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Cite This: ACS Chem. Biol. 2018, 13, 2758−2770 pubs.acs.org/acschemicalbiology

Quantitative Live-Cell Kinetic Degradation and Mechanistic Profiling

of PROTAC Mode of Action
Kristin M. Riching,† Sarah Mahan,† Cesear R. Corona,‡ Mark McDougall,‡ James D. Vasta,†
Matthew B. Robers,† Marjeta Urh,† and Danette L. Daniels*,†
†
Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States
‡
Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States
*
S Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: A new generation of heterobifunctional small

molecules, termed proteolysis targeting chimeras (PROTACs),
targets proteins for degradation through recruitment to E3 ligases
Downloaded via 83.132.123.215 on September 20, 2019 at 14:51:11 (UTC).

and holds significant therapeutic potential. Despite numerous

successful examples, PROTAC small molecule development remains
laborious and unpredictable, involving testing compounds for end-
point degradation activity at fixed times and concentrations without
resolving or optimizing for the important biological steps required
for the process. Given the complexity of the ubiquitin proteasomal
pathway, technologies that enable real-time characterization of
PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving
guidance of chemical structure−activity relationship. Here, we present an innovative, modular live-cell platform utilizing
endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-
terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely
quantifying degradation rates, maximal levels of degradation (Dmax), and time frame at Dmax. These degradation metrics show
specific PROTAC and family member-dependent responses that are closely associated with the key cellular protein interactions
required for the process. Kinetic studies show cellular ternary complex stability influences potency and degradation efficacy.
Meanwhile, the level of ubiquitination is highly correlated to degradation rate, indicating ubiquitination stemming from
productive ternary complex formation is the main driver of the degradation rate. The approaches applied here highlight the
steps at which the choice of E3 ligase handle can elicit different outcomes and discern individual parameters required for
degradation, ultimately enabling chemical design strategies and rank ordering of potential therapeutic compounds.

T argeting key drivers of disease for loss or degradation has

been a desirable outcome for numerous therapeutic
treatments and is often achieved indirectly by modulation of
upstream signaling pathways, transcriptional programs, and/or
epigenetic events.1−3 The first examples of compounds
affecting degradation were demonstrated many years ago
using small molecules or peptides to bridge interactions
between a target protein with components of the ubiquitin
proteasomal system (UPS).4−8 More recently, the approach
has been refined for efficacy and efficiency, comprising a
heterobifunctional compound termed proteolysis targeting
chimera (PROTAC), also known as SNIPER or degrono-
mid.2−6,9−15 Chemically, these compounds consist of a small
molecule target binder or inhibitor on one side which is
chemically linked to an E3 ligase recruiter compound on the
other side1,2 (Figure 1A). PROTACs induce degradation by
simultaneously binding the target protein and the E3 ligase
complex proteins, bringing the target protein into proximity for
ubiquitination and targeting it for degradation through the
UPS1,2 (Figure 1A). While there are hundreds of E3 ligases in
eukaryotes and hundreds more proteins involved in active E3
ligase complexes, very few have known binding compounds
which could be used as recruiter molecules for PROTAC
design.4,5,12,14,16−20 Of those, currently only two proteins, von
Hippel Lindau (VHL) and Cereblon (CRBN), both of which
are substrate adaptor components of larger E3 Cullin-RING
complexes, have shown significant and broad success against a
diverse set of targets.14,17,21−31
Chemical conversion of previously characterized inhibitors
to PROTAC degradation compounds have shown therapeutic
advantages, including improved potency and prolonged
pharmacodynamics,14,21,23,24,28,30,32−35 and in some cases
have unexpectedly introduced specificity not observed in the
parental pan-selective inhibitor.26,30,36,37 Despite these early
successes, multiple challenges still exist in the functional
characterization of PROTACs, including assessment of cellular
permeability, ternary complex formation (target:PROTAC:E3
ligase component), functional ubiquitination, and degradation.
To optimize the likelihood of finding an active compound, an
extensive chemical series is typically developed with two main
Received: July 25, 2018
Accepted: August 23, 2018
Published: August 23, 2018

© 2018 American Chemical Society 2758 DOI: 10.1021/acschembio.8b00692

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Figure 1. Generation of HiBiT-tagged endogenous BET family members. (A) Schematic showing strategy for monitoring PROTAC-mediated
degradation of endogenously tagged HiBiT-BET family in live cells. HiBiT was inserted via CRISPR/Cas9 to the N-terminus of BET family
proteins BRD2, BRD3, and BRD4 in a HEK293 cell line stably expressing LgBiT, which complements with HiBiT to form the luminescent
NanoBiT luciferase. Following treatment with PROTACs, protein degradation is monitored by the loss of luminescence. Ternary complex
formation or ubiquitination is measured with NanoBRET using the HiBiT-BET protein complemented with LgBiT as an energy donor and
HaloTag fused to an E3 ligase component or ubiquitin as the respective energy acceptor. (B) HiBiT blot of CRISPR edited clonal cell lines
expressing endogenous HiBiT-BRD2, HiBiT-BRD3, or HiBiT-BRD4. Single isoforms of BRD2 and BRD3 were found, while both short and long
isoforms of BRD4 were detected. (C) PROTAC-induced degradation of HiBiT-BET family members after 4 h treatment with 1 μM dBET1 or
MZ1. Data are represented as mean RLU values (n = 3) of representative experiments. (D) Bioluminescence imaging using an Olympus LV200
microscope of endogenously tagged HiBiT-BRD4 in LgBiT expressing HEK293 cells treated with 1 μM MZ1 for 2 h.

