Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs

Article abstract of DOI:10.1021/jacs.9b13907

Proteolysis targeting chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including substoichiometric degradation of targets. Their scope, though, is still limited to date by the requirement for a sufficiently potent target binder. A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. Using Bruton's tyrosine kinase (BTK) as a clinically relevant model system, we show efficient degradation by noncovalent, irreversible covalent, and reversible covalent PROTACs, with <10 nM DC50's and >85% degradation. Our data suggest that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to ibrutinib and exhibit potent degradation of BTK in patient-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs. These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.

Full text of DOI:10.1021/jacs.9b13907

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Article
Efficient targeted degradation via reversible
and irreversible covalent PROTACs
Ronen Gabizon, Amit Shraga, Paul Gehrtz, Ella Livnah, Yamit Shorer, Neta
Gurwicz, Liat Avram, Tamar Unger, Hila Aharoni, Shira Albeck, Alexander
Brandis, Ziv shulman, Ben-Zion Katz, Yair Herishanu, and Nir London
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b13907 • Publication Date (Web): 05 May 2020
Downloaded from pubs.acs.org on May 5, 2020
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Efficient targeted degradation via reversible and irreversible covalent PROTACs
Ronen Gabizon1,*, Amit Shraga1,*, Paul Gehrtz1, Ella Livnah1, Yamit Shorer2, Neta Gurwicz3, Liat
Avram4, Tamar Unger5, Hila Aharoni5, Shira Albeck5, Alexander Brandis6, Ziv Shulman3, Ben-
Zion Katz7, Yair Herishanu7, Nir London1,#
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1 Dept. of Organic Chemistry, The Weizmann Institute of Science, Rehovot, 7610001, Israel.
2 Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.
3 Dept. of Immunology, The Weizmann Institute of Science, Rehovot, 7610001, Israel.
4 Dept. of Chemical Research Support, The Weizmann Institute of Science, Rehovot 7610001,
Israel.
5 Structural Proteomics Unit, Department of Life Sciences Core Facilities, The Weizmann
Institute of Science, Rehovot 7610001, Israel.
6 Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 7610001, Israel
7 Department of Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel and Sackler
Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.
* equal contribution
# To whom correspondence should be addressed, nir.london@weizmann.ac.il
Keywords: PROTACs, reversible covalent, cyanoacrylamides, BTK, targeted degradation
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Abstract
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PROteolysis Targeting Chimeras (PROTACs) represent an exciting inhibitory modality
with many advantages, including sub-stoichiometric degradation of targets. Their scope,
though, is still limited to-date by the requirement for a sufficiently potent target binder. A
solution that proved useful in tackling challenging targets is the use of electrophiles to allow
irreversible binding to the target. However, such binding will negate the catalytic nature of
PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They
possess the potency and selectivity associated with the formation of the covalent bond, while
being able to dissociate and regenerate once the protein target is degraded. Using Bruton’s
tyrosine kinase (BTK) as a clinically relevant model system, we show efficient covalent
degradation by non-covalent, irreversible covalent and reversible covalent PROTACs, with
<10 nM DC50’s and >85% degradation. Our data suggests that part of the degradation by our
irreversible covalent PROTACs is driven by reversible binding prior to covalent bond
formation, while the reversible covalent PROTACs drive degradation primarily by covalent
engagement. The PROTACs showed enhanced inhibition of B cell activation compared to
Ibrutinib, and exhibit potent degradation of BTK in patients-derived primary chronic
lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited
enhanced selectivity towards BTK compared to non-covalent and irreversible covalent
PROTACs. These compounds may pave the way for the design of covalent PROTACs for a
wide variety of challenging targets.
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Introduction
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PROteolysis TArgeting Chimeras (PROTACs) are receiving increasing attention as a
new therapeutic modality, as was recently underscored by the first PROTAC, ARV-110, to
enter clinical trials1. PROTACs are comprised of a protein target binding moiety, a linker and
an E3 ubiquitin ligase binder2,3. Upon binding, the PROTAC induces the formation of a ternary
complex between the target and E3 ligase4,5,6,7 resulting in the ubiquitination and degradation
of the target. Compared to traditional inhibition of the target protein, targeted degradation has
several important advantages, including the elimination of all levels of protein function,
enhanced selectivity8,9,10,11,12, longer duration of action due to the need to resynthesize the
target13, and degradation by sub-stoichiometric amounts of PROTAC14.
