REVIEW

High-throughput screening assays for the

identification of chemical probes
James Inglese, Ronald L Johnson, Anton Simeonov, Menghang Xia, Wei Zheng, Christopher P Austin & Douglas S Auld

High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas
of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or
enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses
factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field.
We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes,
cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust,
informative and economically feasible HTS assays.

A variety of strategies (Fig. 1) have been used for HTS assays, includ-
ing the measurement of catalytic activity from a purified enzyme1,
a reconstituted complex2,3, a cellular extract4,5 or a phenotype6–9 in intact
cells. Configuring assays to function within the constraints imposed by
HTS differentiates an HTS assay from traditional laboratory assays, as
outlined in Table 1. The 96-well microtiter plate, originally designed to
facilitate serological studies in virology10, was later recognized as a suit-
able replacement for cuvette, tube and dish-based assays. Combined with
spectrophotometric plate readers, the microtiter plate quickly became
the standard for performing parallel assays for rapid preliminary eval-
uation of compound bioactivity. During the 1990s, the tremendous
increase in chemical libraries and assays with sensitivities amenable to
miniaturization further drove the parallel processing concept that today
is practiced in the 96-, 384- or 1,536-microwell plate formats, among
others. ‘High throughput’ is a relative term, but it is generally defined as
the testing of 10,000 to 100,000 compounds per day, accomplished with
mechanization that ranges from manually operated workstations to fully
automated robotic systems11,12. Assays designed for the purpose of HTS
attempt to integrate biological fidelity with enabling assay and screening
technologies. Subsequently, understanding the pharmacological impact
of the assay design is critical to the identification of biologically relevant
compounds from HTS.
Assay design for HTS
An effective HTS strategy considers both the primary and subsequent
secondary assay designs carefully. First, is the nature of the response to
be measured clearly defined: does the signal increase, decrease, change
in nature or location or belong to part of a more complex response? Can
the response be measured via a single parameter, or is a multiparametric
output possible? For example parameters that provide counter-screen
US National Institutes of Health Chemical Genomics Center, National Institutes
of Health, 9800 Medical Center Drive, Bethesda, Maryland 20892-3370, USA.
Correspondence should be addressed to J.I. (jinglese@mail.nih.gov).
Published online 18 July 2007; doi:10.1038/nchembio.2007.17
information can guide active compound selection by differentiating
selectivity among related targets13, or by distinguishing the inhibition
of a cellular pathway from a cytotoxic response in a cell-based assay14–16.
Second, is the stimulus dependent only on the compound being tested,
or is the response to the compound conditioned by another stimulus,
such as a fully efficacious concentration of agonist (when screening for
an antagonist), a subefficacious concentration of agonist (when screen-
ing for a potentiator) or a permissive temperature? In conditional assays,
compound activities independent of the conditional stimulus can be
false positives or can act outside the pathway of interest (Fig. 1d). Third,
does the duration of the response occur within seconds upon application
of the stimulus, thereby requiring a rapid read (for example, a calcium
transient), or within minutes or hours of the stimulus, thereby permit-
ting endpoint or multiple time point measurements? These parameters
will define the basic format of the primary HTS.
Follow-up assays, also referred to as ‘secondary screens’ or ‘counter
screens’, should be planned before the HTS. It is essential to view the
primary HTS as the initial step of an integrated process. Follow-up tests
validate compounds targeting the intended biological interaction and
assist in the elimination of compounds that generate a positive signal
via other mechanisms. False positives can be anticipated based on the
assay design (for example, fluorescent compounds in an assay with
a fluorescent readout) or eliminated by testing in an independent or
‘orthogonal’ assay that uses a different methodology or biological read-
out of target activity. As an example, cyclic AMP (cAMP), an important
cellular second messenger, can be measured with an output response
signal either directly or indirectly proportional to cellular [cAMP]
(Fig. 2). cAMP assays showing inversely proportional responses to
changes in [cAMP] in principle can function in an HTS-confirmatory
assay sequence in which selected candidates identified in the HTS are
then tested for initial validation of activity. Such ‘preliminary confirma-
tion’ strategies using HTS assays in sequence (or parallel) can greatly
lower the retesting of false positives in substantially lower throughput
secondary assays that are generally more complex and designed to be a
more physiological measure of the biological system under study17.

466 VOLUME 3 NUMBER 8 AUGUST 2007 NATURE CHEMICAL BIOLOGY

 REVIEW

performance18. The Z factor’s unitless scale

PEP ADP
(ranging from 0 to 1) allows comparison of
different assays and screens using the control
wells (Z') and sample wells (Z) of the plate.
b Comparison of Z' and Z in assay validation has
Pyruvate ATP + luciferin been used to determine compound screening
a concentration, as assays in which Z' > Z pre-
dict percent of actives greater than the <1%
hν
target value for single concentration screening
GT
P-γ
S paradigms. If compounds are absent from the
sample field (for example, DMSO only), a low
GPCR Z score may be diagnostic of a faulty reagent
hν
dispense or contamination problem in the
sample wells, for example.
An equally significant component of assay
or validation is assay sensitivity—the accuracy
hν P GFP and reproducibility of potency measurements
of control compounds or conditions. The
GTP-γS minimum significance ratio (MSR, Table 2) is
GFP hν
Eu used to measure the reproducibility of potency
P
values and defines the statistically significant
potency range that can be measured in an
c assay19,20. During HTS, the MSR can be calcu-
d lated from titrations of control compounds on
some or all assay plates in the screen to track
assay sensitivity variation, which is often an
indicator of reagent stability.

Detection of biological responses in HTS

The photon, which is created by either a fluo-
rescent or luminescent mechanism, is the
Figure 1 Assay categories and methodologies. (a) An isolated enzyme catalyzes turnover of a principal output in HTS assays. As the most
profluorescent blue- or red-emitting substrate. (b) A coupled-enzyme assay in which the ATP product, widely used HTS detection modality (Table 3;
generated by the pyruvate kinase–catalyzed reaction (ribbon structure), is detected by a “reporter” see Supplementary Table 1 online for addi-
enzyme (firefly luciferase) to create a luminescent output (hν). (c) A membrane preparation is used to tional detail), fluorescent energy is highly
measure the activity of a GPCR through the ligand-dependent activation of associated G proteins as tunable, which allows the development of a
measured by the binding of a fluorescent europium (Eu3+)-labeled GTP-γS analog. (d) A phenotypic
variety of fluorescent sensors21. Fluorescence
assay designed to probe a signaling pathway dependent on coactivation of an extracellular receptor
(blue) and an intracellular protein (yellow). The intracellular protein is activated by a pharmacological can be bright and occur on a timescale ranging
agent (, blue dashed arrow) as a prerequisite to revealing potentiators (compounds that are active from 10−9 s (for typical organic fluorophores)
only in the presence of the costimulus) of the pathway, as measured by the nuclear translocation of to 10−4 s (for lanthanide cryptates), thereby
a GFP-labeled protein. Such potentiators could act by inhibiting proteins that repress the pathway allowing for light’s many optical properties
(black dashed arrows). to be exploited by a number of detection
methods22. For example, the application of
plane-polarized light in fluorescence polar-
Regardless of the approach taken, the HTS assay protocol must be ization (FP) can be used to measure a probe’s rotational perturba-
simplified from its original form (Table 1), and the following param- tions, thereby enabling simple assay designs dependent on a single
eters must be optimized: (i) sufficient sensitivity to enable the identifi- labeled ligand (ref. 23, and ref. 24 and other articles in the same issue).
cation of compounds with low potency or efficacy. (ii) Reproducibility Alternatively, time-resolvable fluorescent trivalent lanthanides such as
and stability of the biological response between wells and plates. This Eu3+ and Tb3+ can be used to increase assay sensitivity by suppress-
parameter is dependent on both assay reagents and hardware (such as ing background fluorescence from assay components and library
dispensers, incubators and detectors). (iii) Accuracy of the positive and compounds25,26. Further, fluorescent probes can transfer energy to
negative controls as compared with the known pharmacology of the tar- other chromophores or fluorophores in assays that measure distance-
get. (iv) Economic feasibility (frequently measured as cost per well). dependent fluorescence or fluorescence quenching via resonance energy
Validation of an assay protocol determines its suitability for HTS and transfer (FRET or QFRET) mechanisms25–27.
involves two parts: performance and sensitivity18 (Fig. 3). Several sta- For cellular assays in particular, chemical28,29 and genetically encoded27
tistical parameters are used to evaluate assay performance. Signal win- fluorescent probes allow customized, multiplexed or direct measurement
dow and Z factor (Table 2) are calculations that gauge the fold response of events from biological components present at low concentrations30.
between maximum and minimum output signals and the precision of Coupled with scanning microplate cytometers31 or microscope-based
this response within a plate and across plates. The signal window mea- imaging systems, these probes are moving HTS from population-aver-
sures the statistically significant difference between the maximum and aged outputs toward the enumeration of individual cellular events8,9,
minimum signal, but it is not as reliable as Z factor for predicting assay thereby enhancing the information content obtainable from each well.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 8 AUGUST 2007 467

