The biology of chemical space

THE BIOLOGY OF CHEMICAL SPACE
BY JOHN WATSON, PH.D., TRACY WORZELLA, M.S., AND BRAD LARSON, B.A., PROMEGA CORPORATION Introduction
enzymes, leading to nearly universal screening of the majormembers of this drug-metabolizing enzyme family.
The sequencing of the human genome was presented to thepublic as a watershed moment in science that would have a Initial in vitro assays were designed to understand the ADME significant impact on human health. The challenge now is (Adsorption, Distribution, Metabolism, Elimination) properties leveraging our knowledge of the linear DNA sequence to of compounds, including their impact on cytochrome P450 understand the complex world of human biology. Small- enzymes (1). More recently, assays for understanding the molecule drug discovery companies have been at the forefront in vitro toxicity profile of lead compounds have been in using molecular methods to tease out relationships between integrated into most secondary screening programs (2). In specific biomolecular targets and their roles in disease addition to assays designed to test a chemical’s effect on a processes. Those efforts have evolved over the last decade generic target, in vitro methods are also used to understand to the point where in vitro bioassays are routinely used to the selectivity of a compound within a particular target class prioritize new chemical entities prior to animal testing. The (3). For example, kinase profiling has found significant utility intersection between selection and optimization of chemical for understanding the potential off-target effects for this leads and in vitro biology is helping pharmaceutical scientists important group of therapeutics. Importantly, recent results better understand the biology of chemical space.
suggest that the therapeutic value of some kinase inhibitorsis due to inhibition of multiple kinases (4). These recent A common misconception is that the biological effect of a results highlight the importance of screening a large number particular drug is mediated by interaction with a single of targets early in the drug discovery process. molecular target. The reality is that virtually all drugs havecomplex interactions with a large number of biomolecules Chemical Structure and Biological Targets
within the human body. In aggregate, these interactionsdetermine the efficacy and adverse effects of a particular Efforts are now underway to quantify the relationship between compound. However, testing all potential drug leads on certain chemical classes and their effect on in vitro biology.
human subjects to reveal these effects is not possible.
Most large pharmaceutical companies now have databases Historically, animal models were the only surrogate systems on how particular chemical structures affect different biologi- available prior to human clinical trials. However, animal cal targets. This information is driving rational drug design testing is relatively expensive, not feasible for testing large during lead optimization. Often, the challenge is selecting the numbers of compounds, and raises ethical concerns. In vitro best assay to include in the secondary screening program. tools provide a useful alternative for understanding the bio- Two major factors that affect the decision-making process logical properties of potential pharmaceutical compounds.
are assay development costs and the correlation of assayresults with the in vivo biology of the compound. While the Developing In Vitro Assays for HTS
utility of any individual assay is difficult to quantify, the con- The use of in vitro assays for large-scale screening of sensus of most drug discovery biologists is that the pattern chemical libraries expanded significantly in the 1990s. seen when comparing large numbers of related assays run Initial work focused on high-throughput primary screening of on a number of compounds from a particular chemical pharmaceutical companies’ proprietary compound libraries.
series shows significant correlation across the series. By CHEMICAL
The number of leads generated after initial screens had comparing in vitro data with in vivo data generated during to be pared down before animal testing. This led to the clinical studies for compounds in a given series, pharmaceu- development of secondary screening assays designed to tical companies can select structures that are more likely to select compounds with the most drug-like characteristics. be successful in patients. Table 1 shows a profiling screenusing two steroids and two calcium channel blockers, which A number of highly publicized adverse drug incidents have PROFILING
were compared in ten different bioassays. In general, similar also influenced the use of in vitro assays. In particular, the compound classes show comparable profiles. The real value failure of the antihistamines terfenadine and astemizole due of these kinds of data comes from comparing a large to inhibition of the hERG channel has driven pharmaceutical number of structurally related compound groups using a companies to assay for inhibition of this ion channel earlier suite of related assays, such as multiple cytotoxicity assays.