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Efficient degradation typically requires high affinity binding to the target as well as
optimized linker geometry, to optimize the ternary complex formation. However, many targets
such as transcription factors15,16, protein-protein interfaces17,18, or challenging enzyme classes
such as GTPases19, are recalcitrant to ligand discovery. This limits the applicability of
PROTACs against such targets. A possible solution to this problem is to introduce an
electrophile that will allow covalent binding to the target. However, irreversible binding may
reduce potency by negating the catalytic nature of the PROTAC activity. While several
covalent PROTACs have been developed and degrade their target successfully20,21,22, there are
examples in which the introduction of irreversible binding reduces the potency of
PROTACs23,24
.
Theoretically, reversible covalent PROTACs can benefit from both the enhanced
potency, selectivity, and long duration of action that accompany covalent bond formation25,26
,27, without compromising the sub-stoichiometric activity of PROTACs. In this work we set
out to test this hypothesis by the design of cyanoacrylamide-based reversible covalent
PROTACs. To this end, we selected Bruton’s tyrosine kinase (BTK), which is an established
target for non-covalent PROTACs23,28,29,30,31,32, and systematically tested a series of reversible
covalent PROTACs along with their irreversible covalent and non-covalent PROTAC analogs.
Our work resulted in a highly potent and selective reversible covalent PROTAC (RC-3), as
well as insights into the effect of covalent bond formation kinetics on the degradation by
covalent PROTACs.
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Results
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We devised a modular scheme for the synthesis of cyanoacrylamide-based PROTACs
(see methods and SI). Using this route, we synthesized a series of 12 reversible covalent
PROTACs targeting Cysteine 481 in BTK (Supp. Table 1). These are based on the scaffold of
the covalent BTK binder - Ibrutinib - as the protein targeting moiety33, and PEG-based linkers
with varying length (Fig. 1). We used two approaches: in the first, we synthesized an alkyne-
functionalized BTK-binding cyanoacrylamide and an amine-functionalized E3 binder, and
linked them in one-pot reactions using azide-PEG-NHS esters of varying lengths. In the second
approach, we directly functionalized thalidomide with various PEGs and formed the
cyanoacrylamide in a final condensation step.
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We incubated K562 or Mino cells with 1 µM of the compounds for 24 hours and
measured the abundance of BTK by western blot (Supp. Fig. 1). Several of the tested
PROTACs displayed clear BTK degradation and could serve as potential starting points for
optimization. We selected compound RC-1, which is based on a PEG6 linker and displayed
consistent prominent levels of degradation in both cell lines, as a starting point for this study.
Based on compound RC-1 we synthesized additional compounds (Fig. 1), RC-2 with a CH2
group replacing the oxygen nearest the -carbon, their analogous acrylamides IR-1 and IR-2,
and the non-covalent analog NC-1. We also synthesized the cyanoacrylamide RC-3 (Fig. 1),
replacing the C hydrogens with methyl groups. Similar dimethylated cyanoacrylamides were
reported to have improved cellular permeability34.
Figure 1. Structures of reversible covalent, irreversible covalent and non-covalent BTK PROTACs
described in this study. The electrophilic moieties are highlighted in red.
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We evaluated the ability of the compounds to induce degradation of BTK in human cell
lines. We incubated Mino cells with the compounds and followed BTK levels after 24 hours
by western blot (Fig. 2, Supp. Fig. 2). The non-covalent PROTAC NC-1 showed the highest
degradation potency with DC50 = 2.2 nM (Maximal degradation - Dmax = 97%). The irreversible
acrylamides IR-1 and IR-2, and the cyanoacrylamide RC-3 followed closely with DC50’s
under 10 nM and Dmax near 90%. The cyanoacrylamides RC-1 and RC-2 were less potent.
Similar trends were observed in Ramos cells (Supp. Fig. 2A). We conducted metabolomics
studies to estimate if cellular penetration and stability may contribute to the relative potencies
(Supp. Table 2). NC-1 and RC-3 reached an effective concentration which was ~2 times higher
than IR-2 and ~10 times higher than RC-2. Therefore, the lower potency of RC-2 can at least
in part be explained by lower permeability or stability.