variables: linker composition and the E3 ligase complex
recruitment handle.2−4,12,16,19 Compounds are then screened
for function by assessing target protein levels ± PROTAC
treatment at a given time and various concentrations using
end-point techniques such as Western blots or mass
spectrometry. These approaches, however, cannot be done in
live cells, lack adaptability for high-throughput screening, and
are difficult to perform in a quantitative fashion. More
importantly, if degradation is not observed in these measure-
ments, they provide no information as to the point of failure or
which steps in the process could benefit by compound
optimization through further structure−activity relationship
(SAR) efforts.
In this study, we present a modular live-cell platform to
monitor the critical steps of PROTAC mode of action (Figure
1A). At the core of the platform, we combine CRISPR/
Cas938,39 endogenous tagging and luminescent technology40,41
to kinetically measure target protein levels with high precision
and without disruption of native expression levels or
transcriptional regulation (Figure 1A). Coupling this technol-
ogy with optimized bioluminescence resonance energy transfer
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(NanoBRET)42,43 allows for kinetic measurements of intra-
cellular protein interactions along the degradation pathway
such as ternary complex formation, ubiquitination, and
PROTAC-target engagement (Figure 1A). In these studies
we apply this approach to characterize the cellular mechanism
of action of two PROTACs, dBET1 and MZ1, which were
shown to degrade bromodomain and extra-terminal (BET)
family members BRD2, BRD3, and BRD4. 14,30 Both
compounds contain the pan-BET inhibitor JQ1,44 serving as
the target-recruiting moiety, conjugated via an amide bond on
the same attachment point.14,30 Similar to other BET family
PROTACs, both MZ1 and dBET1 demonstrate greater
potency as degradation compounds relative to parental BET
inhibitors.14,17,30,32,34,35,44−46 These two PROTACs were
chosen for this study due to availability, the use of the same
JQ1 ligand with identical conjugation but to different E3
handles, and because they are reported to have differential
degradation patterns.14,30,37 dBET1, which recruits CRBN
through a thalidomide handle, shows equivalent degradation
between BRD2, BRD3, and BRD4.14 Recent structural studies
with related variants of dBET1 show noncooperative binding
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Figure 2. BET family member kinetic degradation profiles and quantitation of degradation parameters. (A) HEK293 cells containing endogenously
tagged HiBiT-BET family member and coexpressing LgBiT were treated with an 8-point concentration series of 1 μM dBET1 or MZ1 added at t =
0. Luminescence (RLU) was continuously monitored in 5 min intervals over a 24 h time period in the presence of a stabilized furimazine substrate,
Endurazine. Profiles are plotted as mean fractional RLU values by normalizing to DMSO control. Error bars are represented as SEM of n = 3
experiments. (B) Representative schematic and equations to determine degradation parameters: degradation rate, Dmax and time at Dmax. Summary
of all degradation parameters tabulated for each BET family member treated with the various concentrations of dBET1 and MZ1 from kinetic
degradation profiles in (A). The initial degradation portion of each concentration curve was fit using GraphPad Prism to a one-component
exponential decay model (red line), and best fit parameters ƛ (degradation rate) and plateau (minimum remaining fraction) were obtained. The
maximum degraded fraction, Dmax, was calculated as 1− plateau. Time at Dmax was defined as the amount of time during which protein levels
remained below the threshold plateau + 10% of Dmax. (C) Degradation rate and (D) Dmax values expressed as percent degradation from (B) plotted
against PROTAC concentration for each family member and PROTAC. (D) DC50 values were obtained by fitting Dmax values to a variable slope
dose−response model in GraphPad Prism. Variability is expressed as SD of the mean from n = 3 experiments. (E) Time at Dmax, as determined by
the formula in (B), for HiBiT-BET family members treated with 1 μM dBET1 or MZ1 PROTAC. Data are represented in singlicate taken from the
combined (n = 3) kinetic traces.

