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Developing predictive assays: The phenotypic screening "rule of 3"

Article  in  Science translational medicine · June 2015

DOI: 10.1126/scitranslmed.aab1201 · Source: PubMed

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 Developing Predictive Assays: The Phenotypic Screening “Rule of 3”

Fabien Vincent1*, Paula Loria1, Marko Pregel2, Robert Stanton3, Linda Kitching4, Karl Nocka5,
Regis Doyonnas1, Claire Steppan1, Adam Gilbert3, Thomas Schroeter,1 Marie-Claire Peakman1
1
Hit Discovery and Lead Profiling, Pfizer, Groton, CT 06340, USA.
2
Rare Diseases Research Unit, Pfizer, Cambridge, MA 02139, USA.
3
Worldwide Medicinal Chemistry, Pfizer, Groton, CT 06340, USA.
4
Neusentis Research Unit, Pfizer, Cambridge CB21 6GS, UK.
5
Immunology and Inflammation Research Unit, Pfizer, Cambridge, MA 02139, USA.

*
Corresponding author. Email: Fabien.Vincent@pfizer.com;

One-sentence summary: Not all phenotypic assays are created equal; evaluating the disease
relevance of the assay system, stimulus and readout can help design the most predictive ones.

Abstract

Phenotypic drug discovery approaches can positively impact the translation of preclinical
findings to patients. However, not all phenotypic assays are created equal. A critical question
then follows: what are the characteristics of the optimal assays? We analyze this question and
propose three specific criteria related to the disease relevance of the assay—system, stimulus,
and endpoint—to help design the most predictive phenotypic assays.

PHENOTYPE DRIVE

Over the past decade, several commentaries have referred to a productivity crisis within the
pharmaceutical industry (1).(1, 2) A potential culprit is an overreliance on the industrialization
of the drug discovery process coupled with a reductionist approach to disease biology focusing
on single, insufficiently validated targets.(3, 4) Recent analyses have indeed confirmed that a
majority of high-profile studies reporting novel potential therapeutic targets contain
irreproducible results and are potentially drawing incorrect conclusions on the link between
these targets and specific diseases.(5, 6)
It is during this time of collective industry soul-searching that Swinney and Anthony
published a thought-provoking review on the origins of newly approved medicines, revealing
the prominence of phenotypic approaches in the discovery of first-in-class, small molecule
drugs.(4) Although an updated survey produced lower percentages of success, the difference in
numbers appears to be mostly attributable to semantic distinctions in the definition of
phenotypic screening and does not overturn the conclusions of the first study in our opinion.(7)
Here, we define phenotypic assays as those compound screening systems which focus on the
modulation of a disease-linked phenotype in a target-agnostic manner in contrast to those
centering on a unique protein (as is the case in target-based drug discovery). This success of
phenotypic approaches, rather unexpected in an era focused on target-based drug discovery,
has been ascribed to the unbiased nature of such screens.(4, 8) Generally, phenotypic assays
are more physiologically relevant than target-based ones as they are minimally cell-based, if not
tissue- or whole-organism–based. Furthermore, they offer the possibility of identifying
compounds acting through either unknown targets or unprecedented molecular mechanisms of
action (MMOA) for known targets. For example, a phenotypic screen using live bacteria led to
Linezolid, member of a novel antibiotic class preventing the initiation of protein translation.(9)
Similarly, the antiepileptic Lacosamide was identified through testing in an in vivo rat model of
epilepsy; following its FDA approval, it was documented to promote slow inactivation of
voltage-gated sodium channels rather than directly blocking them.(10, 11)
With the pharmaceutical industry embracing phenotypic screening anew, a key question
has emerged that has received comparatively scant attention in the scientific literature.(12, 13)
Given that a research program will likely be entirely based on the results of its initial hit
identification screen, the assay itself can be expected to play a critical role in the fate of this
endeavor. What then, are the characteristics of the best phenotypic assays, those most likely to
lead to compounds and mechanisms that successfully translate to patients?

