored in a 90/10 DMSO/water mixture at 4 °
C and observed sequentially over a 24-month period. This paper found that 85% of samples were found in excess of 85% pure -comparable to the values found for our stored copies described here. Similarly, we did not see an overall detrimental trend in the library QC data for the hydrated-DMSO samples compared to samples prepared with DMSO alone in either working or stored copies.
Correlation of LC-MS results with CYP assay results
We next examined if outlier compounds that showed anomalous potencies correlated with lower QC results. Of the 51 outliers observed from the CYP 1A2 assay in the DMSO-working copy, 31 (60%) were found by LC-MS as having a decrease in purity between samples from the stored copy and the working copy, with 5 compounds of the 51 not found in both plate copies. From a copy of 207 compounds showing decreases in potency, the number of compounds where a decrease in purity was measured by LC-MS totaled 84 (41%), while 100 (50%) were characterized as 100% pure both before and after the experiment. The remaining compounds were either not found by LC-MS in either plate copy (n=20) or showed an apparent increase in purity (n=3). For the CYP 2D6 assay against the DMSO copies, out of the 74 total outliers observed, 32 were found to have a decrease in purity (43%), 33 were found at full purity in both plate copies (45%), 5 showed an apparent increase in purity, and 4 were not found by QC in either copy. In the hydrated-DMSO copies, we noted that of the 44 outliers observed in the CYP 1A2 experiment, only 10 (23%) compounds showed a decrease in purity, with 28 (64%) at full purity in both stored and working plate sets. Three were not found by QC, and another 3 showed an apparent increase in purity. A similar distribution was found in the CYP 2D6 experiment, where out of 58 outliers, 14 (28%) compounds decreased in purity, and 39 (67%) were found at full purity in both stored and working plate sets. Two were not found, and 3 showed an apparent increase in purity. From this, it is evident that changes in compound purity measured with LC-MS do not necessarily predict variable CYP assay performance, perhaps due to degradation products acting as inhibitors of the CYP enzyme. However, this result also underscores the need to measure concentration in analytical library QC procedures as this would allow one to address losses in potencies due to precipitation of the compound.
The working library plates in this experiment used four-column flanking regions (the first four columns were reserved for controls) and four trailing rows (compound free/DMSO-only) along the long dimension of the plate. A plate-wise comparison of wells with outlier compounds at any point during the experiment did not show an obvious pattern, e.g., a tendency for compounds stored on plate edges to display high activity variability (data not shown). However, we note that edge-effects may have be more readily determined if a P450 isozyme such as CYP 3A4 was used as was recently demonstrated by Turner and co-workers39
in a study of nanospots formatted to 1,536 and 3,456-well plates.
We have shown how the reproducibility of potency values can change over time due to sample storage conditions. From this study we would recommend storage of 1,536-well compound library plates at room temperature for < 4 months. The Prestwick library used in this evaluation contains known drugs. Although there is certainly an increased effort to make compound libraries more “drug-like”, the optimal storage time may be different for other types of compound collections. The spot-checking of compound libraries using P450 assays as described here may serve as a useful tool for QC in addition to analytical chemistry profiling methods. Finally, our study found that differences, as measured with either traditional QC methods or two robust bioassays, between hydrated-DMSO and DMSO storage were small. Therefore, at least for collections of compounds with “drug-like” properties, the addition of water to the DMSO will likely have minimal detectable effects on bioassay results.