between BET family members with CRBN.47 In contrast,
MZ1, which recruits VHL through a VHL ligand handle,48
shows preferential degradation of BRD4.30 Supporting
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structural and biophysical studies indicate cooperative binding
between VHL and a bromodomain of BRD4 in the presence of
MZ1.37 These structural and biochemical studies yield
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considerable and meaningful insights into the stability and
formation of ternary complexes in vitro but are done with
isolated BET bromodomains and limited components of the
respective multiprotein E3 ligase complexes.37,47 Full-length
BET family members are large proteins, however, each
contains two bromodomains capable of separately binding
compound.44,45 Therefore, in the cell, positioning of the family
members within the E3 ligase complexes could be very
different, potentially leading to critical differences in
degradation activities as well. Here, we complement the
biochemical work and provide additional live-cell context by
characterizing the kinetic mechanistic differences mediated via
the two possible E3 ligase ternary complexes as well as further
understanding of family member specificities. Significant
insights into the degradation process and differential modes
of action of MZ1 and dBET1 are revealed and also show how
these technologies could be applied broadly to enhance
understanding of any PROTAC or process which impacts
protein homeostasis.
■ RESULTS AND DISCUSSION
Generation of Endogenously Tagged HiBiT-BET
Family Members. Protein overexpression can impart defects
on normal protein turnover and homeostasis; therefore, we
developed a means to monitor protein levels in real-time
without disruption of endogenous expression or regulation. To
this end, we used CRISPR/Cas9 genome editing38,39 to
append an 11 amino acid peptide, termed HiBiT,41 to the N-
terminus of BET family members natively expressed in
HEK293 cells, BRD2, BRD3, and BRD4 (Figure 1A and
Supporting Information Table 1). The HiBiT peptide, which
shows high efficiency for CRISPR insertion given its small size,
has pM affinity for and spontaneously complements with the
18 kDa LgBiT protein, forming the luminescent and bright
NanoBiT luciferase41 (Figure 1A). LgBiT can be introduced
for luminescent detection of HiBiT fusion proteins in a variety
of ways, but to enable live cell detection, we performed
CRISPR endogenous tagging in HEK293 cells stably
expressing LgBiT. CRISPR clonal selection was performed,
and genomic analysis by Sanger sequencing revealed
homozygous HiBiT allelic insertions for BRD3 and BRD4,
and a heterozygous insertion for BRD2 (data not shown).
Confirmation of size and expression for each HiBiT-BET
family member was confirmed by SDS-PAGE with luminescent
blotting as described in the Materials and Methods. Single
bands for BRD2 and BRD3 were observed, while both long
and short isoforms of BRD4 were detected, as expected given
the placement of HiBiT on the N-terminus (Figure 1B).
Comparative luminescence expression levels of each family
member showed similar expression levels of BRD2 and BRD4,
while expression of BRD3 was approximately 60% lower
relative to these (Figure 1C and Supporting Information Table
2). End-point analysis of each endogenously tagged HIBiT-
BET family protein treated with 1 μM dBET1 or MZ1 showed
substantial degradation at 4 h (Figure 1C). To show specificity
of degradation, HiBiT-BRD4 cells treated with either dBET1
or MZ1 for 3 h were then treated with a 10-fold molar excess
of JQ1 (Supporting Information Figure 1). This resulted in
increased rates of recovery of BRD4 in the presence of JQ1,
the parental competitive binding inhibitor which does not
induce degradation (Supporting Information Figure 1). To
assess proper localization and response to PROTAC treatment,
bioluminescence imaging using an Olympus LV200 micro-
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scope showed highly uniform expression, nuclear localization,
and rapid degradation of HiBiT-BRD4, as evidenced by loss of
luminescence over 2 h in the presence of 1 μM MZ1 (Figure
1D). Together, these data show expected physiological and
PROTAC responses of the endogenously tagged HiBiT-BET
proteins, indicating insertion of the HiBiT tag at their
respective genomic loci did not impact function.
Determination of Degradation Profiles and Quanti-
fication of Key Degradation Parameters. To investigate
the time-dependent effects of PROTAC-mediated degradation
on each BET family member, comprehensive degradation
profiles of biological triplicates were generated by monitoring
luminescence in 5 min continuous intervals over a 24 h period
(Figure 2A). Treatment of HiBiT-BRD2, BRD3, and BRD4
with an 8-point dilution series ranging from 1 nM to1 μM
dBET1 or MZ1 showed this concentration window resulted in
a broad range of responses from very little degradation to near
complete degradation as detected by luminescence (Figure
2A). Strikingly, each family member showed a unique
signature, which was both PROTAC- and concentration-
dependent (Figure 2A). The degradation profiles in general
contained three phases: an initial degradation curve showing
loss of the target, a Dmax, and then an eventual upward rise
indicating target recovery. Recovery curves were highly
variable, often multiphasic, and not quantifiable. Fortunately,
other parameters such as initial degradation rate, Dmax, and
time at Dmax as depicted in Figure 2B were all amenable for
quantitation from these profiles.
To determine degradation rate and D max at each
concentration, only the starting curve representing the time
frame to maximal degradation was considered, as defined in
Figure 2B. This initial curve fit a single-component exponential
decay model, yielding parameters for cellular degradation rate
and Dmax for concentrations of each PROTAC and BET family
member summarized in Figure 2B. Trends showed that
increasing concentration of PROTAC increased rate of
degradation, eventually reaching a plateau at higher concen-
trations (Figure 2C). This trend indicates that beyond the
plateau, concentration of compound is not rate-limiting.
Moreover, it is expected that very high concentrations will
become inhibitory, consistent with observed hook effects for
PROTACs17,30 Degradation rate plots revealed emergence of
family member differences, particularly for MZ1 treatment,
which showed fast degradation of BRD2 and BRD4, while
BRD3 exhibited much slower degradation (Figure 2C). Such
family differences were not observed with dBET1, which
showed similar rates of degradation for BRD2, BRD3, and
BRD4 (Figure 2C). These results demonstrate how not only
PROTAC-mediated recruitment to either VHL or CRBN E3
ligase complexes can impact the degradation rate, but also how
different family members can show varying rates to the same
PROTAC (Figure 2C).
Despite having differences in the initial degradation rate,
family members were able to achieve similar Dmax if given
enough time, and total amount of degradation increased with
increasing concentrations of PROTAC (Figure 2A and B).
Previous dose response curves (DRCs) to determine cellular
DC50 potency of PROTAC compounds have calculated
degradation at fixed time points,14,30,37,47 overlooking the
time dependency to reach Dmax (Figure 2A). We find that for
any given family member at any concentration, the time at
which Dmax is reached varies greatly at each concentration
(Figure 2A). Therefore, DRCs at fixed times could easily
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misrepresent DC50 potency values depending on the time
chosen for the measurement. For example, measuring
degradation of BRD2 at 2 h would show either PROTAC is
potent for degradation, but at 20 h it would appear that neither
PROTAC had any effect, because degradation had already
occurred and transitioned to recovery (Figure 2A). With these
understandings and to remove the impact of time, we sought a
more accurate method to calculate DC50 values by determining
Dmax at every concentration irrespective of time (Figure 2B and
D). This approach reflects and takes into account the actual
cellular response and the true capacity for degradation.
Comparison of DC50 values calculated in this fashion allowed