UNKNOWN UNKNOWNS

A critical interpretation of recent, major discoveries in both basic and disease biology is that
they probably reveal the extent of what we have yet to learn in these areas. For example, the
existence of mammalian stem cells was an unexpected finding, the ramifications of which are
hard to overstate.(14) Similarly, the discovery of non-coding RNAs and the explosive growth of
the epigenetic field in general, highlight how a basic area of biology has only recently become
better understood.(15) Even well-established dogmas, such as the presumed lack of
neurogenesis in the adult human brain, have been overturned recently.(16)
By definition, target-based drug discovery postulates a direct link between the
modulation of a target through a given MMOA and the resolution or mitigation of a disease
state. However, the high rate of failure of target-based projects due to lack of efficacy indicates
that this underlying hypothesis is often incorrect.(2) The target selection process usually
employs a reductionist approach whereby a hypothesized link from the disease to the target is
built using progressively simpler systems, with the assumption of translation to the clinic at
each step (Figure 1). These assumptions, some known and others unknown to the researchers,
are validated during the drug discovery process as compounds are tested in progressively more
complex systems up to, ultimately, patients. Unrecognized assumptions made during target
selection, or “unknown unknowns”,(17) may then play a substantial role in the documented
high project failure rate.
Figure 1. Target selection and project progression: making and validating translation assumptions.
Symbolic representation of the path followed during target selection, where a relationship is
hypothesized between a specific disease state and a molecular mechanism of action modulating the
activity of a single target, and project progression where this hypothesis is tested in progressively more
disease relevant models.

By design, phenotypic approaches have the potential to mitigate our incomplete

understanding of physiology and disease biology. To fully deliver on that promise, it follows that
those phenotypic assays most faithfully replicating the disease state will incorporate fewer
incorrect assumptions and will consequently be more likely to deliver clinically relevant leads.
In other words, the value of a phenotypic assay lies entirely in its ability to predict the
successful translation of a given compound and/or MMOA to patients. Practically, this
statement can be used as a guiding philosophy to evaluate the potential value of proposed
assays in a resource-constrained environment. From a technical standpoint, three criteria stand
out as helpful in this evaluation: assay system, stimulus, and readout.
We present the least to most relevant assay characteristics using a simple stop-light
analysis in Table 1 and discuss each criterion in this Perspective. For the first criterion, we
propose, rather obviously, that the physiological relevance of the assay system employed in the
phenotypic screen is of critical importance.

Table 1. Analyzing the three key features of phenotypic assays

Criterion Lowest relevance Medium relevance Highest relevance

Assay system Cell-based system Cell-based assay with Assay system with
with single protein expression of multiple clear link to disease
overexpressed and/or proteins (e.g., iPSC-derived
generic cell line recapitulating proper cells with disease-
background (e.g., biological system or linked mutations,
 human embryonic with relevant cells patient-derived
kidney line HEK-293) (e.g., primary, iPSC- primary cells) or
derived, or aiming to replicate
immortalized primary physiology beyond
cells with endogenous 2D, single cell type
expression of target) systems (e.g., 3D
systems, cell co-
culture models, cells
with physiologically
relevant mechanical
stimulation)
Stimulus Limited relevance to Physiologically No exogenous
disease (e.g., relevant, stimulus (e.g.,
chemical cellular multipronged intrinsic disease
injury) stimulation (e.g., stimulus already
cytokine cocktail for provided in patient-
inflammatory derived cells)
diseases)
Readout Distal (e.g., gene Proximal (e.g., Functional
expression) disease-linked protein manifestation of
biomarker) disease as opposed to
biological (e.g.,
muscle contraction)