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Figure 2: Efficient BTK degradation in cells.
A. Western blot evaluation of BTK levels in Mino cells in response to various concentrations of RC-2, IR-2,
NC-1 and RC-3, after 24h incubation.
B. Quantification of BTK levels in (A) by normalization to the -actin house-keeping gene in Mino cells. DC50
and Dmax were calculated by fitting the data to a second order polynomial using Prism software.
C. A summary of the DC50 and Dmax values for the PROTACs in Mino cells describe in (A) and Supp. Fig. 2.
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We followed the rate of BTK degradation facilitated by this compound series via a time
course experiment in Ramos and Mino cells (Supp. Fig. 3). The rates of degradation correlated
well with the DC50 observed after 24 hours, with NC-1, IR-1, IR-2 and RC-3 degrading BTK
within 2-4 hours, while RC-2 and RC-1 required 6-24 hours to reach maximum degradation.
To validate the mechanism of PROTAC mediated degradation of BTK, Mino cells were
pre-treated for 2 hours with either Ibrutinib or thalidomide-OH, and subsequently treated with
the PROTACs for an additional 24 hours. Both Ibrutinib pre-treatment as well as thalidomide-
OH, hindered BTK degradation (Fig. 3A). In contrast to the covalent PROTACs, degradation
by the non-covalent NC-1 was only slightly hindered by thalidomide. In addition, RC-1m, a
methylated thalidomide analog of RC-1, no longer able to bind CRBN, lost all activity (Supp.
Fig. 4), further suggesting CRBN mediated degradation. We treated Mino cells with
Bortezomib, a proteasome inhibitor35, for 1 hour before treatment with the PROTACs and
assessed BTK levels after an additional 4 hours. Bortezomib significantly inhibited
degradation, suggesting proteasome-dependent degradation (Fig. 3B).
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Figure 3: PROTAC mediated BTK degradation is hindered by Ibrutinib, thalidomide and by proteasome
inhibition.
A. Mino cells were either pre-treated for 2 hours with Ibrutinib/thalidomide-OH or untreated, before treatment
with a BTK PROTAC for 24 hours. Subsequently BTK levels were measured via western blot.
B. Mino cells were treated for 1 hour with Bortezomib to inhibit proteasome-dependent degradation, then
PROTACs were added for 4 hours, followed by measuring BTK levels via western blot.
To assess the PROTACs efficiency in a clinically relevant model we tested their ability
to induce BTK degradation in primary cells from chronic lymphocytic leukemia (CLL)
patients. The PROTACs displayed potent degradation with DC50’s < 100 nM, with NC-1 and
IR-2 reaching higher degradation levels than RC-2 and RC-3 (Fig. 4).
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Figure 4: Degradation of BTK in patients-derived CLL cells
Patients-derived primary CLL cells were treated with BTK PROTACs for 18 hours, followed by measuring BTK
levels via western blot. M-IGHV/UM-IGHV: Mutated/Unmutated immunoglobulin heavy chain variable region
(IGHV) gene.
While we observed potent degradation by covalent PROTACs, it was still not clear if
and how the covalent bond contributes to the degradation process. Due to the high non-covalent
binding affinity of Ibrutinib to BTK, it is possible that the degradation is induced primarily by
reversible binding that occurs prior to covalent bond formation. A second related question was
what is the dissociation rate of the covalent complexes formed by the cyanoacrylamides, and
whether this rate can support catalytic degradation to the same degree as non-covalent binding.
To answer these questions, we performed several experiments to evaluate the formation of
covalent complexes with BTK at the timescale and concentration range observed for
degradation, as well as their dissociation kinetics.
First, we tested the degradation activity of the PROTACs against overexpressed wild
type BTK and the C481S mutant, which cannot form covalent complexes (Fig. 5). As expected,
the degradation by non-covalent NC-1 was only mildly affected by the mutation. However, the
covalent PROTACs IR-1, IR-2 and RC-2 also showed low sensitivity to the mutation. In
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contrast, degradation by RC-3 was severely impaired by the mutation, indicating an important
role for covalent engagement in the degradation process by RC-3.