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Table 1 Differences in allowed parameters between laboratory “bench top” and HTS assays
Parameter Bench top
Protocol May be complex with numerous steps, aspirations, washes
Assay volume 0.1 ml to 1 ml
Reagents Quantity often limited, batch variation acceptable, may be unstable
Reagent handling Manual
Variables Many—for example, time, substrate/ligand concentration, compound,
cell type
Assay container Varied—tube, slide, microtiter plate, Petri dish, cuvette, animal
Time of measurement Milliseconds to months
Measurements as endpoint, multiple time points, or continuous
Output formats Plate reader, radioactivity, size separation, object enumeration,
images interpreted by human visual inspection
Reporting format “Representative” data; statistical analysis of manually curated dataset
aSpecial reagent dispensers required. bIdeally available in milligram quantity with analytical verification of structure and purity.
The absence of excitation light energy in the generation of a lumi-
nescence signal greatly reduces compound interference in HTS (Fig. 4).
Although the signal strength can be significantly lower than fluores-
cence, the background is negligible, which allows luminescence-based
assays to have an enormous dynamic range, on the order of 1,000 times
more sensitive than equivalent fluorescence-based assays32. Both che-
miluminescence and bioluminescence use a chemical reaction to create
light. In the case of bioluminescence, an enzyme (typically from the
firefly Photinus pyrali) generates light through its action on a luciferin
substrate, analogs of which have been used to develop assays for cyto-
chrome P450s, proteases and monoamine oxidases32. Further, the active
site of the P. pyrali luciferase has been mutated to spectrally vary the
luminescence33, thereby allowing the use of multiple luminescent sen-
sors in an assay15.
Luciferase reporters are prevalent in HTS assays because of convenient
detection, ample sensitivity and their ubiquitous occurrence in academic
research. Dual luciferase reporter assays with distinct substrate speci-
ficities, kinetics or emission maxima have been useful for identifying
activities specific to the signaling pathway of interest. Frequently, the
second reporter is used to normalize gene expression or discriminate
cytotoxic compounds34,35. Luciferase reporters can be combined with
other detection formats as well, for instance in tandem with a green
fluorescent protein (GFP) reporter14, β-galactosidase or Alamar blue16
to assess cytotoxicity.
The β-lactamase reporter offers particular advantages for HTS. For
example, the β-lactam–coupled coumarin-fluorescein substrate, CCF4/
AM, allows a ratiometric read that helps minimize variation in cell num-
ber or substrate concentration because the emission maxima of the cleaved
and intact β-lactamase substrates (460 and 530 nm, respectively) are dis-
tinct (reviewed in ref. 36). In addition, the uncleaved substrate can be
used to assess cytotoxicity, as fluorescence is detected only if the substrate
is taken up by living cells and de-esterified37,38.
A hybrid system involving both bioluminescence and fluorescence
is represented by BRET-based assays39 (Table 3 and Supplementary
Table 1). Here luciferase luminescence stimulates a proximal fluorescent
protein emission, thereby bypassing the need for exogenous excitation
light. However, this technique may be more challenging to optimize for
HTS assays because of lower S:B ratios and higher background from
weakly associated target proteins40.
In addition, radioactive decay in conjunction with the scintillation
proximity assay (SPA) format is applicable to a host of targets41, and
low-cost absorbance assays have proven reliable in measuring analytes
(for example, H+ or Pi), cellular staining or cell growth. However, in
microtiter plates, absorbance measurements can have low S:B ratios
468
HTS
Few (5–10) steps, simple operations, addition only preferred
<1 µla to 100 µl
Sufficient quantity, single batch, must be stable over prolonged period
Robotic
Compoundb, compound concentration
Microtiter plate
Minutes to hours
Measurements typically endpoint, but also pre-read and kinetic
Plate reader—mostly fluorescence, luminescence and absorbance
Automated analysis of all data using statistical criteria
owing to the short path length that results from low volumes, though
several assays have been implemented in 1,536-well plates42,43.
Variations on the use of these technologies in the design of assays are
impressive, and a discussion of each is beyond the scope of this paper.
Assays suitable for HTS comprise a subset of these and must meet the
requirements outlined in Table 1. The next section highlights the well-
exploited assay formats.
Assays for specific targets
Historically, screening assays have been performed on a limited set of
targets, many involving purified proteins. Approximately two thirds of
therapeutic targets are comprised of enzymes and receptors44. More
recently, HTS labs are increasing attention on cell-based high-content
screens (HCS) that analyze biological events at subcellular resolution8,9; a
2006 survey indicated that roughly half of HTS assays are cell-based and
that the use of HCS is rapidly growing, particularly in secondary screen-
ing45. The types of proteins for which drugs have been successfully devel-
oped are relatively restricted. About half of experimental and marketed
drugs target five main protein families: G protein–coupled receptors
(GPCRs), kinases, proteases, nuclear receptors (NRs) and ion channels46.
Because of the great interest in these protein classes for drug develop-
ment, many approaches and technologies (Table 3 and Supplementary
Table 1) have been developed to assay these targets, and in some cases
they are broadly applicable to other target classes as well.
Enzymes. Enzymes are comprised of six categories: oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases. They catalyze a large
number of chemical transformations. In cases in which the enzyme, cofac-
tor and substrate can be obtained in an isolated, active form, the devel-
opment of an HTS assay generally hinges on the availability of reagents
and technologies to enable substrate or product detection. Here, protein
kinases and proteases will serve to illustrate various approaches to HTS
assay design for enzymes.
Protein kinases. Protein kinases mediate the most prevalent post-transla-
tional modification in cells, phosphorylate an array of targets in signaling
cascades and cycles, and are important for therapeutic intervention, par-
ticularly oncology47. In this section we discuss biochemical approaches to
protein kinase assays, as cell-based assays are covered in the later sections
on cellular signaling. The biochemical approaches can be divided into
two categories: generic assays independent of subfamily (for example,
tyrosine versus serine/threonine) and antibody-based formats that detect
an epitope within the phosphorylated product. The latter are generally
used for a particular subfamily or specific protein kinase.
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Protein kinases catalyze phosphoryl group transfers from ATP
to hydroxyl groups of amino acid side chains of serine, threonine or
tyrosine. An easily implemented assay format that has been used suc-
cessfully in HTS detects ATP depletion via luciferase, an ATP-depen-
dent photoenzyme48,49. A disadvantage of this approach is that 50%
conversion of the ATP is required to achieve a two-fold S:B. This may
require increased enzyme levels for sufficient signal, thus decreasing
sensitivity and increasing reagent costs. Alternatively, kinase activity
can be measured through ADP production. One assay format uses an
ADP-specific antibody and labeled ADP to generate an FP competition
binding assay to monitor ADP product formation50. A coupled enzyme
strategy for ADP detection that yields a fluorescent readout has also
been described51.
A number of methods measure kinase activity by the detection of
phosphorylated peptide. Several formats use micron-sized beads or
particles to partition the product phosphopeptide from ATP substrate
or peptide. Radiometric filter binding remains the standard for kinase
selectivity studies. For HTS, biotinylated 33P-phosphopeptide is typi-
cally captured by streptavidin-coated SPA beads41 or scintillant-treated
microtiter plates (for example, FlashPlates). In immobilized metal-ion
affinity-based fluorescence polarization (IMAP), transition metal-che-
lated particles bind fluorescently labeled phosphopeptides with high
affinity, thereby enabling an antibody-independent FP format for the
detection of phosphopeptide products52. Recently IMAP has been
adapted to FRET-based systems in which size limitations on the poly-
peptide substrate (typically <10,000 MW) are relaxed53. A similar system
has been developed based on fluorescence quenching through an iron-
phosphate interaction54. However, for HTS the ratiometric nature of
IMAP is an advantage of this system over fluorescence quenching.
Antibody-based formats that detect a specific phosphorylated peptide
or sequence capitalize on the many phosphospecific antibodies available
and on nonseparation technologies, including TR-FRET, ALPHA and
FP. Such assays are advantageous in targeting kinase activity dependent
on, for example, hierarchical phosphorylation or kinase cascades55. Also,
multiplexed readouts of kinase activity and cellular phosphoproteins are
possible with these formats56.
A unique system that assays the kinase inactive state relies on ATP
binding and uses enzyme fragment complementation (EFC) for detec-
tion57. Here the general kinase inhibitor, staurosporine, is linked to a
enzyme donor (ED) fragment of β-galactosidase that when displaced
from the kinase ATP pocket restores β-galactosidase activity through α
complementation with the acceptor enzyme (EA) component58.
A final general point, illustrated with kinases, relates to assay designs
that recapitulate as closely as possible the native target to uncover new
mechanisms of compound action. As most biochemical kinase assays
have used only the catalytic domain for HTS, ATP site–directed inhibitors
are the primary class of agents identified. However, with a TR-FRET–
based HTS of Akt-family kinases, Barnett et al.59 discovered a pharmaco-
logically unique class of compounds using the full-length kinases. These
compounds stabilized the inactive conformation of Akt1 and Akt2 via a
site formed only in the presence of the pleckstrin homology (PH) domain
of the kinase59, thereby allowing a unique mode of selectivity between
Akt1 and Akt2 versus Akt3, which is not inhibited by the chemical class
owing to its lack of a PH domain.
Proteases. As well-established drug targets60, proteases have received
considerable coverage in terms of assay formats and reagent kits.
Profluorescent or FRET-based substrates are commonly used to assay
proteases as purified proteins or in cell lysates. Typically, probes are
coupled to a two-to-five-amino-acid peptide comprising a protease
cleavage site and become fluorescent after liberation by protease cleav-
age13,61. Though ideally suited to HTS, a shortcoming of this approach
is that the fluorogenic peptide is often insufficient in length to cover the
entire binding pocket.