Criterion #1: Disease relevance of the assay system

Although the majority of phenotypic screens were conducted in cell lines (often tumor-derived)
as recently as a few years ago, awareness is increasing in the field of the importance of
physiological relevance in the assay systems. Intuitively, one would expect native cells, such as
primary cells and induced pluripotent stem cell (iPSC)–derived, differentiated cells, to be more
representative of human physiology.(18)
Multiple examples support this claim. The karyotype of a cell represents one of its most
fundamental and defining characteristics. A large number of tumor-derived cell lines display
significant genetic abnormalities with some extreme examples bearing in excess of 100
chromosomes as opposed to the expected 46. By that measure, the widely used human
monocytic THP-1 cell line would fare well considering its overall diploid character 2A).(19)
Nonetheless, triploidy is observed for four chromosomes and monoploidy for another, along
with the entire deletion of chromosome X and significant chromosomal rearrangements. A
simple question pertains: is this a monocyte? In other words, can we expect a faithful
representation of all of the functions of a primary human monocyte from such a cell?
Furthermore, it is increasingly clear that most proteins do not function in isolation inside cells
but instead partake in multi-protein complexes for signaling and metabolic purposes. G
protein–coupled receptors provide excellent examples of how the pharmacology of a small
molecule can be significantly influenced by both the expression level of the target and the
presence of relevant protein binding partners.(20) For example, the angiotensin II type I (AT1)-
receptor/ 2C- adrenergic receptor heterodimer activates a unique signaling pathway that is
not engaged by either homodimer.(21)
Accordingly, phenotypic projects can benefit from using more native cellular systems, as
there may be substantial differences in the hits and mechanisms identified (Figure 2). The cystic
fibrosis area supports this approach. There is poor overlap in the activity of compounds that
correct the F508del cystic fibrosis transmembrane regulator (CFTR) trafficking defect between
different cell lines overexpressing the same mutated CFTR protein; (22) consequently, the field
increasingly relies on patient-derived primary bronchial epithelial cells to generate efficacy and
potency data with greater clinical relevance than cell lines.(23, 24) In agreement with these
published results, we have ourselves documented a poor pharmacology correlation between
the cell lines and patient-derived cells. Another report further stresses that as protein
expression and active signaling pathways differ between cell types, so will the hits that are
identified in a screen. In this example, researchers studying familial dysautonomia observed
that compounds identified with patient-sourced, iPSC-derived neural crest cells displayed
altered pharmacology when tested against fibroblasts and lymphoblasts bearing the same
disease-causing mutation and were inactive in neural crest cells from a healthy donor.(25)

Figure 2. Pitfalls associated with the use of less relevant cellular systems. Symbolic representation of a
phenotypic assay system comprising stimulus, cell and assay readout. The Venn diagram displays a
hypothetical partial overlap of hits and mechanisms identified in parallel phenotypic screens conducted
with native cells and the cell line.

Although the use of primary or patient-derived cells invariably raises concerns of donor
variability, the examples above support the notion that using a cell line, often derived from
cancerous tissue, may have overridingly negative consequences on the outcome of a
phenotypic screen. As cells from several donors can be used to validate hits and mechanisms,
we have taken the approach that primary and patient-derived primary cells should be used by
default whenever possible (Table 1). In general, use of human cells and tissue is preferable
because identical genetic mutations in human and rodents can lead to inconsistent phenotypes
in vivo, indicating imperfect replication of some signaling pathways in nonhuman systems.(26)
The cellular microenvironment, including the presence of different cell types and a 3D
setting, can also provide critical inputs required for the proper development of relevant cellular
phenotypes. A 3D system with iPS-derived neurons carrying mutations linked to Alzheimer’s
disease was recently published.(27) Although the β-amyloid plaques–Tau neurofibrillary tangles
hypothesis has long been the backbone of research in this area, the iPSC model provided, for
the first time, a demonstration of the entire pathway in native human cells. Similarly, the local
tissue environment influences gene expression in resident macrophages and therefore
promotes context-dependent functions contributing to tissue homeostasis.(28)