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Figure 5: Degradation of overexpressed BTK and BTK C481S in U2OS cells
A. Transfected U2OS cells were treated with 100 nM PROTAC for 24 hours, followed by measuring BTK levels
via western blot.
B. Quantification of normalized BTK levels in (A).
Second, we tested the ability of the compounds to covalently bind and inhibit BTK.
We performed an in vitro kinase activity assay with both wild type BTK and the C481S mutant,
(Fig. 6A, B). The assay was performed with a preincubation period of two hours, equivalent to
the timescale of degradation induced by the PROTACs in cells (Supp. Fig. 3). As expected,
Ibrutinib was both highly potent against the WT and sensitive to the mutation, with a 74-fold
reduction in potency, indicating efficient covalent engagement. The non-covalent NC-1
showed very mild sensitivity to the mutation, with < 2-fold reduction in affinity. The
acrylamide IR-2 did not inhibit BTK more potently than NC-1 and also showed only slight
sensitivity to the mutation, indicating inefficient covalent bond formation, possibly due to the
lowered reactivity of the -substituted acrylamide. On the other hand, the cyanoacrylamides
RC-2 and RC-3 were an order of magnitude more potent than NC-1 and also highly sensitive
to the mutation with 68-fold and >1000-fold reduction in potency, respectively, indicating rapid
covalent binding to BTK on the timescale of degradation. All the PROTACs tested except RC-
3 exhibited sub-100 nM binding to BTK even after mutation of C481. This may explain why
only degradation by RC-3 was significantly impaired by the mutation.
We also used LC-MS to directly observe the formation of covalent complexes with
recombinant BTK and measure their dissociation kinetics (Figure 6C, D, Supp. Fig. 5A). LC-
MS measurements with 2 µM BTK and 3 µM compound indicated covalent labeling by all
compounds except NC-1, with RC-3 forming the complex extremely fast, followed by RC-2
and IR-2, in agreement with the data from the kinase activity assay. We should note that the
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preincubation of the compounds with 5 mM GSH did not significantly affect protein binding
of neither reversible nor irreversible covalent binders (Supp. Fig. 5B).
To test whether the formation of the covalent adducts is reversible and estimate the
timescale of the exchange, we added 40 µM Ibrutinib to the samples after formation of the
adducts and incubated at 37°C. For the acrylamides IR-1 and IR-2, no Ibrutinib adduct was
observed even after 28 hours of incubation. However, only 80-85% of protein appeared to be
labeled by the PROTACs (Supp. Fig. 5A). This may indicate that in fact IR-1/2 have stably
labeled 100% of the protein, thereby preventing Ibrutinib binding, but dissociation during the
separation or ionization process on the LC-MS may have generated the observed free protein
peak.
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In contrast, for the cyanoacrylamides RC-2 and RC-3, the addition of Ibrutinib led to
the gradual displacement of the PROTAC by Ibrutinib, confirming the reversibility of the
cyanoacrylamide covalent binding. The exchange of the cyanoacrylamide was slow, on the
order of 10-20 hours. The non-covalent PROTAC NC-1 forms no covalent adduct and is
rapidly exchanged by Ibrutinib (100% Ibrutinib labeling by 4 µM in 1 hour at room
temperature; Supp. Fig. 5A), indicating very rapid binding and dissociation kinetics.
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Figure 6: All PROTACs are potent BTK inhibitors in vitro, and the cyanoacrylamides show slow
dissociation kinetics, and sensitivity to the C481S mutation.
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A. In vitro kinase activity assay using wild type BTK (0.6 nM BTK, 5 μM ATP)
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B. Summary of IC50 values for the PROTACs against wild type BTK and C481S BTK.
C. Time course LC-MS binding assay (3 µM compound + 2 µM BTK at room temperature).
D. Ibrutinib competition assay, validates reversible binding by cyanoacrylamides. 40 µM Ibrutinib was added to
the preformed complex, incubated at 37°C, and the different species were quantified by LC-MS.