for rank ordering potency, with MZ1 showing BRD4 > BRD3
> BRD2 and with dBET1, BRD3 > BRD4 > BRD2 (Figure
2D). The window of family member potencies exhibited a
broader range with MZ1 (9X) as compared to dBET1 (4X),
but significant differences in degradation capabilities only
emerged at very low concentrations of PROTAC (Figure 2D),
matching previous studies with the endogenous proteins
analyzed by Western blotting.30
As other PROTAC cellular studies have utilized ectopic
expression of fluorescent proteins to determine compound
degradation potencies,47 we wanted to examine whether this
approach influenced the kinetic degradation rate and Dmax. To
do so, comparative experiments were performed with Nano-
Luc40 (the parental luciferase of the HiBiT+LgBiT comple-
mentation technology) fused to BRD4 and transiently
transfected in HEK293 cells. In comparison to the profile for
endogenous HiBiT-BRD4 treated with MZ1 in Figure 2A,
ectopically expressed NanoLuc-BRD4 treated with MZ1 shows
a significantly different degradation signature (Supporting
Information Figure 2A). Overall, the degradation window was
compressed, showing only a small population of the NanoLuc-
BRD4 was degraded, even at high concentrations of MZ1
(Supporting Information Figure 2A). Determination of the
degradation rate showed degradation of the ectopic NanoLuc-
BRD4 was slower (Supporting Information Figure 2B), and
the DC50 showed a 30× reduction in apparent potency
(Supporting Information Figure 2C). Moreover, the profiles
showed more rapid protein recovery, not matching the
prolonged time at Dmax observed for endogenously tagged
BRD4 (Figure 2A). Because constitutive expression from a
non-native promoter lacks the relevant epigenetic and
transcriptional control found at the endogenous target loci
and ectopic expression will produce excess target protein levels
in the cells, degradation profiles cannot be meaningfully
interpreted (Supporting Information Figure 2A), and quanti-
tative parameters will be skewed (Supporting Information
Figures 2B and C).
While the degradation rate and DC50 values are readouts of
efficiency and potency, they do not capture the time at Dmax in
the cells, which speaks to the time of sustained compound
efficacy. To quantify this time frame, we defined the time at
Dmax as the period in which protein levels were within a
threshold window of 10% of Dmax: (plateau + 0.1(Dmax))
(Figure 2B). Calculation of time at Dmax in this fashion showed
this parameter increased with increasing concentration of
PROTAC, with a few outliers were observed at the extremely
low levels of degradation where the threshold window
approached the level of experimental noise (Figure 2B).
From this analysis, we found that MZ1 treatment showed
longer times at Dmax for all BET family members as compared
to dBET1 treatment at 1 μM (Figure 2E), with similar trends
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found at both 100 nM and 10 nM treatments (Supporting
Information Figures 3A and B). Furthermore, the time at Dmax
for each family member followed the trends in family member
potency for a particular PROTAC (Figure 2D and E). For
example, BRD4 showed greatest MZ1 potency and greatest
time at Dmax, while BRD2 had lowest MZ1 potency and
shortest time at Dmax, despite having a fast initial degradation
rate (Figure 2C, D, and E).
These data suggest many factors play a role in time at Dmax
and recovery. The finding that BET family members show
longer times at Dmax after treatment with MZ1 as compared to
dBET1 (Figures 2A, B, and E and Supporting Information
Figure 3) support previous reports of reduced cellular
compound stability of dBET1.14 The differential family
member response however to the same PROTAC (Figure
2A, B, and E and Supporting Information Figure 3) indicate
other events are impacting these phases. This is most apparent
for BRD2, which shows faster recovery as compared to BRD3
and BRD4 (Figure 2A); therefore, we investigated whether
inhibition alone of BET proteins could lead to compensatory
changes in BRD2 protein levels. To do this, we performed
kinetic analysis of HiBiT-BRD2 after treatment with JQ1
alone. In these studies, we saw a rapid and significant increase
in HiBiT-BRD2 levels (Supporting Information Figure 4A),
not observed for HiBiT-BRD3 and HiBiT-BRD4 (Supporting
Information Figures 4B and C) similarly treated with JQ1,
indicating potential feedback on BRD2 synthesis as a
consequence of BET inhibition. These results and others
that have shown a similar effect on endogenous BRD2 by mass
spectrometry49 suggest that the rapid increase in BRD2
following BET family degradation may be due to compensa-
tory feedback mechanisms, and demonstrate how these can
outcompete degradation to drive faster recovery of specific
family members over others.
Kinetic Monitoring of Ternary Complex Formation
and Ubiquitination. To relate the degradation profiles to the
specific interactions in the degradation pathway, we inves-
tigated the kinetics of intracellular ternary complex formation.
To do so, we utilized proximity-based NanoBRET technol-
ogy42 comprising the same endogenously tagged HiBiT-BET
family members complemented with LgBiT as luminescent
energy donors, and ectopically expressed HaloTag-CRBN or
HaloTag-VHL fusions as energy acceptors. (Figure 1A). Due
to its ratiometric nature,42 NanoBRET is particularly suited for
these studies as loss of the donor (target protein) will not
impact the readout of ternary complex formation (Supporting
Information Figure 5), and is capable of detecting transient
interactions. To first demonstrate ternary complex specificity
by NanoBRET, we examined ternary complex formation of
ectopically expressed NanoLuc-BRD4 with either HaloTag-
VHL or HaloTag-CRBN in the presence of either PROTAC.
Only the specific pairings of NanoLuc-BRD4 with the
respective E3 ligases and PROTACs, showed a dose-depend-
ent increase in ternary complex formation, as measured by
NanoBRET (Supporting Information Figure 5A). While
detection of ternary complexes within short periods of time
(30−60 min) was possible, longer term kinetic analyses were
challenging due to the significant loss of the target proteins. To
mitigate this loss and study the ternary complex stability over
several hours, MG132, a proteasomal inhibitor, was added to
prevent degradation, and resulted in a more robust assay
window (Supporting Information Figures 5B and C). Because
MG132 can be toxic to cells, we tested cell viability by
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Figure 3. NanoBRET ternary complex formation and ubiquitination of BET family members. (A and B) HEK293 cells containing endogenously
tagged HiBiT-BET family members and expressing LgBiT were transiently transfected with either HaloTag-VHL or HaloTag-CRBN (A) or
HaloTag-Ubiquitin (B), which encodes for the first ubiquitin repeat, amino acids 1−76, of the polyubiquitin-B precursor protein. Forty-eight hour
post-transfection, NanoBRET ratios were calculated to measure intracellular ternary complex formation (A) or target ubiquitination (B) in the
presence of 1 μM dBET1 or MZ1 (t = 0). Kinetic monitoring of NanoBRET was performed for 6 (A) or 2 (B) h in the presence of both stabilized
furimazine substrate, Vivazine, and NanoBRET618 fluorescent ligand. Experiments were also set up for all assays without NanoBRET618
fluorescent ligand as a control and allowed for subtraction of background within the NanoBRET assay, as detailed in the Materials and Methods.
Data are represented as fold increase in NanoBRET by normalizing to DMSO control. Variability expressed as SEM from n = 3 experiments. (C)
HEK293 cells containing endogenously tagged HiBiT-BET family members and expressing LgBiT were treated at the indicated times with 1 μM
dBET1 or MZ1 PROTACs. Cells were lysed with digitonin and incubated for 10 min with both primary polyclonal anti-Ubiquitin and Alexa-594
fluorescent secondary antibody to determine NanoBRET ratios. Data are represented as fold increase in NanoBRET by normalizing to t = 0 time
point. Variability expressed as SEM from n = 3 experiments. (D) Degradation rate at 1 μM PROTAC from Figure 2C is plotted against
ubiquitination fold increase at the 1 h time point shown in (C).