Criterion #2: Disease relevance of the stimulus

Most phenotypic assays require a stimulus to achieve the production of the desired phenotype.
We suggest that careful consideration should be given to this parameter in terms of its
relevance to the indication of interest (Table 1).
The stimulus applied to the assay system will direct the engagement of specific signaling
pathways, which in turn will substantially bias the mechanisms and targets which may be
identified in the screen. The ideal stimulation would thus be derived from an accurate and
complete understanding of the disorder’s root causes. As discussed previously, we likely lack
such knowledge in many cases. Our suggestion would be to sidestep this conundrum through
the use of highly disease-relevant biological systems which intrinsically contain the appropriate
stimulus such as patient-derived cells or iPSC-derived cells incorporating specific disease-
causing genetic alterations.(23-25)
In cases where the above strategy cannot be employed, a stimulus will need to be
chosen, taking into account the fact that the root causes of many disorders involve more than a
single causative agent or mutation. The optimal phenotypic assays will attempt to appropriately
recapitulate this activation complexity through the use of relevant, synergistic stimuli. Examples
of this approach can be found in the suite of primary cell assays addressing inflammatory and
autoimmune disorders used to profile the ToxCast library.(29) A contrario, the use of less
relevant stimuli, such as broad cytotoxicants (e.g., high levels of H2O2) to recreate a cellular
injury of interest, runs a significant risk of missing relevant mechanisms while capturing some
which would not translate to patients (Figure 3A, left).(30) Similarly, selecting a relevant
activation method downstream of the disease-causing inputs would theoretically result in the
upstream mechanisms being lost to researchers as potential therapeutic avenues (Figure 3A,
right).
A

Figure 3A. Pitfalls associated with the use of less disease relevant stimuli. Symbolic representation of
the consequences derived from choosing either a non-disease relevant stimulus or a stimulus engaging
only part of the disease relevant pathways.

Criterion 3: Proximity of the assay readout to the clinical endpoint

The relationship between the assay readout and the clinical endpoint constitutes the last
evaluation criterion. Specifically, we propose that more mechanisms with the potential to
translate to clinical efficacy will be identified as the assay readout moves closer to the clinical
endpoint, from basic gene or protein expression to functional or macrophysical manifestations
of the disease (Table 1).
The track record of gene expression readouts such as reporter gene assays is lackluster
with respect to phenotypic drug discovery: no recent, first in class, small-molecule drug has
originated from such an assay.(7) A potential explanation is that mechanisms influencing gene
expression represent only a fraction of all mechanisms affecting a given phenotype (Figure 3B).
A study comparing hit rates for compound libraries using broad gene expression and
multiplexed cytological readouts found only partial overlap between the two sets of hits.(31) An
in house study aimed at discovering novel mechanisms leading to the upregulation of ApoE
secretion compared confirmed hits obtained in the same cellular system using reporter gene
and ELISA readouts. While the reporter gene assay successfully identified compounds providing
large increases in ApoE secretion, it missed half of the overall hit set, i.e., those compounds
providing lower but still substantial effects. Furthermore, transcriptional mechanisms have the
potential to affect a broader set of cellular pathways, and might thus carry greater safety
attrition risk.
B

Figure 3B. Pitfalls associated with the use of less disease relevant assay readouts. Symbolic
representation of the mechanisms captured by different assay readouts for a hypothetical screen aiming
to increase the activity of a given protein. Gene expression assay readouts limit the number and type of
mechanisms which can be uncovered in a phenotypic screen. PTM, post-translational modification.

When translational biomarkers, endpoints that can be monitored clinically to predict

efficacy in patients, can be used as assay readouts they offer data more proximal to clinical
endpoints.(13) Mechanisms discovered to influence a given biomarker in vitro would be
expected to display a similar effect in vivo and may provide a therapeutic benefit to patients.
However, though helpful in many situations, biomarkers are not a panacea in drug
development as they have proven difficult to identify and validate for a range of
indications.(32)
As a result, focusing on functional, potentially macrophysical (e.g., muscle contraction)
readouts reproducing key in vivo disease phenotypes may be more productive. First, the more
downstream the readout is, the more mechanisms modulating this phenotype are captured
during the screen (Figure 3B). Second, with an endpoint closely related to the desired clinical
readout, fewer assumptions are built into the assay leading to compounds and mechanisms
which are more likely to translate to patients. Indeed, phenotypic screening with functional
readouts extends back decades to Sir James Black, who exploited ex vivo assays utilizing
contraction of heart tissue to drive the development of beta blockers.(33)