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To assess their proteomic selectivity, we incubated the PROTACs at 50 nM or 100 nM
for 24 hours with Ramos cells, and followed the change in protein abundance via quantitative
label-free proteomics (Fig. 7, Supp. Fig. 6). In agreement with the western blot analysis, BTK
was efficiently degraded by all the PROTACs we tested. NC-1, IR-1, IR-2 and RC-2 also
degraded the known Ibrutinib off-targets CSK, LYN and BLK33, while several other off-
targets, such as LCK and PLK1, were not significantly affected. NC-1 showed the highest
degradation potency against BTK, in agreement with western blot analysis. On the other hand,
the only significant off-target of RC-3 was BLK (a covalent off-target of Ibrutinib) with no
activity against the non-covalent off-targets CSK and LYN, representing enhanced selectivity,
and in agreement with the reduced non-covalent affinity of RC-3 to BTK. No other significant
off-targets were detected consistently (Supp. Dataset 1).
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Figure 7. Proteomic analysis reveals high selectivity for both covalent and non-covalent BTK PROTACs.
Ramos cells were incubated with each PROTAC (50 nM) or DMSO in quadruplicates for 24h, and were then
subjected to label-free quantitative proteomics analysis. Each graph plots the Log2 fold-change of proteins in the
treated samples compared to the DMSO controls (X-axis) vs. the -log(p-value) of that comparison in a student's
T-test (Y-axis).
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Lastly, we assessed the ability of the PROTACs to abrogate the activation of primary
mouse B cells in response to B cell receptor stimulation. For this purpose, primary B cells were
treated with anti-IgM for 18 hours 36,37, followed by staining for CD86, a B cell activation
surface marker (Fig. 8; Supp. Fig. 7). The inhibition of B cell activation correlated well with
the BTK degradation activity, with NC-1 and IR-2 showing the strongest effect, followed by
RC-3 and RC-2. NC-1 and IR-2 displayed superior inhibition compared to Ibrutinib,
underscoring the benefit of targeted degradation compared to inhibition alone. The
cyanoacrylamides RC-3 and RC-2 required higher concentrations to reach maximal activity
but also displayed superior activity to Ibrutinib at 1 µM.
Figure 8: PROTACs inhibit B cell receptor signaling more potently than Ibrutinib.
Dose response curves for B cell response after anti-IgM induced activation and treatment with BTK PROTACs
or Ibrutinib for 24 hours. The Y-axis shows normalized CD86 Mean fluorescence intensity, where 100%
activation is cells stimulated with anti-IgM, and 0% activation is unstimulated cells.
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The motivation for developing reversible covalent PROTACs lies in the combination
of the advantages encompassed by covalent binding, such as increased potency and selectivity,
while maintaining the reversibility that is considered important for the catalytic nature of
PROTAC efficacy. Several previous studies reported non-covalent PROTACs against
BTK28,29,30,31,32 and some indicated that irreversible binding might be detrimental to the activity
of covalent PROTACs23. In this work we tested whether cyanoacrylamide reversible covalent
binders could serve as potent PROTACs. Our results show that both acrylamides and
cyanoacrylamides can function as potent and selective PROTACs, including in patient-derived
cell lines (Fig. 4), with the irreversible IR-2 being amongst the most potent BTK PROTACs
reported to date. Still, the non-covalent PROTAC NC-1 outperformed IR-2.
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Since Ibrutinib displays nM binding even without covalent bond formation38 (Fig. 6B),
non-covalent BTK PROTACs can be very potent23,39,28, and adding irreversible covalent
binding would primarily be expected to reduce potency due to the loss of catalysis. Indeed, the
noncovalent NC-1 was the most potent PROTAC we tested, similarly to Tinworth et al23.
However, very potent degradation was also observed with acrylamide PROTACs such as IR-
2. We observed that IR-2 forms covalent bonds slowly relative to the rate of degradation, most
likely due to the lower reactivity of substituted acrylamides, and therefore much of its activity
may have been derived from reversible binding. Tinworth et al.23 tested irreversible covalent
PROTACs based on CRBN and IAP binders, which were inactive and were also substituted
acrylamides. These PROTACs harbored a piperazine moiety in the linker, attached one carbon
away from the acrylamide group, which may affect reactivity and PROTAC binding. However,
in vitro kinase assays using wild type and mutant BTK had similar results to those reported
here, with their acrylamide PROTAC exhibiting essentially the same IC50 towards BTK as the
non-covalent counterpart. Therefore, the covalent PROTACs tested by Tinworth et al. most
likely also have formed covalent bonds inefficiently, and their inactivity may have resulted
from issues such as permeability, stability or an unfavorable geometry. Conversely, Xue et al.22
recently developed unsubstituted acrylamide BTK PROTACs that covalently engaged BTK
and degraded it the cell, albeit not to 100%. Along with our study this indicates that
measurement of the relative rates of covalent bond formation and degradation is needed to
estimate how covalent binding affects PROTAC activity.