a c
Gαi-coupled response Gαs-coupled response
EFC

100 100 100

EA
Activity (%)

Activity (%)

75 75 75
Activity (%)

ED

50 50 50

25 25 25

S
hν 0 0 0
Log[compound] (M) Log[compound] (M) Log[compound] (M)
TR-FRET b
hν 100 100 100
D A
Activity (%)

Activity (%)
Activity (%)

75 75 75

50 50 50

D 25 25 25
A
0 0 0
Log[compound] (M) Log[compound] (M) Log[compound] (M)

ED-labeled cAMP Acceptor-labeled cAMP Cellular cAMP

GPCR Gi AC [cAMP] AC Gs GPCR

Figure 2 Influence of assay design on the measured biological response. (a) An EFC-based competitive immunoassay illustrates an output signal that is
directly proportional to intracellular [cAMP]. (b) A TR-FRET output signal is indirectly proportional to intracellular [cAMP]. (c) The resulting responses for
hypothetical assay of Gi-coupled (left) and Gs-coupled (right) GPCRs are shown for the EFC (top) and TR-FRET assays (bottom) for compounds acting as
either agonists (dotted lines) or antagonists (solid lines). Assays having opposing signal outputs in response to the same analyte; here [cAMP] is an example
of an assay pair in which one could function in HTS and the other in folow-up confirmation and false positive detection.

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Table 2 Equations for determining assay performance or sensitivity

Parameter Equation Comment
Coefficient of variation σ × 100 A measure of the precision relative to the mean value, calculated for the maximum and
%CV = —
µ minimum signals. An acceptable limit is <15%.
Signal to noise µmax – µmin A measure of the signal strength. This equation is sometimes given asa: µmax – µmin
S:N =
σmin σmax2 + σmin2
Signal to background µmax Usually calculated using control compounds, acceptable value >2-fold.
S:B =
µmin

Signal window µmax – µmin – 3(σmax + σmin) The significant signal between max and min controls, acceptable value >2-fold b.
SW =
σmax

Z' factor (3σmax + 3σmin) Representation of the SW using a score where a value >0.5 represents an acceptable
Z' = 1– assay. Z' is measured in the absence of library compounds; Z is in the presence.
µmax – µmin

Minimum significance ratio Smallest potency ratio between two measurements that is statistically significant
MSR = 102σd
(with 95% confidence).
Cheng-Prusoff IC50 Used to derive the Ki from the IC50 under conditions of competitive inhibition.
Ki =
(1 + [S]/Km)

Cheng-Prusoff IC50 Used to derive the Ki from the IC50 under conditions of competitive binding.
(ligand-binding) Ki =
(1 + [L]/Kd)

σ, s.d. of the assay signal; µ, mean of the assay signal; σd, s.d. of the difference in log potency; max, maximum signal; min, minimum signal. aThis is a redundant description of
the signal window equation. bSome assays, such as those with ratiometric data, may perform adequately with as little as a 20% change in signal (for example, see ref. 111) or with
0 < Z ′ < 0.5.

Although there are numerous ways to measure endoprotease activity,
assays for exoproteases that recognize C- or N-terminal residues are far
less available62. The hydrolytic activity of the protease has been used
to process simple chromogenic esters and amides, but assays based on
these substrates generally lack sensitivity for miniaturized HTS assays.
One approach is to use protease-mediated “epitope unmasking,” in
which cleavage by the exoprotease reveals a binding site for a detection
reagent. Here, sensitive time-resolved FRET (TR-FRET)-based formats,
for example, can be applied63.
Cellular FRET-based methods have been developed using fluorescent
proteins or dyes linked by a protease consensus sequence. When expressed
or taken up by cells, cleavage of the probe can be measured by a change in
fluorescence upon activation of proteases such as caspase-3 (ref. 64) and
hepatitis virus (HCV) NS2/3 (ref. 65). Similarly, cells expressing a fusion
of GFP and alkaline phosphatase coupled through an NS3/4A cleavage site
were used to monitor the activity of this HCV protease by the detection
of secreted alkaline phosphatase66. In an approach based on cell viability,
procaspase-3 was modified to contain a β-secretase cleavage motif that
triggered apoptosis in 293T cells upon expression of β-secretase. β-secre-
tase inhibitors were shown in this assay to promote cell survival67.
Nuclear receptors. NRs are transcription factors that are characterized
by common modular features including a DNA-binding domain (DBD)
and a ligand-binding domain (LBD). NRs are regulated by endobiotic
ligands such as hormones and metabolites, or through xenobiotics68.
The human genome encodes ~48 NR members of known and unknown
function (orphan NRs) that are actively being studied in drug discovery
and basic research. Consequently, the ligand or DNA-binding properties
of isolated domains, and the cellular translocation and gene transactiva-
tion properties of NRs, have been featured in assays designed for HTS
(ref. 69 and other articles in the same focus issue).
Classical competition binding assays for NRs can be divided into
radiometric and fluorometric assays. SPA formats have been developed
for NRs such as peroxisome proliferator-activated receptor70, and FP has
been used for well-characterized receptors such as the steroidal NRs, in
which a fluorescent conjugate of a known receptor ligand and a purified
LBD are prepared71.
Using GFP fusions of NRs8, cell imaging assays have been described
that measure ligand-dependent, cytoplasm-to-nucleus translocation
of the glucocorticoid receptor (GR)72 or engineered NR chimeras73.
Translocation assays using GFP fusions of GR have been applied to
1,536-well systems (see ref. 8; PubChem BioAssay ID (AID) 450).
Additionally, EFC assay formats have been described to measure NR
translocation72,74 (PubChem AID 451). However, none of the aforemen-
tioned assays can distinguish the functional effect of active compounds
on NR transactivation.
To distinguish between agonist and antagonistic activity of com-
pounds, the targeting of NRs has been addressed with TR-FRET
ligand-dependent coregulator recruitment assays75, constructed with
either coactivators or corepressors to identify agonists and antagonists.
Additionally, cell-based reporter gene assays provide among the most
sensitive means to screen for functional activity. These assays fuse NR
response elements to reporters such as luciferase76, β-lactamase, and
secreted alkaline phosphatase (ref. 69 and other articles in the same
focus issue).
G protein–coupled receptors. GPCRs are targets of intensive drug
development by the pharmaceutical industry, and numerous, well-estab-
lished HTS assay methods exist77,78 and continue to be developed79.
Technologies to measure ligand-receptor binding41,80 and the functional
consequence of receptor activation77 have enabled screens that target
the rich pharmacology of this receptor superfamily, including assays to
differentiate full, partial and inverse agonists, antagonists and allosteric
modulators81, and to identify ligands for orphan GPCRs8,82.
GPCRs broadly orchestrate a wide range of cellular events and pro-
cesses for which HTS assays have been configured, including guanine
nucleotide exchange5,83, modulation of second messenger concentra-
tions84,85, ion channel activity86, protein phosphorylation56, protein
trafficking8,87,88, pigment dispersion89, gene transcription6 and cell
proliferation90, among others. The assays have different advantages and
limitations, but collectively they illustrate the sophistication possible
in HTS assay design given the current state of the art. For example,
GPCRs that activate intracellular Ca2+ stores can be assayed using cal-
cium-sensitive dyes (for example, Fluo-3 and Fura-4) and rapid-inject