CellTiter-Glo after 10 μM MG132 treatment over 6 h and
found no impact on cell number (Supporting Information
Figure 5D). We then performed kinetic analysis of ternary
complex formation in the presence of MG132 for 6 h with
endogenously tagged HiBiT-BET family members comple-
mented with LgBiT. Complexes between all BET family
members and CRBN in the presence of dBET1 showed very
rapid association kinetics, peaking at 1 h and then either
holding steady or declining (Figure 3A). In contrast,
complexes of BET family members with VHL and MZ1
exhibited slower association kinetics, yet continued to increase
over time showing longer complex stability (Figure 3A). The
prolonged stability of the ternary complexes mediated by MZ1
compared to dBET1 for all BET family members agreed well
with the trend that MZ1 treatment resulted in longer time at
Dmax (Figures 2A and E), indicating ternary complex stability
directly influenced the duration of degradation efficacy.
Because our readout of ternary complex formation is only a
measurement of binding and not activity of the complex, we
measured downstream target ubiquitination. Intracellular
ubiquitination kinetics were measured by NanoBRET using
the same HiBiT-BET family members as donors, but paired
with HaloTag-Ubiquitin as an acceptor. Robust and rapid
ubiquitination of BRD2 and BRD4 was observed with MZ1
treatment, while BRD3 showed both slower kinetics and
reduced ubiquitination levels (Figure 3B). For BET family
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members treated with dBET1, ubiquitination signals were
similar in fold change over time and in general slower as
compared with MZ1 treatments (Figure 3B). As an orthogonal
approach, NanoBRET was also used to probe endogenous
ubiquitination of HiBiT-BET family members, utilizing a
polyclonal ubiquitin primary antibody and Alexa594-con-
jugated secondary antibody as an acceptor. These data show
identical trends to those observed in the live-cell kinetic
experiments with HaloTag-Ubiquitin (Figure 3C). The
different NanoBRET ratios obtained for the same target
treated with either MZ1 or dBET1 indicate that ubiquitination,
either in pattern or extent, is different dependent on the E3
ligase complex to which it is recruited. The degradation rates
determined in Figure 2C show striking similarity to the trends
in ubiquitination for respective family members and
PROTACs. Plotting degradation rates against the relative
increase in ubiquitination at the same concentration of
PROTAC revealed a positive and linear correlation, indicating
the step of ubiquitination is the predictive step of degradation
and controls the rate (Figure 3D).
Lytic and Live Cell Target Engagement and Cell
Permeability Assessment. Given the large molecular size of
heterobifunctional degraders, a significant step in the process
of chemical optimization is characterization of cell perme-
ability, target occupancy, and binding affinities to both the
target and E3 ligase complex. To facilitate these studies, we
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wanted to transition from monitoring protein:protein inter-
actions to protein:small molecule interactions. To this end, we
applied NanoBRET technology using ectopic NanoLuc fusions
as donors and fluorescently labeled small molecule tracers as
acceptors.43 This approach interrogates the separate events of
PROTAC binding to either target or E3 ligase component
(Figure 4A), with a key advantage in that it can be performed
in both live cell or lysate formats to assess permeability.43
Competitive displacement of the VHL tracer molecule
revealed identical binding affinities between MZ1 and a related
potent monovalent VHL binder, VH298,48 in lytic format
(Figure 4B), in agreement with biophysical measurements in
vitro which showed identical binding affinity with recombinant
Figure 4. NanoBRET target engagement with PROTAC compounds.
(A) Schematic showing NanoBRET target engagement strategy with
either E3 ligase component or BRD4 using NanoLuc fusions as
energy donors and fluorescent small molecule tracers as energy
acceptors, which can be competitively displaced by unlabeled
compounds. (B and C) Competitive displacement profiles of
HEK293 cells transiently transfected with NanoLuc-VHL and
incubated with the VHL fluorescent tracer in the presence of serial
titrations of either MZ1 or VH298 in digitonin-permeabilized lysate
(B) or live cells (C). (D) Live cell competitive displacement profiles
of HEK293 cells transiently transfected with NanoLuc-BRD4 and
incubated with the BRD4 fluorescent tracer in the presence of serial
titrations of either MZ1, dBET1, or JQ1 in live cells. Data are
represented as NanoBRET ratios normalized to zero compound.
Error bars are expressed as SD of the mean (n = 3) of a representative
experiment. (E) NanoBRET kinetic monitoring of IC50 values for
JQ1, dBET1, and MZ1 binding to BRD4 in live cells. Data are
expressed as singlicate IC50 values from RLU (n = 3) of a
representative experiment. (F) Tabulated IC50 values for indicated
target, PROTAC, and assay format.
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VHL protein (Kd = 66 nM for MZ137 and 90 nM for
VH29848). These results, however, were not recapitulated in
live cells, with MZ1 showing a >40-fold reduction in binding
affinity relative to that of VH298 (Figures 4C and F).
Similar live cell target engagement studies were done with
NanoLuc-CRBN and a CRBN tracer and showed a 10-fold
reduction in binding of dBET1 as compared to that of
thalidomide (Supporting Information Figures 6A−C). To
interrogate the live cell binding affinities and kinetics to BRD4
by these PROTACs, competitive displacement studies were
done using a BET family tracer with NanoLuc-BRD4. Results
showed dBET1 and MZ1 had an 8- and 15-fold right-shifted
reduction in binding affinity, respectively, compared to that for
JQ1 in live cells (Figures 4D and F). Intracellular kinetics were
performed and found that both PROTACs had significantly
slower target engagement compared to JQ1 in live cells and
apparent IC50 values which decreased over time (Figure 4E).
Together, these results indicate reduced and slow cellular
permeability of MZ1 and dBET1, yet they remain highly
efficacious for degradation at substoichiometric binding affinity
concentrations, supporting a catalytic mechanism of action by
these PROTACs.
Response of cMyc in MV4;11 Cell Lines to Inhibitors
and PROTAC Treatments. One of the primary consequences
and desired outcomes of BET family inhibition is down-
regulation of key oncogenic targets, including cMyc.44,45 To
quantitatively study cMyc levels post-treatment with BET
inhibitors as compared to PROTACs, we tagged endogenous
cMyc with HiBiT on the C-terminus using CRISPR/Cas9 in a
relevant leukemia line, MV4;11 (Supporting Information Table
1). Treatment of these cells with 10 nM JQ1, dBET1, or MZ1
over a 24 h time period showed differential levels of cMyc
expression knockdown, from minimal loss with JQ1, to 50 and
80% loss with dBET1 and MZ1, respectively (Figure 5A).
Correlation to cell viability assays at each time point
revealed that only treatment with MZ1 resulted in measurable
cellular death at this concentration (Figure 5B). At a higher
concentration, both compounds showed significant reduction
in cMyc expression and concurrent cellular death (Figures 5C
and D). While cMyc is one of many transcriptional targets
impacted by BET inhibition44,45 and PROTAC-mediated
degradation,14 the use of HiBiT endogenous tagging as a
downstream reporter of function allowed for understanding of
the amount of loss needed to elicit the desired phenotypic
outcome.
■ CONCLUSION
Generation of heterobifunctional PROTAC compounds, either
de novo or from known inhibitors, presents an exciting new
direction for drug discovery.1,2,10,12 BET family PROTACs,
including those studied here, have shown advantages over BET
inhibitors and hold great promise for potential treatment of a
wide variety of diseases, including cancer and inflamma-
tion.14,17,30,32,34,35,44,45 In this work, we further understanding
of the cellular mechanism of action of dBET1 and MZ1 using a
suite of cell-based assays combined with CRISPR/Cas938,39
endogenous tagging, allowing for precise and quantitative
kinetic analysis of key degradation events. Importantly, we also
assess which stages of the degradation process are impacted by
recruitment to different E3 ligase complexes, a critical
consideration and variable in the development of PROTAC
compounds. Currently the primary E3 ligase components
utilized are VHL and CRBN, but active research efforts are
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ACS Chemical Biology Articles