In parallel to this publication, Guo et al.34 have also reported cyanoacrylamide-based
BTK degraders, using a different linker design. For that series of PROTACs the
cyanoacrylamides were much more potent than the equivalent noncovalent and acrylamide
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PROTACs, which they attribute to significantly higher cell penetration of the
cyanoacrylamides. Their study thus support the use of reversible covalent PROTACs but
makes it difficult to draw conclusions regarding the role of the covalent bond in the
degradation. Here, RC-3 and NC-1 penetrated the cells to a similar degree, and both bind BTK
reversibly, with RC-3 showing much better IC50 (Fig. 6A). However, NC-1 is still a more
efficient BTK degrader. We suggest two hypotheses for this discrepancy: First, the non-
covalent NC-1 has a much less rigid linker than IR-2 and RC-3, with free rotation around the
bond proximal to the amide linkage. This flexibility may aid the PROTAC in adopting the
optimal configuration for the ternary complex formation and for ubiquitination, or increase the
stability of the interaction of the BTK-PROTAC complex with the E3 ligase5,7, which is likely
more relevant to degradation efficiency, and may also explain the ability of NC-1 to compete
with thalidomide (Fig. 3A) compared to the other PROTACs. Second, the non-covalent NC-1
has a rapid binding and dissociation equilibrium – in the presence of preincubated NC-1,
Ibrutinib labels BTK fully within 1 hour (Supp. Fig. 4). Therefore, NC-1 can bind BTK in the
cell, promote the formation of the ternary complex to induce ubiquitination, and quickly
dissociate to bind more BTK molecules, even before the ubiquitinated BTK undergoes
proteasomal degradation. The cyanoacrylamides tested here dissociate in timescales of 10-20
hours, similar to the residence times observed for other cyanoacrylamide inhibitors32.
Therefore, they can only be recycled after the bound BTK molecule has been degraded,
resulting in less efficient catalysis.
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While RC-3 was not as potent as NC-1 in BTK degradation, it did have a significant
advantage in selectivity. The addition of the cyanoacrylamide with the geminal dimethyl group
greatly diminished the reversible binding affinity (which was observed for other
cyanoacrylamide inhibitors of BTK27), while maintaining potent covalent binding. This
significantly reduced the activity against the noncovalent off targets LYN and CSK.
We conclude that reversible covalent PROTACs hold promise for selective degradation
of challenging targets for which no high affinity reversible ligand is available, and these are
the targets where the benefits of covalent PROTACs are likely to be most evident.
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Methods
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General outline of reversible covalent PROTAC synthesis
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To synthesize reversible covalent PROTACs, we prepared PEG-monotosylates of
different lengths and coupled them to 4-OH-thalidomide to generate thalidomide-PEG-OH
constructs (Supplementary Material). These were oxidized to aldehydes, followed by an aldol
condensation with the BTK inhibitor cyanoacetate to generate the cyanoacrylates. During the
condensation, the ether linkage nearest the cyanoacrylate in RC-1 and RC0a-j was frequently
cleaved and higher molecular adducts were formed, as observed by LC/MS measurements. In
the synthesis of RC-2 and RC-3 (where the last ether linkage was replaced with a CH2 group
or C(CH3)2), the condensation was considerably slower with reduced unwanted side reactions
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and higher yield (see Supplementary Material for synthesis procedures). H and C NMR
spectra were recorded on a 11.7T Bruker AVANCE III HD spectrometers. Chemical shifts are
reported in ppm on the δ scale downfield from TMS and are calibrated according to the
deuterated solvents (see supplementary material).