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imaging platforms84. However, GPCRs not naturally coupled to [Ca2+]
modulation could not be assayed by this means until the development
of so-called universal adaptors, molecular engineered G proteins91 or
promiscuous G proteins92, to reconfigure such GPCRs to stimulate Ca2+
signaling. Today GPCR second messengers can be assayed by the direct
measurement of cAMP85 (Fig. 2) or inositol phosphate species57,93.
Assays based on GPCR internalization are independent of G protein
subtype, unlike second messenger formation, and have been applied suc-
cessfully to analyze a number of GPCRs using either standard microtiter
plate readers94 or fluorescence microscopy8,9. The use of the adaptor
protein β-arrestin 2, fused with GFP, permits an assay design that gen-
erally does not require modifications to the target receptor’s N or C
terminus. Such phenotypic assays enabled with automated high-content
image analysis are bridging cell biology with HTS in a way that will have
a major impact on chemical biology, as discussed below.
Ion channels and transporters. Ion channels are membrane-spanning
proteins that regulate electrochemical gradient-driven movement of
inorganic ions such as Na+, K+, Ca2+ and Cl– into or out of cells. HTS
technologies aimed at this target class are under rapid development95.
The existence of multiple responsive states, including closed, open and
inactive, to which drugs respond differently, add to the pharmacological
diversity and assay development challenges96.
Ligand binding, ion flux, and fluorescent probe-based assays are the
established methods for ion channel HTS (Table 3 and Supplementary
Table 1). Competition binding measures the affinity of the ligand-
channel protein interaction but is dependent on the availability of
radiolabeled ligands. As with GPCRs, binding assays do not detect the
functional consequence of compound interactions with ion channels.
For these reasons, cell-based functional assays are more relevant to ion
channel screening at both the primary and secondary levels.
The calcium-sensing fluorophores Fluo-3, Fluo-4 and Fura-2 have
been used extensively in HTS for voltage- and ligand-gated channels
and for GPCR-induced intracellular calcium release95. The fluorescence
intensity of these probes inside cells increases proportionally with the
elevation of intracellular free calcium concentration97. The addition
of opaque dyes to the assay buffer eliminates the need for cell washing
steps, as only intracellular fluorescence of adherent cells is detected in
bottom-read measurements, thereby improving compatibility with HTS
protocols95. Voltage-sensing probes track changes in membrane poten-
tial and have been used in several assay formats, such as FRET-based and
positional voltage sensors98.
Ion flux measurements using radioactive tracers such as 86Rb+, 45Ca2+,
22Na+ and 36Cl– are established assays of ion channel activity. However,
the medium removal steps and considerable radioactivity limit this
approach for HTS. Atomic absorbance spectrometry using nonradio-
active tracer ions has recently permitted significant improvements to
the ion flux assay for HTS99.
Patch-clamp electrophysiology remains the standard for measuring
ion channel activity, as the potency of certain compounds, especially
those with state- or voltage-dependent mechanisms, may appear signifi-
cantly less potent when using other assay methods (for example, fluo-
rometric or rubidium ion flux). Automated patch-clamp instruments
represent a remarkable development in technology, increasing through-
put from approximately ten compounds per week to hundreds95,100.
However, further improvements are needed in seal quality, throughput,
and cost before this technology can replace other formats such as fluo-
rescence-based assays for HTS.
The broad examination of drug candidates for ion channel activity has
become critical in the assessment of a candidate’s safety profile. In addi-
tion to the functional inhibition of ion channels, the folding and traf-
NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 8 AUGUST 2007
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ficking of ion channels represents another avenue for the development
of HTS assays analogous to those developed for GPCRs. For example,
an ELISA-based assay has been reported that measures the amount of
cell surface hERG channel to determine drug effects on hERG channel
trafficking101.
Transporters, another category of transmembrane proteins, have an
important role in ion, membrane potential and nutrient homeostasis.
Perturbations in transporter function can result in disease and altered
drug absorption, distribution, metabolism, excretion and toxicity
(ADMET) properties, leading to drug resistance, for example. As a case
in point, multidrug-resistance (MDR) transporters (for example, P-gly-
coprotein) belong to the family of ATP-binding cassette (ABC) proteins
that extrude xenobiotic substances from the cell, including many drugs.
Assays based on ATPase activity and fluorophore transport have been
described102,103. Typical assay formats for amino acid and neurotransmit-
ter transporters rely on radiolabeled substrates (for example, 3H-glycine)
and involve multiple assay steps including medium aspirations. However,
new assay methods are being developed that are suitable for HTS such as
yeast heterologous expression systems104 and anion sensors105.
Assays for pathways and networks, and cellular phenotypes
The complexity of components and their interactions within cellular
networks offers a rich source of new targets to assay using technologies
to examine protein interactions, expression, distribution and stability.
For instance, signaling mediated by Hedgehog, Wnt and Notch proteins
uses unconventional proteins implicated in developmental and disease
processes, making these pathways important therapeutic targets106 that
are assayable with HTS automated fluorescence microscopy methods8.
The following sections describe additional cellular assays but reveal only
a glimpse of the breath of cellular processes amenable to testing by HTS
assays.
Transcriptional readouts. Reporter gene expression is a mainstay for
detection in cellular signaling assays6 and is useful for broadly identi-
fying compounds that modulate the pathway and isolate new targets
within. However, the purposely promiscuous nature of these assays is
disadvantageous if the objective is a probe of a particular component
of the pathway, as ‘target decryption’ can be a formidable undertaking.
A separate critical consideration resides in identifying a cell type that
appropriately reflects the cellular context of the signaling pathway being
assayed by the reporter; both the types and amounts of cell surface recep-
tors and intracellular signaling components can vary widely among the
commonly used engineered cell lines.
Protein interactions and redistribution. Several innovative enzymatic
and image-based methodologies have provided new ways to identify
compounds that modulate movement and associations of protein com-
ponents within living cells. These approaches fall into several general
categories: reporter complementation, resonance energy transfer and
protein redistribution. Low-affinity associations form the basis of pro-
tein complementation assays (PCA), in which two proteins known to
associate are separately fused to N- and C-terminal halves of a bifur-
cated reporter protein. Upon interaction of the two sentinel proteins,
the halves of the reporter (now in close proximity) reconstitute a func-
tional enzymatic or fluorescent reporter. Commonly used PCA reporters
include β-lactamase, GFP, YFP and luciferase27,40,107. This methodology
is attractive because it can be adapted to many protein-protein interac-
tions and is amenable to screening and profiling, as demonstrated with
YFP-based108 and β-lactamase109 reporters. Alternatively, high-affinity
β-galactosidase α complementation has been used to monitor protein
movement. Here the ω fragment is targeted to a specific subcellular
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Table 3 Assay technologies