GO FORTH AND SCREEN

Admittedly, the above criteria will prove difficult to attain in certain cases. However, such
phenotypic assays do exist and have proven their translational value. For example, cystic
fibrosis drug discovery has benefited from the use of patient-derived airway epithelial cells,
offering a compelling assay system that carries an intrinsic disease stimulus. These cells,
coupled with readouts measuring channel activity and the airway surface liquid they release,
have provided an outstanding in vitro model of the disease, enabling the development of the
first drugs improving lung function in patients bearing the G551D and F508del mutations.(23,
24) Similarly, monitoring Plasmodium cell proliferation in primary human erythrocytes led to
the discovery of KAE609. This novel antimalarial agent exerts its effects through an
unprecedented target, ion channel PfATP4, and recently demonstrated efficacy in patients.(34)
Finally, an in house phenotypic screen run using ATP-stimulated primary human monocytes
with IL-1 secretion as readout led to the development of clinical candidate CP-456,773 for
inflammatory disorders. Interestingly, this compound was recently characterized as an inhibitor
of NLRP3 inflammasome formation, a signaling pathway yet to be identified at the time of the
screen.(35)
Sourcing of primary human cells and tissues, especially patient-derived, for phenotypic
screening is crucial given the benefits derived from their use. Their availability has increased
noticeably over the past few years through both commercial and not for profit sources such as
academia-industry collaborations.(36) For the most part, such assays are unlikely to be high
throughput. However, although a thorough discussion of compound library size for phenotypic
screening is outside the scope of this perspective, it is noteworthy that lacosamide was
discovered in the 1990’s using an in vivo epilepsy model with extremely low throughput.(10)
This suggests that disease relevance has the potential to trump compound throughput as the
critical assay parameter in phenotypic drug discovery.
To increase the odds of clinical translation of compounds and mechanisms identified by
phenotypic screening, assays should strive to replicate the disease of interest in terms of the
assay system and stimulus while ideally employing a miniaturized version of the clinical
endpoint as the assay readout. This approach would minimize the number of assumptions
which are implicitly and explicitly made at project initiation and thus partly mitigate our
incomplete understanding of human physiology and disease. Accordingly, phenotypic screening
may be considered a more humble way of conducting drug discovery, and, with the right assay
system, one more likely to succeed.