Western Blotting
Ramos (ATCC, CRL-1596), Mino (ATCC, CRL-3000) or K562 (NCI-60) cell lines
were counted and diluted to 106 cell/ml, using 1 mL per well in a 24-well plate, and U2OS
(ATCC HTB-96) cells over expressing human BTK WT or C481S mutant were grown using 2
mL in a 6-well plate (see supplementary methods). Cells were incubated with 1% DMSO or
compound in indicated concentrations for 24 hours unless indicated differently or cells were
left untreated. Lysates were prepared as previously described29, and samples were measured
for total protein quantification by Bicinchoninic Acid (BCA) assay (#23225 ThermoFisher
Scientific). supplemented with 4x loading buffer including 20 mM DTT, heated to 70°C for 10
minutes and loaded into 4% SDS-PAGE gel, run for 45 minutes at 140 mV, then transferred
into nitrocellulose membrane (Biorad) using Trans-Blot Turbo transfer system (Biorad).
Membrane was stained with Ponceau (Sigma) to validate transfer for 10 minutes in gentle
agitation then de-stained for 1 hour with MQ water. Membrane was blocked with Licor
blocking buffer (LIC927-70001) for 1 hour, washed three times for 5 minutes with TBS-T and
incubated with primary antibody BTK (D3H5) Rabbit mAb (CST; 8547 S) overnight (16
hours) at 4°C, washed three times for 5 minutes with TBS-T and incubated with primary
antibody against β-actin (CST; 3700) for 1 hour at 25°C. Membrane was washed three times
for 5 minutes with TBS-T and incubated with a fluorescent secondary antibodies Anti-Mouse
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IgG Atto 488 (Sigma; 62197) and Anti-Rabbit-IgG Atto 647N (Sigma; 40839) for 1 hour, then
washed three times for 5 minutes with TBS-T, dried and immediately imaged and analysed
using Licor odyssey CLx. Prism (GraphPad) software was used to calculate degradation levels,
and we used second order polynomial fit to estimate DC50 and Dmax values.
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In vitro Activity Assays for BTK (Carried out by Nanosyn, Santa Clara, CA)
Test compounds were diluted in DMSO to a final concentration that ranged from 2 μM
to 11.3 pM, while final concentration of DMSO in all assays was kept at 1%. The compounds
were incubated with BTK for 2 hours in a 2x buffer containing the following: 1.2 nM BTK,
100 mM HEPES pH = 7.5, 10 mM MgCl2, 2 mM DTT, 0.1% BSA, 0.01% Triton X-100, 20
μM Sodium Orthovanadate and 20 μM Beta-Glycerophosphate. Reaction was initiated by two-
fold dilution into a solution containing 5 μM ATP (50 μM for C481S) and substrate. The
reference compound Staurosporine was tested in a similar manner.
In vitro BTK Binding assays
Binding experiments were performed in Tris 20 mM pH = 8, 50 mM NaCl, 1 mM DTT.
BTK kinase domain was diluted to 2 uM in buffer, and 3 uM PROTAC was added by adding
1/100th volume from a 300 µM solution. The PROTACs were incubated with BTK at room
temperature for various times. For testing by LCMS, 24 µl of the solution were mixed with 6
µl of 2.4% formic acid, and 10 µl were injected to LCMS.
For binding reversibility experiments, the PROTACs were incubated with BTK for 2
hours at room temperature, followed by addition of 40 µM Ibrutinib (by addition of 1/100th
volume of a 4 mM solution in DMSO). The samples were incubated with Ibrutinib at 37°C for
various times and tested by LCMS as described before. For the noncovalent PROTAC NC-1,
Ibrutinib was added to the complex at a concentration of 4 µM and incubated for 1 hour at
room temperature.
For binding experiments in the presence of glutathione, freshly dissolved reduced
glutathione was incubated at 6.14 mM with 4 M compounds for 30 minutes at room
temperature in Tris 20 mM pH = 8, 50 mM NaCl. At this point BTK was added to a
concentration of 2 M (diluting the GSH to 5 mM and the compounds to 3.25 M) and LC-
MS was used as described previously to follow the covalent labeling of BTK.