Technology Description Refs.
AAS Atomic absorption spectroscopy for measurements of ion transport/flux (primarily focused on K + channels with Rb+ as tracer); 99
96-/384-well microtiter plates enabled with, for example, the ICR 12000.
Absorbance Follows Beer’s Law; trans- or epi-absorbance; via fluorescence attenuation. 42,43
ALPHA Amplified luminescent proximity homogeneous assay; donor-acceptor beads of ~200 nm diameter; λex 680 nm (laser); singlet 56,149
oxygen reacts with acceptor bead within ~200 nm distance; λem 520–620 nm.
Bioluminescence Renilla or firefly luciferases used in reporter gene assays; analogs of D-luciferin used to expand biochemical assays for 32
proteases, P450s, monoamine oxidases.
BRET Bioluminescence resonance energy transfer; useful for protein-protein interactions; Renilla luciferase donor luminescence (480 39,150
nm) stimulates fluorescence of GFP/YFP acceptor; follows dipole-dipole interaction 1/r 6 (~5 nm maximum).
Bla/CCF4 FRET-based assay using β-lactamase and β-lactam-containing substrate (for example, CCF4); λex 395 nm, λem 460/530 nm; 36,38,151
ratiometric reporter gene assays and other formats.
DELFIA Dissociation-enhanced lanthanide fluorescent immunoassay/time-resolved fluorescence; ELISA-like format using lanthanide 25
chelate-labeled antibody; τf ~500 µs; separation-based enhanced fluorescence; variations using labeled ligands; extremely
sensitive.
ECL Electrochemiluminescence assay. Uses microcarbon electrode plates allowing electrical oxidation of a [Ru(phen) 3]2+ label. 17
Multiplex applications in ELISA format.
EFC Enzyme fragment complementation. Based on β-galactosidase α complementation; high-affinity complementation. Applied to 57,74
both cell-free and cell-based systems.
FLT Fluorescent lifetime; time-domain method (~ns) measures the rate of fluorescent decay following excitation; promises to 146
eliminate fluorescence compound interference; only recently applied to some microplate systems.
FP Fluorescence polarization; polarized light reports change in probe ligand τc; fluorophores of τf ~ 5 ns needed to measure ligand 23,24
of MW < 1,500 binding to a receptor of MW ≥ 10,000; widely applied, ratiometric measurement.
FRET Fluorescence resonance energy transfer; donor-acceptor pair as in EDANS-DABCYL peptides or fluorescent proteins (for 22,53
example, CFP/YFP). Signal distance dependence ~5 nm maximum.
Fluorescence Measurement of fluorescence intensity including the use of chromophores for quenching as in profluorescent protease 22,152
substrates (~5 nm maximum distance); widely applied; superquenching varies in that nonequimolar amounts of donor
fluorophore and quencher are used. Includes kinetic modes of detection with timescales of approximately minutes to hours.
Fluorescence Requires fluorescent imaging plate reader or equivalent (for example, FLIPR, FDS6000, VIPR); timescale ~seconds; GPCR 84,98
(rapid kinetic) functional assays using Ca2+-sensitive dyes, or steady state ion channel activity using voltage-sensitive dyes.
GFP and variants Cell-based imaging microscopy/cytometry assays; translocation, redistribution, protein-protein interactions, reporter gene 27,112
assays; see below.
HT electrophysiology High-throughput electrophysiology; measurement of ion channel currents with microaperture arrays such as the planar 98,99,153
patch-clamp electrode (for example, PatchXpress 7000A, IonWorks Quattro).
PCA Protein complementation assay; recombinant split reporters (for example, GFP, luciferase, β-lactamase) fused to interacting 40,108
proteins; phenotypic assays; low-affinity complementation.
SPA, FlashPlate Scintillation proximity assay; nonseparation-based radiometric detection using solid supports; typical isotopes include 35S, 33P 41
or 3H.
TR-FRET Time-resolved FRET; also known commercially as HTRF/LANCE; nonseparation-based format using a lanthanide cryptate donor 25,26,53
fluorophore of long fluorescence lifetime (τf ~ 500 µs); detection distance ~9 nm max.
τf, fluorescent lifetime; τc, rotational correlation time.

region and the α-peptide-tagged translocating protein restores β-galac-
tosidase activity once α and ω combine74.
In the resonance energy transfer methods FRET and BRET (Table 3
and Supplementary Table 1), donor and acceptor (or sensor) proteins
are fused to the target proteins of interest. In BRET, a signal is generated
when a bioluminescent donor enzyme is brought into proximity with
an acceptor fluorophore, the photon emission of which is measured39.
In FRET, energy is transferred from a donor to an acceptor fluorophore
when the two proteins come into close proximity, which is manifested
as a change in wavelength of the emitted fluorescence. FRET-based
sensors use spectrally distinct fluorescent proteins such as engineered
GFP and YFP (reviewed in ref. 27). Intracellular indicators of specific
kinase activity have been described using phosphorylation-activated
FRET sensors engineered from fluorescent protein pairs110, or for
insulin signaling via PIP3 recruitment of labeled Akt2 PH domains111.
Additionally, a cell-based HTS using BRET to detect CCR5-β arrestin
association identified inhibitors of CCR5-mediated signaling94.
FRET, BRET, PCA and similar methods have been combined with
subcellular imaging to produce highly successful and versatile HTS
assays for monitoring protein movements within cells9,110. The develop-
ment of technology platforms for automated cellular imaging, together
with sophisticated object recognition algorithms, has made the screen-
ing of cell-based protein redistribution assays routine8,9. For instance,
assays monitoring the redistribution of Akt1 (ref. 112), MAPK2 (ref.
113) and ERF1 (ref. 114) have been used for HTS. Indeed, MacDonald
et al.108 report the use of 49 PCAs to monitor protein redistribution or
interactions for profiling small-molecule activities.
Protein stability. The addition of degradation and destabilization
domains to reporter proteins has been useful for assaying a number
of cell processes (such as ubiquitin-mediated degradation and the cell
cycle) and enabling screening of unstable proteins such as the cis-act-
ing viral NS2/3 protease65. For pathways in which a particular protein
is controlled through degradation, destabilization domain GFP sensors
allow a measure of pathway activity. For instance, fusing the degrada-
tion and localization domains of cyclin B1 onto GFP conferred onto the
sensor the equivalent turnover and localization as endogenous cyclin
B1, thereby allowing high-content screening for cell cycle modulators8.
Similarly, fusion of the IκBα destabilization domain to luciferase was
used to identify modulators of NFκB signaling15. Also, coupling of

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the degradation domain of ornithine decarboxylase to the fluorescent
protein ZsGreen was used to discover new proteosome inhibitors115.
Fusing only the degradation domain of a particular protein, as opposed
to the entire protein, to a sensor is advantageous in that it minimizes
the pathway interference that can be caused by overexpression of the
full-length protein8.
Assays for rare disease targets. With over 1,800 genetic associations with
human disease now known116, the number of human diseases for which
molecularly based assays could be developed is large. Occasionally a
rare disease is caused by mutation of an immediately druggable tar-
get, and even more rarely one that may be ameliorated by a clinically
approved drug117. More commonly however, the genes responsible for
rare diseases have obscure or entirely unknown functions. Some com-
mon causes of human genetic diseases are listed below and illustrate
the challenge of assay development for HTS in this arena:
(i) Nonsense mutations resulting in premature termination and
absence of protein expression (for example, phenylketonuria, spinal
muscular atrophy (SMA) and cystic fibrosis): assays to identify com-
pounds that compensate for the defect, for example by upregulating
an alternative pathway or pseudogene as in SMA118, or promoting
readthrough of premature stop codons119.
(ii) Missense mutations resulting in a potentially functional but mis-
folded or mistrafficked protein (for example, cystic fibrosis): assay needs
to discover compounds that act as molecular chaperones to facilitate
correct folding and trafficking of the protein to its proper location120.
(iii) Synonymous mutation that introduces a new splice donor or
acceptor site, thereby altering splicing and producing an abnormal
protein (for example, Hutchinson-Gilford Progeria Syndrome and
β-thalassemia): assays to identify compounds that alter splicing121.
(iv) Motif expansion or duplication mutations that result in expres-
sion of a protein with toxic or dominant negative function (for example,
Huntington’s disease): assay needs to identify molecules that downregu-
late the expression of the toxic protein or interfere with its function122.
These urgent and challenging biological problems will be an impor-
tant frontier for HTS assay development in the next decade.
Assay optimization for HTS
A balance between statistical performance and assay sensitivity facili-
tates the identification of the broadest range of compound potencies
and efficacies from HTS. Assays are generally configured to measure one
or more of the following changes: (i) concentration of a substance (for
example, enzyme substrate or product, second messenger or reporter
gene product); (ii) concentration of a receptor-ligand complex or (iii)
distribution of cellular markers. In addition, different methodologies
measuring the same biological process can have discordant determi-
nations of apparent compound potency and efficacy37,123–125, which
emphasizes the need for selecting the proper assay format.
In general, four types of assays can be defined based on the acceptable
limits of Z' factor and assay sensitivity (Fig. 3a). Conditions leading
to satisfactory performance (Z' factor > 0.5) should be identified that
minimally impact assay sensitivity of a control ligand or stimulus (Fig.
3a, quadrant i). With this in mind, the optimal assay will not always
have the highest Z' factor, as some very robust assays (Z' scores > 0.8)
can have poor sensitivity (Fig. 3a, quadrant iii). Conversely, assays with
lower Z' factors may have superior sensitivity (for example, Fig. 3a,
quadrants i and ii) and may be rescued for HTS using replicate or
concentration-dependent formats126. Because potent compounds can
be identified in poorly designed assays, the irrational exuberance of
observing such high-potency or high-efficacy compounds is often dis-
placed by the realization that the activity was artifactual in nature, and
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a careful analysis of the screening results ensues to identify compounds
with reliable biological activity, many times with lower (µM) potency.
Thus, the need for assay sensitivity is critically important to identify
compounds with half-maximal inhibitory concentrations (IC50s) in
the vicinity of the screening concentration, as these are false-negative
candidates in assays having poor sensitivity.
Ideally, HTS assays are designed with substrate (S) or ligand (L) con-
centrations below their Km or Kd, so that the observed potency (IC50)
approximates the intrinsic potency Ki (see equations in Table 2; this
relationship differs for more complex mechanisms or when determin-
ing functional activity of receptors such as Kb values127–129). Though
increasing either [S] or [L] improves the overall Z' factor of the assay,
doing so could potentially increase the IC50 values above the threshold
used to identify actives in the screen (Fig. 3b,c), thereby preventing
them from being identified as positives. Catalytic systems (for example,
enzyme assays) should be designed for the minimum substrate con-
version needed to achieve an acceptable Z' factor. Although shifts in
IC50s can be less than two-fold for enzyme assays operating at conver-
sions upward of 80%130, high turnover significantly affects the ability
to detect inhibitors with potencies near the screening concentration
(Fig. 3d–f) and lowers the assay sensitivity for enzyme activators.
Many HTS technologies are inherently unable to detect low-affin-
ity complexes (Kd ~ 1 µM) without resorting to high ligand or tar-
get concentration, or engineering in a high-affinity interaction. The
various assays developed to screen for inhibitors of the protein-protein
interaction between the p53 tumor suppressor protein and its negative
regulator (DM2) illustrate these issues (see Supplementary Table 2
online for assay details). In one assay the measurement of a p53-DM2
interaction with a competition-binding FP assay131 required 1 µM DM2
to allow sufficient complex formation (~50% bound) for a detectable
signal. This configuration limits the observable compound potency
range, thereby rendering the assay unreceptive to weak compounds and
making it difficult to determine the rank order of potent compounds.
Conversely, high-affinity interactions resulting from enhanced avid-
ity can increase the apparent affinity of the binding partners many fold
through multiple receptor-ligand interactions. This “Velcro effect” is
evident as a rightward shift in IC50 values of competing control ligands.
For example, returning to the p53-DM2 interaction, Kane et al.132
developed a TR-FRET–based assay for this protein-protein interac-
tion that required only nanomolar protein concentrations, in contrast
to the micromolar level needed in the previous FP format. However,
enhanced affinity due to multivalent interactions in the resulting com-
plex (Supplementary Table 2) decreased the sensitivity of the assay as
judged by the higher-than-expected IC50 of unlabeled competitor p53
protein. The potential for such avidity effects through surreptitious poly-
valent interactions should always be considered carefully when examin-
ing the benefits and limitations of the assay133. Further, irrespective of
affinity, assay conditions requiring a high fraction of bound ligand (for
example, most FP-based assays) cannot rely on the Cheng-Prusoff cor-
rection to estimate the Kd or Ki of control or test compounds. Rather,
accurate approximations can be made using equations describing the
complete equilibria of the competing components134. In another bio-
chemical format, the thermal shift assay135, which measures a ligand’s
ability to stabilize a protein’s thermal denaturation, has been used to
investigate compound binding to the p53 binding domain of DM2
directly. Though thermal shift technology at present is relatively low-
throughput, requiring comparatively large amounts of target protein,
it is one of the few function- or ligand-independent methods to screen
for small molecule–protein interactions that may provide a means to
identify small molecules with protein complex disruption potential
(Supplementary Tables 1 and 2).
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a b c 12 1.0
1
100
10
0.8
i iii