Figure 5. PROTAC modulation of cMyc-HiBiT expression and viability in MV4;11 cells (A and B) MV4;11 cells containing endogenously tagged
cMyc-HiBiT were treated 10 (A) or 100 (B) nM JQ1, dBET1, or MZ1. (C and D) Cell viability was measured by CellTiter-Glo following
treatment with 10 (C) or 100 (D) nM for each respective compound. All data are represented as RLU normalized to DMSO control. Error bars are
expressed as SD of the mean (n = 3) of a representative experiment.

Figure 6. Model of degradation phases and contributing mechanisms Schematic showing the three phases of degradation: initiation of degradation,
degradation maximum (Dmax), and recovery correlated to the key mechanistic processes listed in black for each phase. The target protein response
to PROTACs at each phase is represented pictorially, showing first introduction of PROTAC and target protein, followed by loss of target protein,
and then recovery. Listed in red are the parameters identified to regulate each phase with arrows depicting the processes which to monitor or
optimize.

underway to identify other efficacious E3 ligase components to
serve as recruiters.2−4,12,16,19 This will result in an expansion of
options in the future, directly translating to increased chemical
development and furthering the need for robust and relevant
technologies to efficiently profile and triage PROTAC cellular
activity.
To fully characterize and compare PROTAC-mediated
degradation via the VHL or CRBN E3 ligase complexes,
quantifiable parameters of the cellular degradation profile were
determined in these studies. Across all BET family targets
studied here, we found that the MZ1/VHL recruitment system
elicits faster rates of degradation, slower, but more stable
ternary complex formation, enhanced ubiquitination, greater
compound potency, and longer-lasting degradation efficacy as
compared to the dBET1/CRBN system. Because both
compounds contain JQ1 as the target specific handle, we
conclude these differences are directly attributed to ternary
complex stability and relative positioning within the different
E3 ligase systems of each BET target protein for ubiquitina-
tion. Our ternary complex kinetic data correlate well with
recent structural studies of bromodomains of BRD4 proteins in
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complex with MZ137 or related dBET compounds47 and their
respective E3 ligase partners. In the case of MZ1/VHL,
positive cooperativity in binding was observed,37 in agreement
with our finding that these complexes were more stable over
time in cells. In contrast, for related dBET compounds with
CRBN, a lack of cooperativity as well as multiple
conformations have been reported,47 suggestive of more
dynamic and transient ternary complex formation for this
system. Indeed, we see this behavior mirrored inside the cell
with complexes forming quickly but not showing increasing
stabilization over time with any of the BET family members.
Our studies were done in HEK293 cells, and it is worth
noting that degradation profiles could vary in other cell lines
that may have different expression levels of E3 ligase
components or BET family members. However, we would
not expect overall trends to be different because ternary
complex stability and activity of specific complexes would likely
be the same. Because thorough studies comparing functional
outcomes imparted by use of different E3 ligase handles have
not been previously performed, it is difficult to predict whether
changes in the E3 recruitment system will be favorable or
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ACS Chemical Biology
unfavorable for any given target. Additional BET family
PROTACs,17 including more potent versions of dBET1, have
been reported.47,50 It would be intriguing to determine which
cellular steps and parameters contribute to the improvement in
potency for this class of compounds, and then translate this
understanding to help guide further chemical design as well as
to enhance SAR for BET degraders in general.
More broadly, we seek to use the functional relationships
determined from analysis of dBET1- and MZ1-mediated
degradation to build a mechanistic map of primary
contributing events for each phase of the PROTAC cellular
degradation profile; degradation initiation, Dmax, and recovery
(Figure 6). This model can be used as a guide for compound
rank-order profiling, design, and optimization, showing which
measurements will be most predictive in improving any given
phase in degradation. The important contributors to the initial
degradation phase are PROTAC compound permeability,
binding affinities to targets and E3 components, and
recruitment not simply into a ternary complex, but a
productive complex to achieve ubiquitination. Ultimately the
level of target ubiquitination, and not necessarily the efficiency
of ternary complex formation dictates the rate of degradation.
For Dmax, we find, perhaps not unexpectedly, that this phase is
related to the long-term stability of the ternary complex and is
also highly dependent on any downstream cellular feedback to
loss of the target which would promote recovery. This
interplay is most evident in our data for BRD2, which shows
fast rate of degradation, stabilized ternary complex formation,
but by far the shortest time at Dmax. We found this to be due to
competing upregulation of protein levels, a response stimulated
by JQ1 treatment alone that appeared to be amplified in
response to PROTAC treatment from the added layer of BET
family degradation compared to inhibition. The protein
recovery phase, which is challenging to study given its
multiphasic and more complex profiles, will likely vary greatly
between targets, as we have found for BET family members,
and will also depend on the cellular chemical stability of the
PROTAC compound. Together, these correlations help break
down some of the barriers in properly assessing PROTAC
activity within the highly complex degradation process,
providing useful entry points and important parameters to
monitor for optimization of key steps required for degradation.
Our understanding of the degradation process, particularly
the quantitative and kinetic determination of the three phases
shown in Figure 6, would not have been possible without the
use of the highly sensitive endogenous tagging system,
HiBiT,41 and complementary intracellular protein interaction
studies using NanoBRET.42,43 Prior to our studies presented
here, understanding of PROTAC degradation profiles have
been highly granular with limited quantitation, using primarily
Western blot analysis to observe target loss and recovery at
fixed time points and concentrations. Moreover, other
approaches have focused on monitoring ectopically expressed
fusion tagged proteins or domains in the cells.47 While this
allows for qualitative and visual determination of degradation
as well as study of protein interactions as we have shown here,
its application for quantitative degradation analysis resulted in
alteration of degradation rate, reduced Dmax, right-shifted DC50
values, and non-native recovery profiles. Therefore, to maintain
endogenous expression level and regulation, the pairing of
HiBiT luminescent technology and CRISPR/Cas9 endogenous
tagging was critical. Compared to other tags that could also be
used for endogenous insertion, HiBiT provides additional
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capabilities of high sensitivity over a broad dynamic range.41 In
our studies, we were are able to accurately and continuously
monitor degradation over a 3-log range with high reproduci-
bility, indicating this approach can enable detection of proteins
that have low levels of native expression and/or for
characterization of early development PROTAC compounds,
which might not show fast or robust degradation.
Other approaches are currently also used to study
protein:protein and protein:small molecule interactions but
do not have the same advantages for degradation research as
compared to NanoBRET.42 Given its proximity-based and
ratiometric nature, NanoBRET is suited for studying
interactions that are transient, and measurement of the
interaction is not influenced by changes in the level of donor
if this occurs.42 This is why we have specifically chosen the
target protein as the energy donor in the configuration of the
assay, allowing for determination of complex formation even
with active loss of the target. In contrast, other protein
interaction technologies are reliant on a single readout, such as
fluorescence or luminescent complementation and lack the
ability to discern whether changes in signal are due to the
