Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL

Article abstract of DOI:10.1002/anie.201507634

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: The identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

Full text of DOI:10.1002/anie.201507634

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201507634
German Edition: DOI: 10.1002/ange.201507634
Drug Design
Modular PROTAC Design for the Degradation of Oncogenic BCR-
ABL
Ashton C. Lai+, Momar Toure+, Doris Hellerschmied, Jemilat Salami, Saul Jaime-Figueroa,
Eunhwa Ko, John Hines, and Craig M. Crews*
Abstract: Proteolysis Targeting Chimera (PROTAC) technol-
ogy is a rapidly emerging alternative therapeutic strategy with
the potential to address many of the challenges currently faced
in modern drug development programs. PROTAC technology
employs small molecules that recruit target proteins for
ubiquitination and removal by the proteasome. The synthesis
of PROTAC compounds that mediate the degradation of c-
ABL and BCR-ABL by recruiting either Cereblon or Von
Hippel Lindau E3 ligases is reported. During the course of
their development, we discovered that the capacity of
a PROTAC to induce degradation involves more than just
target binding: the identity of the inhibitor warhead and the
recruited E3 ligase largely determine the degradation profiles
of the compounds; thus, as a starting point for PROTAC
development, both the target ligand and the recruited E3 ligase
should be varied to rapidly generate a PROTAC with the
desired degradation profile.
tial to replace the need for continuous treatment with a cure
for CML.
Recently, our lab and other groups have developed
a small-molecule drug platform that works by protein
degradation and has the potential to address the challenges
faced in current drug development programs.[10–13] Proteolysis
Targeting Chimera (PROTAC) technology utilizes hetero-
bifunctional small molecules whereby one end of the mole-
cule recruits an E3 ubiquitin ligase while the other end
engages the target protein.[14] Upon ternary complex forma-
tion, the recruited E3 ligase ubiquitinates the target, leading
to subsequent degradation by the proteasome (Figure 1A). In
C
hronic myelogenous leukemia (CML) is most often caused
by the loss of autoinhibitory constraints on the c-ABL kinase
domain in the oncogenic fusion protein BCR-ABL. This
constitutively active tyrosine kinase drives uncontrolled
cellular proliferation through STAT5, MAPK, PI3K/Akt,
and CrkL signaling pathways.[1–3] With the advent of tyrosine
kinase inhibitors (TKIs) targeting BCR-ABL, CML has
become a chronic but manageable disease. Imatinib mesylate,
the first TKI developed against BCR-ABL, binds compet-
itively at the ATP-binding site of c-ABL and inhibits both c-
ABL and BCR-ABL, leading to inhibition of cell prolifer-
ation and apoptosis of non-progenitor leukemic cells.[4,5]
Second-generation TKIs (such as dasatinib, bosutinib) were
subsequently developed to treat CML patients with acquired
resistance to imatinib.[6] Despite the remarkable success of
BCR-ABL TKIs, all CML patients must remain on lifelong
treatment owing to persistent leukemic stem cells (LSCs) in
spite of BCR-ABL inhibition. One hypothesis suggests that
BCR-ABL acts as a protein scaffold for compensatory
signaling pathways, allowing LSCs to survive kinase inhib-
ition.[7–9] Therefore, knockdown of BCR-ABL has the poten-
Figure 1. Approach to PROTAC development. A) PROTACs act through
proximity-induced ubiquitination, leading to degradation by the protea-
some. B) Overlay of bosutinib (blue; PDB: 3UE4) onto c-ABL-dasatinib
crystal structure (yellow; PDB: 2GQG). Linkers were attached via the
solvent exposed site (red circle). C) Linkers utilized to connect the
respective TKI to the E3 recruiting ligand.
contrast to inhibitor-based pharmacology, PROTAC technol-
ogy requires only transient binding to any surface of the target
to catalytically induce ubiquitination and degradation; thus,
PROTACs have emerged as a novel therapeutic approach to
target so called “undruggable” proteins and have successfully
been employed to degrade several proteins such as the
estrogen-related receptor alpha,[13] cellular retinoic acid bind-
ing proteins,[15] and BRD4.[10–12] Despite these prior success
stories, there have been no examples of PROTAC-induced
degradation of tyrosine kinases thus far.[13] In this study, we
sought to induce degradation of the BCR-ABL fusion protein
as an archetypical tyrosine kinase implicated in cancer.