in the drug-screening process. Other drugs such as cimeti- The result is a “bar code” for a particular compound and dine (H2 blocker) and mibefradil (calcium channel blocker) compound class. These data are similar to those seen with have struggled due to their interaction with cytochrome P450 The Biology of Chemical Space
Kinase-Glo® Plus Assay
PROFILING
P450-Glo™ Assay
CYP2C19*
CHEMICAL
Pgp-Glo™ Assay
CellTiter-Glo® Assay
Caspase-Glo® 3/7 Assay
Table 1. Inhibition (Green) correlates with chemical structure. Red = noninhibitory or stimulatory. Kinase-Glo® Plus Assay was run using the Poly
E4Y substrate (Sigma Cat.# P0275) for Src kinase or the Kemptide substrate (Cat.# V5601) for PKA. In the Src kinase assay the IC50 was greater
than 100µM for nicardipine and nifedipine; the IC50 for dexamethasone was 2.67µM and for progesterone was 6.48µM. For the PKA assay, the
IC50 values for nicardipine, nifedipine and dexamethasone were 6.14, 0.18, and 0.29µM, respectively. The IC50 for progesterone was greater than
100µM. CYP450 assays were performed using the P450-Glo™ Assay according to the protocols outlined in Technical Manual #TB340. For the
CYP2C19 assay, the IC50 values of nicardipine, nifedipine and progesterone were 0.65, 1.3, and 1.6µM, respectively. Dexamethasone had no
effect on CYP2C19 under the conditions assayed. For CYP2D6, nicardipine had an IC50 of 1.8µM, while nifedipine and progesterone had IC50
values greater than 100µM. Dexamethasone did not appear to affect CYP2D6 activity. For CYP1A2, the IC50 values of nicardipine and nifedipine
were 6.3 and 0.02µM, respectively. Dexamethasone had an IC50 of 38.66µM, and progesterone had an IC50 of 17.77µM. We used a double-
stable HEK293 cell line for the CellTiter-Glo® and Caspase-Glo® 3/7 Assays. Five thousand cells/well were plated, and the drugs were incubated
for 4 hours before the assays were run. *P450-Glo™ Assays for CYP2C19 and CYP2D6 are currently in development.
nucleic acid microarrays where any given data point has less generated by the Molecular Library Screening Center Network value than the aggregate of overlapping oligo hybridizations.
(MLSCN; see the interview with Jim Inglese, Deputy Director, The overall profile often provides useful information for of the NIH Chemical Genomics Center (NCGC), one of the ten predicting the in vivo behavior of a compound.
centers of the MLSCN, on page 22). Publication of the resultsfrom multiple assays using the MLSCN compound library One of the frustrations for academic pharmacologists has should help develop biological profiles useful for under- been the lack of accessiblity to the proprietary in vitro assay standing complex biological questions and validation of new databases held by drug discovery companies. This issue will drug targets. This public NIH Roadmap Initiative will help to likely be alleviated by the development of an annotated public fulfill the promise of the the human genome project. ■ database (PubChem™) containing in vitro assay results References
Caspase-Glo, CellTiter-Glo and Kinase-Glo are registered trademarks of Promega Corporation.
1. Zlokarnick, G., Grootenhis, P.D.J. and Watson, J.B. (in press) P450-Glo and Pgp-Glo are trademarks of Promega Corporation.
PubChem is a trademark of The National Library of Medicine.
Products may be covered by pending or issued patents or may have certain limitations. Please 2. Riss, T.L. and Moravec, R.A. (2004) Assay and Drug Dev.
visit our Web site for more information.
Technol. 2, 51–62.
3. Godl, K. et al. (2003) Proc. Nat. Acad. Sci. USA 100, 15434–9.
4. Editorial (2005) Nat. Biotechnol. 23, 267.

Source: http://au.promega.com/~/media/files/resources/cell%20notes/cn013/the%20biology%20of%20chemical%20space.pdf?la=en