For data analysis, the raw spectra were deconvoluted using a 27000:37000 Da window
and 1 Da resolution. The signal from masses 27000:30000 and 34000:37000 (which contained
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no peaks) was averaged and subtracted from the whole signal. The peaks of each species were
integrated using a 100 Da window in every direction (reducing the window down to 10 Da did
not change the results significantly).
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Supporting Information
 Additional information including detailed experimental methods, description of the
Reversible covalent BTK PROTAC library and its degradation results in two cell lines,
BTK degradation by PROTACs in Ramso cells, data on cellular penetration of
PROTACs, time dependency of BTK degradation, validation of CRBN mediated
degradation, kinetic studies of BTK labeling using LC/MS, and GSH effect on labeling,
proteomics selectivity analysis for the PROTACs at additional concentrations, B cell
receptor signaling inhibition data, detailed synthetic protocols for preparation of
compounds with high resolution mass spectrometry and NMR analysis (PDF).
 Proteomics dataset file: full lists of proteins identified in the proteomics, with
quantification of the changes in abundance and p-values derived from t tests (XLSX).
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Acknowledgments
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We thank Dr. Haim Barr and Dr. Christian Dubiella for critical reading of the
manuscript. S.A. is the incumbent of Prof. David Casson Research Fellowship, N.L. is the
incumbent of the Alan and Laraine Fischer Career Development Chair; N.L. would like to
acknowledge funding from the Israel Science Foundation (grant No. 2462/19), The Rising Tide
Foundation, The Israel Cancer Research Fund, the Israeli Ministry of Science. Technology
(grant No. 3-14763), and the Moross integrated cancer center. N.L. is also supported by the
Helen and Martin Kimmel Center for Molecular Design, Joel and Mady Dukler Fund for
Cancer Research, the Estate of Emile Mimran and Virgin JustGiving and the George
Schwartzman Fund. R.G. was supported by the state of Israel, ministry of Aliyah, Center for
Integration in Science. Y.H. is supported by grants from the Israeli Science Foundation
(1707/19), Israeli Cancer Association and Sackler Faculity of Medicine, Tel-Aviv University.
We thank Dr. Ville Paavilainen for the BTK plasmid. The proteomics work was supported by
the De Botton Protein Profiling institute of the Nancy and Stephen Grand Israel National Center
for Personalized Medicine, Weizmann Institute of Science. We thank Dr. Sergey Malitsky from
the Life Sciences Core Facilities at Weizmann Institute of Science for use of the LC-MS/MS
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Figure 1. Structures of reversible covalent, irreversible covalent and non-covalent BTK PROTACs described in
this study. The electrophilic moieties are highlighted in red.
84x78mm (600 x 600 DPI)
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Figure 2: Efficient BTK degradation in cells.A. Western blot evaluation of BTK levels in Mino cells in response
to various concentrations of RC-2, IR-2, NC-1 and RC-3, after 24h incubation.B. Quantification of BTK levels
in (A) by normalization to the -actin house-keeping gene in Mino cells. DC50 and Dmax were calculated by
fitting the data to a second order polynomial using Prism software.C. A summary of the DC50 and Dmax
values for the PROTACs in Mino cells describe in (A) and Supp. Fig. 2.
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Figure 3: PROTAC mediated BTK degradation is hindered by Ibrutinib, thalidomide and by proteasome
inhibition.A. Mino cells were either pre-treated for 2 hours with Ibrutinib/thalidomide-OH or untreated,
before treatment with a BTK PROTAC for 24 hours. Subsequently BTK levels were measured via western
blot.B. Mino cells were treated for 1 hour with Bortezomib to inhibit proteasome-dependent degradation,
then PROTACs were added for 4 hours, followed by measuring BTK levels via western blot.
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Figure 4: Degradation of BTK in patients-derived CLL cells
Patients-derived primary CLL cells were treated with BTK PROTACs for 18 hours, followed by measuring BTK
levels via western blot. M-IGHV/UM-IGHV: Mutated/Unmutated immunoglobulin heavy chain variable region
(IGHV) gene.
84x100mm (300 x 300 DPI)
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Figure 5: Degradation of overexpressed BTK and BTK C481S in U2OS cells
A. Transfected U2OS cells were treated with 100 nM PROTAC for 24 hours, followed by measuring BTK levels
via western blot.
B. Quantification of normalized BTK levels in (A).
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