Herein, we describe the successful development of the
first PROTACs that induce the degradation of an oncogenic
tyrosine kinase, BCR-ABL. In the development process, we
[*] A. C. Lai,[+] Dr. M. Toure,[+] Dr. D. Hellerschmied, J. Salami,
Dr. S. Jaime-Figueroa, Dr. E. Ko, Dr. J. Hines, Prof. C. M. Crews
Departments of Chemistry; Molecular, Cellular & Developmental
Biology; Pharmacology, Yale University
New Haven, CT 06511 (USA)
E-mail: craig.crews@yale.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507634.
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evolved a synthetic strategy for PROTAC design that
incorporates variations in both warhead and E3 ligase ligands
and allows one to rapidly assess the degradation profiles of
PROTAC families.
phosphorylation of CrkL and STAT5 at higher concentrations
(Supporting Information, Figures S1–S4). Since the imatinib
warhead did not engage c-ABL or BCR-ABL to the same
extent as the parent compound in cell culture, we hypothe-
sized that a more potent inhibitor warhead (bosutinib or
dasatinib) would be a better choice for the next PROTAC
series.
Through a similar synthetic route, we conjugated bosuti-
nib to the VHL recruiting ligand, producing bosutinib-VHL
(BOS-VHL) PROTACs. Despite target engagement as deter-
mined by inhibition of downstream signaling, the BOS-VHL
PROTACs also did not induce degradation of BCR-ABL or
c-ABL (Figure 2A). This finding was consistent across several
To produce BCR-ABL degrader compounds, we conju-
gated BCR-ABL TKIs (imatinib, bosutinib, and dasatinib)
that bind the c-ABL kinase domain, to a known Von Hippel
Lindau (VHL) E3 ubiquitin ligase ligand or to the thalido-
mide derivative, pomalidomide, to recruit Cereblon (CRBN)
E3 ligase.[10,13,16,17] The resulting bifunctional compounds are
expected to bind BCR-ABL by the TKI moiety and VHL or
CRBN via its recruiting ligand. Using the crystal structures of
the c-ABL kinase domain in complex with the TKIs (imatinib,
dasatinib, and bosutinib), we were able to predict the best
position to attach our linkers to the respective TKIs such that
critical binding interactions were not disrupted (Fig-
ure 1B).[18–20] Four different linkers of varying composition
and length were evaluated as a survey of the potential
chemical space (Figure 1C). These linkers contain a mixture
of hydrophobic and hydrophilic moieties to balance the
hydrophobicity/hydrophilicity of the resulting hybrid com-
pounds. The panel of hybrid compounds was then assayed for
retention of binding to c-ABL kinase domain through
KinomeScan (Table 1). All of the compounds lost affinity
for the phosphorylated and non-phosphorylated form of ABL
compared to the parent compound.
Table 1: Selected PROTAC affinities for the ABL kinase domain.[a]
Compound
ABL
ABL
(non-phosphorylated)
(phosphorylated)
Imatinib
0.86
4.3
6.2
0.063
1.4
0.91
0.03
0.92
0.60
36
93
110
0.023
0.63
0.55
0.02
0.47
0.32
IMA-6-2-2-6-VHL
IMA-6-2-2-6-CRBN
Bosutinib
BOS-6-2-2-6-VHL
BOS-6-2-2-6-CRBN
Dasatinib
Figure 2. VHL-based PROTACs. A) BOS-6-2-2–6-VHL, and B) DAS-6-2-
2-6-VHL were incubated with K562 human chronic myelogenous
leukemia cells for 24 h. The concentrations of the parent inhibitors are
1 mm. As determined by immunoblot, degradation of c-ABL can be
observed with DAS-VHL starting at 1 mm; however, no degradation of
BCR-ABL was observed in any of the VHL-based PROTACs.