modulation of the interaction or changes in levels of one or
both partners. A further advantage of NanoBRET is the dual
readout capability, which allows simultaneous monitoring of
both the interaction (protein:protein or protein:small mole-
cule) by calculation of NanoBRET ratios as well as target loss
by direct luminescent monitoring, provided the target protein
is the donor.
The platform of technologies presented here to characterize
the cellular mechanism of action of two BET bromodomain
PROTACs, dBET1 and MZ1, not only advance our under-
standing of these chemical probes, but more broadly provide a
comprehensive analysis to advance PROTAC drug discovery
and cellular activity assessment. Our cellular analysis shows
that both ternary complex stability and subsequent ubiquiti-
nation are key steps, but they influence and regulate different
phases of degradation (Figure 6), correlations not previously
described due to studies done primarily with in vitro assays.
These understandings combined with our technology platform
to study each parameter in live cells will help further
compound optimization, including fine-tuning family member
specificity if desired. While the focus of this work was on
PROTAC characterization, the approach presented here can
be applied to study any process that impacts protein
homeostasis, as demonstrated with cMyc (Figure 5) and in
our other studies of therapeutic targets HIF1α and β-catenin
(data not shown). The diverse capabilities and applications of
these technologies coupled with endogenous tagging via
CRISPR/Cas9 present a highly enabling and physiologically
relevant means to study protein degradation, providing unique
insight into the individual and highly complex mechanisms
regulating the degradation process.
■
MATERIALS AND METHODS
Expression Plasmids. Clones expressing N-terminal HaloTag
fusions of human VHL (NM̅ 000551) and Cereblon (AAH17419.1)
were obtained from Kazusa DNA Research Institute (Kisarazu, Japan)
as pFN21A HaloTag CMV Flexi Vectors (Promega). The N-terminal
HaloTag-Ubiquitin constructed consisted of the first ubiquitin repeat,
amino acids 1−76, of the human polyubiquitin-B precursor
(NM̅ 018955) and was cloned into the pFN28K (Promega) without
appending any additional residues to the native C-terminal sequence.
N-terminal NanoLuc fusions of human VHL (NM̅ 000551), Cereblon
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ACS Chemical Biology
(AAH17419.1), and BRD4, long isoform (NM̅ 058243) were cloned
into pFN31K vectors (Promega).
Reagents and Cell Culture. dBET1 was purchased from Cayman
Chemical, JQ1 was purchased from Xcessbio Biosciences, MZ1 and
VH298 were generously donated by A. Ciulli (University of Dundee),
and MG132 was purchased from InvivoGen. HEK293 and MV4;11
cells were obtained from the American Type Culture Collection and
cultured at 37 °C, 5% CO2 in DMEM (Gibco) or IMDM (American
Type Culture Collection), respectively, containing 10% fetal bovine
serum (Seradigm).
Generation of HEK293 LgBiT Stable Cell Line. HEK293 cells
(8 ×106) plated in a 10 cm2 Petri dish were transfected with 20 μg
LgBiT CMV plasmid for 24 h and grown in 2 mg mL−1 G418
selection antibiotic (Promega) until large colonies were present.
Clones were isolated by limiting dilution into 96-well plates and after
expansion were screened for LgBiT expression by triplicate plating
with one plate receiving HiBiT Lytic Detection Reagent (Promega)
supplemented with purified HiBiT-HaloTag and another plate
receiving CellTiter-Glo (Promega). A clone with high luminescence
relative to CellTiterGlo was expanded from the third plate and
maintained in 800 μg/mL G418.
CRISPR/Cas9-Editing. One nanomole Alt-R CRISPR RNA
(crRNA) and 1 nmol Alt-R trans-activating crRNA (tracrRNA)
were assembled in 50 μL of Nuclease-Free Duplex Buffer (Integrated
DNA Technologies) by incubation at 95 °C for 5 min and cooling to
RT to generate gRNA for BRD2, BRD4, and cMyc. CRISPRevolution
sgRNA EZ kit (Synthego) was used as the gRNA for BRD3. Single-
stranded ultramer DNA oligonucleotides (IDT) were used as the
ssODN donor templates. Ribonucleoprotein (RNP) complexes with
recombinant Streptococcus pyogenes Cas9 with an N-terminal tag
containing a histidine-nuclear localization signal-myc sequence
(Aldevron) were assembled as previously described41 by incubating
100 pmol of Cas9 and 120 pmol of gRNA for 10 min at ambient
temperature. For HiBiT knock-in to BRD2, BRD3, and BRD4, 2 ×
105 HEK293 cells were resuspended in 20 μL of 4D Nucleofector
solution SF, and RNP complexes along with 100 pmol ssODN
template were electroporated into cells with the 4D Nucleofector
System (Lonza) using program CM-130. For HiBiT knock-in to
cMyc, MV4;11 cells were resuspended in 20 μL of 4D Nucleofector
solution SE, and RNP complexes along with 100 pmol ssODN
template were electroporated into cells with the 4D Nucleofector
System (Lonza) using program DJ-100. Immediately following
electroporation, cells were incubated at ambient temperature for 10
min before transferring to a 12-well plate for culturing. Edited pools
were analyzed for HiBiT insertion by assaying for luminescence on a
GloMax Discover (Promega) 48−72 h postelectroporation.
Generation and Validation of Clonal HiBiT Insertions. Clonal
populations of edited cells were obtained by sorting live singlets using
a FACSMelody cell sorter (Becton Dickinson) into 96- or 384-well
plates. Clones were expanded and screened by luminescence, and
genomic DNA was isolated using the Wizard Genomic DNA
Purification kit (Promega) from 8 to 10 clones per edited pool.
Genomic DNA was amplified using primers designed to anneal ∼200
bases either upstream or downstream from the inserted ssODN
template sequence, subcloned into a pF5K vector backbone
(Promega), transformed into JM109 cells (Promega), and 24
individual colonies per clonal population were sequenced by Sanger
sequencing. Clones were selected based on 100% sequence
conformity with no insertions or deletions in the HiBiT-containing
open reading frame.
HiBiT Blotting. HEK293 clonal cells (2 ×105) expressing HiBiT-
BET family members were plated in 24-well dishes overnight and then
lysed in 100 μL of mammalian lysis buffer (Promega) supplemented
with Protease Inhibitor Cocktail (Promega) and RQ1 RNase-Free
DNase (Promega) on ice. One hundred microliters of 2× SDS-PAGE
gel loading buffer (120 mM Tris-HCl, pH 6.8, 1.5 mM bromophenol
blue, 25% glycerol, 200 mM dithiothreitol, and 1% SDS) was added to
each sample and incubated for 5 min at 95 °C. Lysates were separated
by SDS-PAGE and transferred to a nitrocellulose membrane.
Following transfer, the membrane was briefly incubated in TBST
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(20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) with
1 mM dithiothreitol (DTT) before replacing buffer with NanoGlo
Blotting Buffer containing 100 nM purified LgBiT protein (Promega)
and incubating at 4 °C overnight with shaking. NanoGlo Luciferase
Assay Substrate (Promega) was added, and luminescence was
captured on an ImageQuant LAS 4000 digital imaging system (GE
Healthcare Life Sciences).
Bioluminescence Imaging. Clonal HiBiT-BRD4 edited cells
were plated in 8-well glass bottom chamber slides (Nunc Lab-Tek II)
at a density of 8 × 104 per well in 200 μL of growth medium and
incubated at 37 °C, 5% CO2 overnight. Following incubation,
medium was replaced with Opti-MEM (Gibco) containing 20 μM
Vivazine extended release substrate (Promega) and incubated for an
additional 1 h at 37 °C, 5% CO2 to allow equilibration of substrate
and stabilization of luminescent signal. Real-time imaging was
performed using the LV200 bioluminescence imaging system
(Olympus) equipped with an ImagEM X2 EM-CCD camera
(Hamamatsu) and a temperature-controlled stage. Images were
collected for a period of 2 h with cellSens software (Olympus)
using a 60× oil-immersion objective, electron multiplying EM gain of
1200, and 10 s exposure times. Average projections of 10 sequential
images were generated, and dynamic range adjustments and
pseudocolor rendering was performed using FIJI.
Kinetic Live Cell HiBiT Detection. Clonal cell lines edited for
HiBiT insertion to BET family members were plated in white 96-well
tissue culture plates at a density of 2 × 104 cells per well in 100 μL of
growth medium and incubated overnight at 37 °C, 5% CO2. For
experiments with transient expression of NanoLuc-BRD4, 8 × 105
HEK293 cells were first transfected with 0.02 μg of NanoLuc-BRD4
diluted in 2 μg of promoterless carrier DNA using FuGENE HD
(Promega), and the following day, were plated in white 96-well tissue
culture plates at a density of 2 × 104 cells per well in 100 μL of growth