Fold (IC50/Ki)
75 8 Z' factor

Inhibition (%)

Z' factor
0.6
Z' factor

0.5 6
50
0.4
4
ii iv
25 Fold
0.2
2

0 0.0
0 0
3 10 –6 –5 –4 0.2 0.4 0.6 0.8 1.0

Fold (IC50´/IC50) Log[inhibitor] (M) Fraction bound receptor

d e f
12 1.0 12 1.0
100
10 10
0.8 0.8
Z' factor Z' factor

Fold (IC50´/IC50)
Fold (IC50´/IC50)

75 8 8
Inhibition (%)

Z' factor
0.6 0.6

Z' factor
6 6
50
0.4 0.4
4 4
Fold Fold
25 0.2 0.2
2 2

0 0 0.0 0 0.0
–9 –8 –7 –6 –5 –4 0 25 50 75 100 0 25 50 75 100

Log[inhibitor] (M) Conversion (%) Conversion (%)

Figure 3 Assay performance and sensitivity. (a) Assay performance (Z' factor) is plotted against sensitivity, as defined by the ratio of observed (IC50’)
and known (IC50) values of a control compound. i, Optimal performance and sensitivity (Z' > 0.5, IC50’/IC50 < three-fold). ii, Poor performance and good
sensitivity (Z' < 0.5). iii, Good performance and poor sensitivity (Z' > 0.5; IC50’/IC50 > three-fold). iv, Poor performance and sensitivity (Z' < 0.5; IC50’/IC50
> three-fold). (b) Effect of varying the [ligand]/Kd ratio for a binding assay. Concentration-response (CR) curves represent [ligand]/Kd ratios as follows: 0.10,
blue; 1, green; 3, orange; 5, black; and 11, purple. IC50’ was calculated using the Cheng-Prusoff equation (Table 2), with a compound Ki = 5 µM and a
ligand Kd = 10 µM. The gray box is the “observation window” representing observed activity for a 10 µM screening concentration and a hit cutoff of >30%
inhibition. (c) A modeled assay in which between ~30% and 70% bound receptor yields optimal performance. (d) CR curves for an enzyme assay at different
substrate conversions (10%, red; 40%, blue; 60%, green; 80%, orange; 90%, black and 98%, purple) for two reversible inhibitors (solid lines, IC 50 = 100
nM; dotted lines, IC50 = 10 µM). The observation window (gray box) indicates how shifts in IC50’ limit the identification of weak inhibitors, including potent,
low-efficacy compounds (gray dotted curve). (e,f) Shifts in IC50’ were calculated as described130. Two modeled enzyme assays in which either an adequate
performance (for example, two-fold S:B and %CV < 5%) is achieved at 10% conversion (e) or the equivalent performance is not reached until ~60%
substrate conversion (f). Grey regions indicate areas of either poor assay performance or poor assay sensitivity (a, c, e and f).

Owing to recent advances in high-throughput cellular imaging9,136,
phenotypic assays now represent a viable alternative or complement to
biochemical formats for protein-protein interaction HTS assays. Several
cell-based assays have been used to investigate the p53-DM2 interac-
tion108,137, and these may circumvent the affinity and ‘enhanced avidity’
concerns encountered in systems configured with isolated proteins.
Reagent-coated surfaces provide both opportunities and drawbacks
for HTS assays. Formats involving reagent-coated beads or surfaces
enable a physical separation of a bound and free ligand, thereby elimi-
nating filtration steps41,133, or they provide components that comprise
the detection system, as in ALPHA138. However, two issues should be
considered when designing heterogeneous assays using solid supports.
One issue pertains to the “avidity” of the interaction mentioned above
that can occur in bead-based assays in which two binding partners are
coated or tethered to separate beads, which results in multiple binding
sites on the solid support (for example, the Velcro effect). The other
issue is the capacity of the solid support to ‘hold’ a reagent or substrate.
For example, when presenting an enzyme substrate on a solid support
the amount of substrate that can be coated onto a surface can limit the
reaction rate, and is often compensated by increasing enzyme concen-
tration to achieve a suitable signal139. The net result of increasing target
concentration is to increase assay cost and decrease sensitivity140.
Compound interference within HTS assays
Compound-dependent assay interference is a very common cause
of screening artifacts and must be ruled out before any compound
activity is attributed to modulating the biological activity of interest.
Here the inhibition or activation of an assay signal is not caused by the
compound’s effect on the target or pathway of interest, but rather it
is due to the compound’s ability to mimic that effect as a result of its
physicochemical properties (for example, light absorbance) or adventi-
tious biological activities (for example, luciferase inhibition)141,142 (Fig.
4). Cheminformatic approaches have been described that attempt to
identify or remove from screening collections compounds with such
problematic characteristics143, but currently prediction of such prop-
erties is not reliable, and each active compound must be assumed to
be an artifact until proven otherwise. This may be particularly true
for chemical genomics applications, as the allowed functionalities of a
chemical probe are less strict than for a medicinal lead. Thus although
the boundaries of a useful chemical series may be broader for chemical

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genomics applications than for drug development, the potential for
misleading HTS activity is perhaps even greater with ‘probe-like’ than
with ‘drug-like’ compounds.
Compound fluorescence and absorbance are very common causes
of signal amplification or attenuation. Heterocyclic compounds that
dominate screening collections most commonly fluoresce in the
blue-green range, meaning that common assay sensors, such as GFP
and coumarin, as well as the many profluorescent enzyme substrates
that excite or emit near the blue end of the spectrum, are susceptible
to this type of optical interference. Assay responses can differ in
signal strength or brightness and can determine assay sensitivity to
certain classes of interferences, such as attenuation of luminescence
or scintillation by light-absorbing compounds142. Signal brightness
becomes particularly important with regard to compound interfer-
ence when incident light is used to excite a fluorophore. In this case,
assays having emitted light signals that overlap with the fluorescence
from compounds will show increased false positives (Fig. 4a,b). The
amount of compound fluorescence is dependent on the wavelength
of excitation energy used, with blue light being more problematic
than red light for typical heterocyclic compound libraries142,144.