REFERENCES AND NOTES

1. J. W. Scannell, A. Blanckley, H. Boldon, B. Warrington, Diagnosing the decline in pharmaceutical R&D

efficiency. Nature reviews. Drug discovery 11, 191-200 (2012).
2. M. Allison, Reinventing clinical trials. Nature biotechnology 30, 41-49 (2012).
3. F. Sams-Dodd, Is poor research the cause of the declining productivity of the pharmaceutical industry? An
industry in need of a paradigm shift. Drug discovery today, (2012).
4. D. C. Swinney, J. Anthony, How were new medicines discovered? Nature reviews. Drug discovery 10,
507-519 (2011).
5. F. Prinz, T. Schlange, K. Asadullah, Believe it or not: how much can we rely on published data on potential
drug targets? Nature reviews. Drug discovery 10, 712 (2011).
6. C. G. Begley, L. M. Ellis, Drug development: Raise standards for preclinical cancer research. Nature 483,
531-533 (2012).
7. J. Eder, R. Sedrani, C. Wiesmann, The discovery of first-in-class drugs: origins and evolution. Nature
reviews. Drug discovery 13, 577-587 (2014).
8. J. A. Lee, E. L. Berg, Neoclassic drug discovery: the case for lead generation using phenotypic and
functional approaches. Journal of biomolecular screening 18, 1143-1155 (2013).
9. D. N. Wilson, F. Schluenzen, J. M. Harms, A. L. Starosta, S. R. Connell, P. Fucini, The oxazolidinone
antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning. Proceedings of
the National Academy of Sciences of the United States of America 105, 13339-13344 (2008).
10. D. Choi, J. P. Stables, H. Kohn, Synthesis and anticonvulsant activities of N-Benzyl-2-
acetamidopropionamide derivatives. Journal of medicinal chemistry 39, 1907-1916 (1996).
11. A. C. Errington, T. Stohr, C. Heers, G. Lees, The investigational anticonvulsant lacosamide selectively
enhances slow inactivation of voltage-gated sodium channels. Molecular pharmacology 73, 157-169
(2008).
12. U. S. Eggert, The why and how of phenotypic small-molecule screens. Nature chemical biology 9, 206-209
(2013).
13. D. C. Swinney, The value of translational biomarkers to phenotypic assays. Frontiers in pharmacology 5,
171 (2014).
14. J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall, J. M.
Jones, Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 (1998).
15. T. R. Cech, J. A. Steitz, The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-
94 (2014).
16. P. S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A. M. Alborn, C. Nordborg, D. A. Peterson, F. H. Gage,
Neurogenesis in the adult human hippocampus. Nature medicine 4, 1313-1317 (1998).
17. H. Seely, Pieces of Intelligence: The Existential Poetry of Donald H. Rumsfeld. (Free Press 2010), pp. 128.
18. S. J. Engle, F. Vincent, Small molecule screening in human induced pluripotent stem cell-derived terminal
cell types. The Journal of biological chemistry 289, 4562-4570 (2014).
19. M. D. Odero, N. J. Zeleznik-Le, V. Chinwalla, J. D. Rowley, Cytogenetic and molecular analysis of the
acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation. Genes, chromosomes & cancer
29, 333-338 (2000).
20. T. P. Kenakin, A Pharmacology Primer. (Academic Press, Burlington, MA, USA, ed. Second, 2006).
21. M. Bellot, S. Galandrin, C. Boularan, H. J. Matthies, F. Despas, C. Denis, J. Javitch, J. Mazères, S. J.
Sanni, V. Pons, M. H. Seguelas, J. L. Hansen, A. Pathak, A. Galli, J. M. Sénard, C. Galés, Dual agonist
occupancy of AT1-R–α2C-AR heterodimers results in atypical Gs-PKA signaling. Nature chemical biology
11, 271-279 (2015).
22. N. Pedemonte, V. Tomati, E. Sondo, L. J. Galietta, Influence of cell background on pharmacological rescue
of mutant CFTR. American journal of physiology. Cell physiology 298, C866-874 (2010).
23. M. A. Ashlock, E. R. Olson, Therapeutics development for cystic fibrosis: a successful model for a
multisystem genetic disease. Annual review of medicine 62, 107-125 (2011).
24. F. Van Goor, S. Hadida, P. D. Grootenhuis, B. Burton, J. H. Stack, K. S. Straley, C. J. Decker, M. Miller, J.