medium and incubated overnight at 37 °C, 5% CO2. For end point
live cell detection, wells were treated with dBET1 or MZ1 compounds
for the indicated time frames before addition of NanoGlo Live Cell
Substrate (Promega) and reading luminescence on a GloMax
Discover. For continuous live cell detection out to 24 h, medium
was replaced with CO2-independent medium (Gibco) containing 20
μM Endurazine, an extended time-released substrate (Promega), and
plates were incubated at 37 °C, 5% CO2, for 2.5 h before addition of a
3-fold serial dilution of 1 μM final concentration dBET1, MZ1, or
JQ1 compounds. Plates retaining the plate lids were then read every 5
min for a period of 24 h on a GloMax Discover (Promega) set to 37
°C.
Quantitation of Degradation Kinetics. Degradation rate and
degradation plateau were calculated by fitting only the initial
degradation portion of each kinetic concentration curve to the
equation:
y = (y0 − plateau)e−ƛt + plateau
where ƛ = degradation rate in units of hr−1. The degraded fraction,
Dmax, was calculated as 1 − plateau. For each curve, the first few data
points before onset of degradation were excluded from the fits. Time
at Dmax was determined by calculating the length of time during which
the fractional population remained below a threshold defined as
plateau + 0.1(Dmax).
Lytic HiBiT Detection and Viability of MV4;11 Cells. Clonal
cMyc-HiBiT edited cells were replicate plated at a density of 5 × 104
cells per well in solid, white 96-well tissue culture plates (Corning
Costar #3917), and cultured overnight. The following day, cells were
treated with either 10 nM or 100 nM JQ1, dBET1, or MZ1 for the
indicated time frames. Following each treatment time point, one
replicate plate was assayed for luminescence of cMyc-HiBiT by
addition of an equal volume of NanoGlo HiBiT Lytic Reagent
(Promega N3030) to each well, and another replicate plate was
assayed for cell viability by addition of an equal volume of CellTiter-
Glo 2.0 to each well (Promega). Plates were shaken on an orbital
shaker for 10−20 min before reading luminescence on a GloMax
Discover (Promega).
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ACS Chemical Biology
NanoBRET Live Cell Ternary Complex and Ubiquitination.
HEK293 clonal cells (8 ×105) expressing the HiBiT-BET family
members were transfected with FuGENE HD (Promega) and 2 μg of
HaloTag-CRBN, HaloTag-VHL, or HaloTag-UBB in 6-well plates.
For transient NanoBRET experiments with NanoLuc-BRD4, 8 × 105
HEK293 cells were transfected with FuGENE HD (Promega) and
0.02 μg of NanoLuc-BRD4 with either 2 μg of HaloTag-CRBN, or
HaloTag-VHL. The following day, 2 × 104 transfected cells were
replated into white 96-well tissue culture plates in the presence or
absence of HaloTag NanoBRET 618 Ligand (Promega) and
incubated overnight at 37 °C, 5% CO2. The following day, medium
was replaced with Opti-MEM (Gibco) containing 20 μM Vivazine, an
extended time-released substrate (Promega), and plates were
incubated at 37 °C, 5% CO2, for 1 h before addition of DMSO or
1 μM final concentration dBET1 or MZ1 compounds. Plates were
then read every 3 min for a period of 6 h on a CLARIOstar equipped
with an atmospheric control unit (BMG Labtech) set to 37 °C and
5% CO2. Dual filtered luminescence was collected with a 460/80 nm
bandpass filter (donor, NanoBiT-BET protein) and a 610 nm long
pass filter (acceptor, HaloTag NanoBRET ligand) using an
integration time of 0.5 s. Background subtracted NanoBRET ratios
expressed in milliBRET units were calculated from the equation:
ji acceptor channel acceptor channel (no ligand) zyz
mBRET ratio = jjjj
k donor channel (no ligand) z{
− member time at Dmax at 10 and 100 nM PROTAC, BET
donor channel
Fold increase in BRET was calculated by normalizing mBRET ratios
to the average mBRET ratios for DMSO controls.
NanoBRET Ubiquitination ImmunoAssay. Clonal cell lines
edited for HiBiT insertion to BET family members were plated in
white 96-well tissue culture plates at a density of 2 × 104 cells per well
in 100 μL of growth medium and incubated overnight at 37 °C, 5%
CO2. The following day, 1 μM dBET1 or MZ1 was added to the plate
for the indicated time frames before replacing medium with Opti-
MEM (Gibco) containing 200 μg/mL digitonin, 1:200 dilution of
primary anti-Ub antibody (Enzo Life Sciences, BML-PW8810), 1:500
dilution of secondary antimouse Alexa 594 antibody (Cell Signaling
Technologies, 8890), and 20 μM NanoGlo substrate. Additional
control wells received no antibodies (control for background
NanoBRET) or no primary antibody (control for specificity). Plates
were placed on an orbital shaker for 10 min and NanoBRET
measurements were collected on a CLARIOstar (BMG Labtech).
NanoBRET calculations were made according to the equation in the
section: NanoBRET Live Cell Ternary Complex and Ubiquitination.
Fold increase in NanoBRET was calculated by normalizing mBRET
ratios to the untreated, t = 0 wells.
NanoBRET Target Engagement. NanoLuc-CRBN, VHL-Nano-
Luc, and NanoLuc-BRD4 were transfected in HEK-293 cells using
FuGENE HD (Promega) according to the manufacturer’s protocol.
Briefly, NanoLuc/target fusion constructs were diluted into Trans-
fection Carrier DNA (Promega) at a mass ratio of 1:10 (mass/mass),
after which FuGENE HD was added at a ratio of 1:3 (μg DNA: μL
FuGENE HD). One part (vol) of FuGENE HD complexes thus
formed were combined with 20 parts (vol) of HEK-293 cells
suspended at a density of 2 × 105, followed by incubation in a
humidified, 37 °C/5% CO2 incubator for 20 h. Following transfection,
cells were washed and resuspended in Opti-MEM. NanoBRET assays
were performed in white, 96-well plates (Corning 3600) at a density
of 2 × 104 cells/well. All chemical inhibitors were prepared as
concentrated stock solutions in DMSO (Sigma-Aldrich) and diluted
in Opti-MEM (unless otherwise noted) to prepare working stocks.
For end point analysis of target engagement, cells were equilibrated
for 2 h with energy transfer probes and test compound prior to
NanoBRET measurements. NanoBRET tracers were prepared at a
working concentration of 20× in tracer dilution buffer (12.5 mM
HEPES, 31.25% PEG-400, pH 7.5). VHL NanoBRET Tracer was
added to the cells at a final concentration of 1 μM, CRBN NanoBRET
tracer was added to cells at a final concentration of 0.5 μM, and
NanoBRET BRD Tracer-02 was added to cells at a final concentration
of 0.25 μM. To measure NanoBRET in live cells, NanoBRET
Articles
NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega)
were added according to the manufacturer’s recommended protocol,
and filtered luminescence was measured on a GloMax Discover
luminometer equipped with 450 nm BP filter (donor) and 600 nm LP
filter (acceptor) using 0.3 s integration time. For real-time target
engagement analysis, NanoBRET NanoGlo Substrate and Extrac-
ellular NanoLuc Inhibitor (Promega) and 1× NanoBRET tracer were
added to the cells 30 min prior test compound addition. To measure
NanoBRET in permeabilized cells, digitonin was added to the cells to
a final concentration of 50 ug/mL and Extracellular Nluc inhibitor
was omitted during the detection step. Milli-BRET units (mBU) are
calculated by multiplying the raw NanoBRET values by 1000.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.8b00692.
Tables of CRISPR guide and template sequences and
comparative HiBiT-BET family expression; figures of
kinetic competition of dBET1 and MZ1 with JQ1,
zz1000
impact of ectopic expression on degradation, BET family
family protein level profiles after JQ1 inhibition, ternary
complex specificity and impact of proteasomal inhib-
ition, and NanoBRET target engagement of dBET1 with
NanoLuc-CRBN, (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*Tel: +1-608-274-4330; Fax: +1-608-277-2601; E-mail:
danette.daniels@promega.com.
ORCID
Danette L. Daniels: 0000-0002-7659-3020
Notes
The authors declare the following competing financial
interest(s): All authors are employees of Promega Corporation
and Promega Corporation is the commercial owner by
assignment of patents of the HaloTag, NanoLuc, NanoBRET
target engagement, NanoBiT, and HiBiT technologies and
their applications.
■
ACKNOWLEDGMENTS
We wish to thank A. Ciulli for providing MZ1 and VH298
compounds, T. Machleidt and M. Schwinn for guidance on
HiBiT CRISPR/Cas9 editing and development of LgBiT
stable cell line, M. Slater for advice on CRISPR clonal
selection, C. Zimprich for help with target engagement, T.
Worzella for Glo-Max Discover instrumentation support, and
G. Tarpley for thoughtful advice and continued support.
■
REFERENCES
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