a b c

Excitation light

hν hν

Emission
light

Positives False positives

Figure 4 Compound effects on assay outputs. (a–c) The effect of different types of compound interference within a library (colored slices of the pie charts) on
the scoring of actives. Interfering compounds are represented as dark spots arranged as a shadow cast by assay output signal, and individual spots represent
genuine actives in assays using different modes of detection (blue or red fluorescence, or luminescence). In assays in which blue spectrum excitation is
used (a), significant interference from compound emission is possible, whereas for red-shifted excitation light (b), interference is typically reduced. Also
shown below are example fluorescent compounds 5 and 6, excited by blue and red light, respectively. (c) Luminescence assays do not use an external
excitation source, which results in negligible interference by compound fluorescence unless compounds or sample impurities absorb light (for example, inner
filter effect by 6). (d) Example molecules that illustrate different interference mechanisms. Biotin (1) analogs such as 1a disrupt biotin-avidin complexes
commonly used in assay designs (for example, PubChem AIDs 595 and 632). The imidazole (2) derivative 2a can coordinate with Ni2+ and interfere with
polyhistidine-tagged proteins at µM concentrations. Nickel-chelated microspheres are used in some instances to tether polyhistidine-tagged proteins (for
example, PubChem AIDs 595 and 632), and can thus be disrupted by compounds similar to 2a at typical screening concentrations. The luciferin (3) analog
3a is an inhibitor of the reporter luciferase. A major source of interference is compound aggregation (see 4), in which detergent-sensitive ‘promiscuous’
inhibitors block assays by forming target-sequestering/denaturing compound aggregates (4a)147.

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To provide experimental data on the fluorescent properties of the
compounds screened in our center, we recently performed spectro-
scopic profiling of ~70,000 compounds from the Molecular Libraries
Small Molecule Repository for eight common wavelengths (unpub-
lished data; PubChem AIDs 587–592). For example, 4-methylumbel-
liferone (4-MU; excitation 385 nm, emission 502 nm) is a commonly
used label in profluorescent substrates for a wide range of hydrolytic
enzymes. We found ~2% of the library fluoresced at a level equivalent to
at least 100 nM 4-MU, which indicates that a screen using this excitation
wavelength would yield many false positives via this mechanism.
Several approaches can minimize interference from fluorescent
compounds, and should be used whenever possible. Separation-based
formats such as ELISA offer an advantage over solution-phase assays,
as compounds are removed before the detection step. However, the
necessary separation steps can make ELISA difficult to miniaturize. For
homogeneous assays, compound fluorescence can be reduced by using
probes that use red-shifted fluorophores61, time-resolved lanthanide-
based fluorescence25,26, pre-reads or multiple time points that permit
background subtraction or rate determinations145. Technologies that
in principle are minimally affected by compound fluorescence, such as
fluorescence lifetime (FLT) spectroscopy146, are in development, but for
now remain unproven in HTS.
Signal attenuation or activation of assay signal can also be caused
by compound aggregation, which can result in target denaturation,
ligand sequestration or light scattering147 (Fig. 4c,d). Like the optical
properties discussed above, aggregation often occurs within the concen-
tration range of the expected biological response and so may be easily
mistaken for true modulation of the target biology. Aggregation effects
are most prevalent at concentrations >1 µM, the hallmark of which is
the elimination of apparent compound activity by the addition of deter-
gents such as Triton X-100, a disruptor of aggregate micellar structures.
Such compounds may show steep Hill slopes, but this alone does not
distinguish aggregation from biologically relevant, stoichiometric, or
cooperative modulation of enzyme activity. The prevalence of aggrega-
tion-dependent enzyme inhibition has been observed to range from 2
to 10%, depending on the concentration of detergent and the chemi-
cal library tested147; importantly, no clear structure-activity relation-
ship has been evident from these studies, making the establishment of
aggregation behavior currently entirely empirical. Results of a large
aggregation screen recently performed at our center can be found in
PubChem (AIDs 585 and 584).
Biological interferences are the second major source of compound-
specific assay artifacts (Fig. 4a). These are due to inhibition (or, less
frequently, activation) of a detection component of the assay such as
inhibitors of enzyme reporters (for example, luciferases or β-lactamases)
or intermolecular interactions (for example, nickel chelate–polyhisti-
dine tag, small-molecule antigen-antibody, biotin-streptavidin). As an
example, inhibition of luciferase by library compounds is common and
has been described for the well-publicized compound resveratrol148.
A recent screen of P. pyrali luciferase showed between 2 and 3% of the
library was capable of interfering with luciferase detection (unpub-
lished data; PubChem AID 411).
Summary
Given the right compound library, assay design becomes the determining
factor for identifying chemical probes using HTS. Before the selection
of an assay format for HTS, thorough consideration of its advantages,
limitations, and (when possible) prior application in HTS is recom-
mended. The investigator’s familiarity with specific technologies should
not form the basis of an assay strategy; rather, an unbiased assessment
of key factors discussed here will increase the probability of a successful
476
HTS assay. Assay sensitivity and susceptibility to compound interference
must also be studied prospectively when designing assays for HTS and
planning follow-up analysis of active samples.
In general, the signal output of an assay should be assessed in con-
junction with consequential controls. For example, protein-protein
interaction assays using purified proteins may have suitable statistical
performance factors but show poor sensitivity, which suggests that cel-
lular assays with phenotypic outputs should be considered as alterna-
tives. When investigating novel target biology, effort should be made
to leverage existing well-developed technologies where possible. For
instance, methods developed to detect ATP and ADP in protein kinase
assays48,51,53 are also useful for lipid49 and metabolic kinases126.
As HTS assay technologies, screening systems, and analytical instru-
mentation such as patch clamping and mass spectrometry evolve, the
interfacing of large compound libraries with sophisticated assay and
detection platforms will greatly expand the capability to identify chemi-
cal probes for the vast untapped biology encoded by genomes. The rami-
fications will allow a reinterpretation of the ‘druggable genome’.
Note: Supplementary information is available on the Nature Chemical Biology website.
ACKNOWLEDGMENTS
We thank D. Leja and J. Fekces for illustrations. The authors are supported by the
Molecular Libraries Initiative of the NIH Roadmap for Medical Research and the
Intramural Research Program of the National Human Genome Research Institute,
NIH.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturechemicalbiology
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Supplementary Table 1: Assay Technologies Additional Entries
Refs. Refs.
Technology Description
Technology Assays
AAS Atomic absorption spectroscopy for measurements of ion transport/flux (primarily 1, 2 3-7
focused on K+ channels with Rb+ as tracer); 96/384-well microtiter plates
enabled with, for example, the ICR 12000.

Absorbance Follows Beer's Law; trans or epi-absorbance; via fluorescence attenuation 8, 9 10

ALPHA Amplified luminescent proximity homogeneous assay; donor-acceptor beads of 11, 12 13-16
~ 200 nm dia.;λex 680 nm (laser); singlet oxygen reacts with acceptor bead within
~200 nm distance; λem 520-620 nm

Bioluminescence Renilla or firefly luciferases used in reporter gene assays; analogs of D-luciferin 17 18-27
used to expand biochemical assays for proteases, P450s, monoamine oxidases
28, 29 30-37
Bla/CCF4 FRET-based assay using β-lactamase and β-lactam-containing substrate (e.g.
CCF4); λex 395 nm, λem 460/530 nm; ratiometric reporter gene assays and other
formats.

BRET Bioluminescence resonance energy transfer; useful for protein-protein 38, 39 40

interactions; Renilla luciferase donor luminescence (480 nm) stimulates
fluorescence of GFP/YFP acceptor; follows dipole-dipole interaction 1/r6 (~ 5 nm
maximum)

DELFIA Dissociation-enhanced lanthanide fluorescent immunoassay/Time resolved 41, 42 43-49

fluorescence; ELISA-like format using lanthanide chelate-labeled antibody; τf ~
500 usec; separation–based enhanced fluorescence; variations employing
labeled ligands; extremely sensitive.

ECL Electrochemiluminescence assay. Uses micro-carbon electrode plates allowing 50 20

electrical oxidation of a Ru(phen)32+ label. Multiplex applications in ELISA
format.
 51, 52 53-58
EFC Enzyme fragment complementation. Based on β-galactosidase α-
complementation; High affinity complementation. Applied to both cell-free and
cell-based systems.