McCartney, E. R. Olson, J. J. Wine, R. A. Frizzell, M. Ashlock, P. A. Negulescu, Correction of the
F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proceedings of the
National Academy of Sciences of the United States of America 108, 18843-18848 (2011).
25. G. Lee, C. N. Ramirez, H. Kim, N. Zeltner, B. Liu, C. Radu, B. Bhinder, Y. J. Kim, I. Y. Choi, B.
Mukherjee-Clavin, H. Djaballah, L. Studer, Large-scale screening using familial dysautonomia induced
pluripotent stem cells identifies compounds that rescue IKBKAP expression. Nature biotechnology 30,
1244-1248 (2012).
26. T. Farfel-Becker, E. B. Vitner, A. H. Futerman, Animal models for Gaucher disease research. Disease
models & mechanisms 4, 746-752 (2011).
27. S. H. Choi, Y. H. Kim, M. Hebisch, C. Sliwinski, S. Lee, C. D'Avanzo, H. Chen, B. Hooli, C. Asselin, J.
Muffat, J. B. Klee, C. Zhang, B. J. Wainger, M. Peitz, D. M. Kovacs, C. J. Woolf, S. L. Wagner, R. E.
Tanzi, D. Y. Kim, A three-dimensional human neural cell culture model of Alzheimer's disease. Nature
515, 274-278 (2014).
28. D. Gosselin, V. M. Link, C. E. Romanoski, G. J. Fonseca, D. Z. Eichenfield, N. J. Spann, J. D. Stender, H.
B. Chun, H. Garner, F. Geissmann, C. K. Glass, Environment drives selection and function of enhancers
controlling tissue-specific macrophage identities. Cell 159, 1327-1340 (2014).
29. N. C. Kleinstreuer, J. Yang, E. L. Berg, T. B. Knudsen, A. M. Richard, M. T. Martin, D. M. Reif, R. S.
Judson, M. Polokoff, D. J. Dix, R. J. Kavlock, K. A. Houck, Phenotypic screening of the ToxCast chemical
library to classify toxic and therapeutic mechanisms. Nature biotechnology 32, 583-591 (2014).
 30. A. Nahirnyj, I. Livne-Bar, X. Guo, J. M. Sivak, ROS detoxification and proinflammatory cytokines are
linked by p38 MAPK signaling in a model of mature astrocyte activation. PloS one 8, e83049 (2013).
31. M. J. Wawer, K. Li, S. M. Gustafsdottir, V. Ljosa, N. E. Bodycombe, M. A. Marton, K. L. Sokolnicki, M.
A. Bray, M. M. Kemp, E. Winchester, B. Taylor, G. B. Grant, C. S. Hon, J. R. Duvall, J. A. Wilson, J. A.
Bittker, V. Dancik, R. Narayan, A. Subramanian, W. Winckler, T. R. Golub, A. E. Carpenter, A. F. Shamji,
S. L. Schreiber, P. A. Clemons, Toward performance-diverse small-molecule libraries for cell-based
phenotypic screening using multiplexed high-dimensional profiling. Proceedings of the National Academy
of Sciences of the United States of America 111, 10911-10916 (2014).
32. J. P. Ioannidis, Biomarker failures. Clinical chemistry 59, 202-204 (2013).
33. J. Black, Nobel lecture in physiology or medicine--1988. Drugs from emasculated hormones: the principle
of syntopic antagonism. In vitro cellular & developmental biology : journal of the Tissue Culture
Association 25, 311-320 (1989).
34. M. Rottmann, C. McNamara, B. K. Yeung, M. C. Lee, B. Zou, B. Russell, P. Seitz, D. M. Plouffe, N. V.
Dharia, J. Tan, S. B. Cohen, K. R. Spencer, G. E. Gonzalez-Paez, S. B. Lakshminarayana, A. Goh, R.
Suwanarusk, T. Jegla, E. K. Schmitt, H. P. Beck, R. Brun, F. Nosten, L. Renia, V. Dartois, T. H. Keller, D.
A. Fidock, E. A. Winzeler, T. T. Diagana, Spiroindolones, a potent compound class for the treatment of
malaria. Science 329, 1175-1180 (2010).
35. D. G. Perregaux, P. McNiff, R. Laliberte, N. Hawryluk, H. Peurano, E. Stam, J. Eggler, R. Griffiths, M. A.
Dombroski, C. A. Gabel, Identification and characterization of a novel class of interleukin-1 post-
translational processing inhibitors. The Journal of pharmacology and experimental therapeutics 299, 187-
197 (2001).
36. A. M. Edwards, C. H. Arrowsmith, C. Bountra, M. E. Bunnage, M. Feldmann, J. C. Knight, D. D. Patel, P.
Prinos, M. D. Taylor, M. Sundstrom, S. G. C. O. S. T.-D. Partnership, Preclinical target validation using
patient-derived cells. Nature reviews. Drug discovery 14, 149-150 (2015).

Competing interests: All authors are employed by Pfizer, Inc.

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