FCS / FIDA Fluorescence correlation spectroscopy / Fluorescence intensity distribution 59, 60 61-65
analysis; single molecule detection technology. Stochastic sampling of individual
labeled ligand fluorescence using confocal optics with illumination and detection
volume of ~1 femtoliter (fL), from a 1 uL sample volume. Enabled with the
Evotec Mark-II system.

FLT Fluorescent lifetime; time-domain method (~ns) measures the rate of fluorescent 66
decay following excitation; promises to eliminate fluorescence compound
interference; only recently applied to some microplate systems.
67, 68 69-78
FP Fluorescence polarization; polarized light reports change in probe ligand τc;
fluorophores of τf ~ 5 ns needed to measure ligand of MW < 1500 binding to a
receptor of MW ≥ 10,000; widely applied, ratiometric measurement.

FRET Fluorescent resonance energy transfer; donor-acceptor pair as in EDANS- 79, 80 81-84
DABCYL peptides or fluorescent proteins (e.g. CFP/YFP). Signal distance
dependence ~ 5 nm maximum.
(see also, Bla/CCF4)

Fluorescence Measurement of fluorescence intensity including the use of chromophores for 80, 85 86-97
quenching as in profluorescent protesase substrates (~ 5 nm maximum
distance); widely applied; superquenching varies in that non-equimolar amounts
of donor fluorophore and quencher are used. Includes kinetic modes of detection
with timescales ~ minutes to hours. Labeled antibodies extensively used in
imaging microscopy (see also ‘GFP and variants’ entry for references)

Fluorescence Requires fluorescent imaging plate reader or equivalent (e.g. FLIPR, FDS6000, 98, 99 99-108
VIPR); timescale ~ seconds; GPCR functional assays using Ca2+ sensitive dyes,
(rapid kinetic)
or steady-state ion channel activity using voltage sensitive dyes.
GFP and variants Cell-based imaging microscopy/cytometry assays; translocation, redistribution, 109-112 18, 110,
protein-protein interactions, reporter gene assays; HTS enabled with microplate-
113-118
compatible microscope-based imagers (e.q., ArrayScan, Opera, Pathway
BioImager, and INCell imagers) and cytometers (below).

HT High-throughput electrophysiology; measurement of ion channel currents with 119-122 123-127

microaperture arrays such as the planar patch-clamp electrode (e.g.,
Electrophysiology
PatchXpress 7000A, IonWorks Quattro). Transporter assays using solid
supported membranes (e.g., SURFE2R) (see also ion flux measurements via
AAS)
“Label Free” See following three entries
Spectrometry Mass spectrometry (e.g., RapidFire and SpeedScreen), high-field NMR (SAR by 128-131 132-135
NMR), fragment based methods using X-ray crystallography.

Optical Refractive index based. Measurement of ligand-binding using prism-coupled 136-138 139, 140
evanescent waves sensors, including surface plasmon resonance (SPR),
resonant waveguide grating (RWG), and resonant mirrors. kon and koff measured.
(e.g., Biacore, Plasmon Imager, IBIS, IAsys, Epic, Octet System). 384-well
microplate-based assays enabled with Epic system.

Non-optical Includes measurements of electrical impedance, quartz crystal acoustic 141-146 147-151
resonance, heat of binding /unfolding, ∆H°, and use of microcantilevers. Systems
for measuring electrical impedance (e.g., RT-CES) are particularly useful for cell-
based assays, and piezoelectric quartz crystal-based resonant acoustic profiling
(e.g., RAP♦id 4), isothermal (e.g. AutoITC) and differential scanning calorimetry
(e.g., VP-Capillary DSC System, MiDiCal) have generally been applied to
measure molecular interactions and enzyme reactions.

Laser Cytometry See following three entries

Acumen-enabled Commonly referred to as ‘Laser-scanning fluorescence microplate cytometry’ 152 153
Based on optical thresholding of fluorescence signal from cellular fluorescence
compared to background fluorescence. Used with adherent or settled cells,
suitable for microplate-based assays. Enabled with the Acumen Explorer eX3
(405, 488, and 633 nm laser excitation).
 FMAT-enabled Fluorometric microvolume assay technology; also known as, Laser-scanning 154, 155 43, 156-162
imaging or laser-scanning cytometry. Relies on ‘macro-confocal’ optical
discrimination of fluorescence signal from accumulation of fluorescent ligands on
a receptor-containing cell or particle over that of unbound ligand in an equivalent
area of surrounding buffer. Used with adherent cells or settled beads, suitable
for microplate-based assays. Enabled with the 8200 Cellular Detection System,
formerly known as the FMAT (633 nm HeNe laser excitation).

Flow-based Flow cytometry detection using laser-induced fluorescence or light scatter from 163-165 166-168
individual cells or beads traveling sequentially in a hydrodynamically focused
laminar flow. The optical configuration permits limited excitation of sample buffer
surrounding the cell only, thus reducing the background signal from, for example,
unbound fluorescent ligand. Dye-encoded beads allow high level of multiplexing.

PCA Protein complementation assay; recombinant split reporters (e.g., GFP, 169, 170 153, 171,
luciferase, β-lactamase) fused to interacting proteins; phenotypic assays; low 172
affinity complementation.
SPA, FlashPlate Scintillation proximity assay; non-separation based radiometric detection using 173 174-186
solid supports; typical isotopes include 35S, 33P or 3H

ThermoFluor Temperature-dependent protein unfolding is monitored using environmentally 187 188

sensitive fluorescent probes that interact specifically with nonnative or unfolded
exposed hydrophobic regions of the target protein. Compounds that bind and
stabilize the protein will shift melting temperature, Tm, to a higher temperature,
destabilizing substances lower the Tm.

TR-FRET Time resolved-FRET; also known commercially as HTRF/LANCE; non- 41, 42, 79 44, 189-201
separation based format employing a lanthanide cryptate donor fluorophore of
long fluorescence lifetime ( τf ~ 500 usec); detections distance ~ 9 nm max.

NOTES: τf = fluorescent lifetime.τc = rotational correlation time.

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Supplementary Table 2. Assays developed to measure the p53-DM2 interaction

Format Technology Components Conditions / Comment Ref:

p53 DM2
Competition FP FITC-[15-30]p53 peptidea GST-[9-149]DM2 • 15 nM FITC peptide 1
Binding (Kd, app =2 uM); low • 1 uM DM2
affinity peptide • 384-well plates
FITC-11, 11 amino acid • Non-Langmuir binding
phage peptide (Kd, app =1
uM)
Competition FP 6-FAM, 6 amino acid Trx-His6-factor Xa • 1 nM FAM peptide 2
Binding p53-derivedb peptide (Kd cleavage site-[17- • 10 nM DM2
= 2 nM) 125]DM2 • 384-well plates
• Non-Langmuir binding.
• Cubic equation to determine Kic
Competition TR-FRET biotin-trx-[1-83]p53; GST-[1-188]DM2; • 10 nM p53 3
Binding streptavidin (SA)-XL665 Eu(K)-anti-GST • 5 nM DM2
• 100 nM SA-XL665,
• 20K B-counts Eu(K)-anti-GST
• 96-well plates
• 35-fold rightward shift in unlabeled
[1-83]p53 IC50d
Phenotypic ‘GRIP’ Protein EGFP-[1-312]p53 PDE4A4-[1-124]DM2 • Internal system controls and Nutlin- 4
distributione 3 assay positive controls.f
• 384-well plates
Phenotypic PCA p53-Venus[2] Venus[1]-DM2 • Transient transfection 5
• 96-well plates

Direct Tm shift not applicable Glycine-[17-125]DM2 • 10 uM DM2 6

Binding ‘ThermoFluor’ • 100 uM fluorescent probe, Dapoxyl
sulfonic acid
• 384-well plates
• FP assay used to confirm actives
Notes:

a. Sequence of 15 amino acid p53 peptide = SQETFSDLWKLLPENN; phage display peptide= SFTDYWRDLEQ
b. Ac-FR-Dpr(6FAM)-Ac6c-(6-Br)WEEL-NH2
c. Uses the physiologically relevant cubic equation to determine Ki values in competition experiment under concentration depletion conditions of
the three starting species (e.g., non-Langmuir binding conditions).
d. Enhanced complex avidity, presumably multivalent interactions.
e. GRIP is an acronym for ‘Green fluorescent protein-assisted readout for interacting proteins.’
f. System controls: RS25344 induces localization of PDE4A4 into compact foci; RP73401 mediates foci dispersion.

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