DAS-6-2-2-6-VHL
DAS-6-2-2-6-CRBN
[a] Affinity values in nm.
different linkers connecting the bosutinib inhibitor and the
VHL recruiting ligand (Supporting Information, Figures S2–
S4). The representative blot shown in Figure 2A is of BOS-
VHL with the linker 1-{2-[2-(hexyloxy)ethoxy]ethoxy}hexane
hereby designated as 6-2-2-6, which refers to the alkyl/ether
composition.
Along with the BOS-VHL series, we also incorporated
dasatinib as the ligand binding warhead. In contrast to the
IMA-VHL and BOS-VHL PROTACs, dasatinib-based
PROTAC (DAS-VHL) induced a clear (> 65%) decrease of
c-ABL at 1 mm PROTAC concentration (Figure 2B). The
apparent decrease of protein degradation seen at higher
PROTAC concentrations (10 mm) has been observed with
other PROTACs and is attributed to the formation of
separate c-ABL-PROTAC and VHL-PROTAC dimers
rather than the c-ABL-PROTAC-VHL trimeric complex
required for productive ubiquitination.[13,21] The c-ABL
degradation seen with the prototype DAS-VHL PROTAC
was consistently observed with PROTACs possessing differ-
Interestingly, within each warhead series, the 1,5-bis-
(hexyloxy)pentane linker (hereby designated 6-5-6) yielded
compounds with the most significant loss (maximum 86-fold)
in binding affinity (Supporting Information, Table S1). Spec-
ulatively, the more hydrophobic nature of 6-5-6 could lead to
the compound folding back on itself; therefore, there could be
a larger entropic cost to binding. Despite some loss in affinity,
all hybrid compounds still bound non-phosphorylated c-ABL
in the low nanomolar range (0.28 nm–24 nm). The bosutinib-
and dasatinib-based PROTACs bound phosphorylated c-
ABL in the high picomolar range (88 pm–1500 pm).
All PROTACs were then tested for c-ABL and BCR-
ABL degradation in cell culture. Surprisingly, no degradation
of BCR-ABL or c-ABL was observed in K562 CML cells
when treated with imatinib-VHL (IMA-VHL) or imatinib-
CRBN (IMA-CRBN) PROTACs, despite the fact that the
PROTACs bound their targets, as evidenced by the reduced
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ent linkers (Supporting Information, Figures S2–S4). Thus, we
where, despite target engagement, no degradation of c-ABL
have found that, independent of simple target binding, the
inhibitor warhead (imatinib, bosutinib, or dasatinib) largely
determines the capability of a PROTAC to induce c-ABL
degradation.
or BCR-ABL was observed. Thus, the inactive BOS-VHL
compounds were converted to active BCR-ABL and c-ABL
degrader compounds by switching to the CRBN E3 ligase. As
demonstrated by these two inhibitor warhead series, the
oncogenic fusion protein BCR-ABL is differentially suscep-
tible to PROTAC-mediated degradation, depending on the
E3 ligase (VHL or CRBN) recruited to the target.
As BCR-ABL degradation can be observed at 25 nm with
the DAS-6-2-2-6-CRBN PROTAC, we next sought to deter-
mine the cellular effects of the PROTAC (Figure 3B). In
a cell viability assay, we determined that DAS-6-2-2-6-CRBN
is active against BCR-ABL driven K562 with a half-maximal
response concentration (EC50) of 4.4 Æ 2.1 nm (Figure 4).
Despite the success of DAS-VHL with the c-ABL
degradation, no degradation of BCR-ABL was seen with
any of the VHL-based PROTACs. This lack of degradation
cannot be attributed to loss of binding affinity, as these VHL-
based PROTACs still bind and inhibit both c-ABL and BCR-
ABL in cell culture (Figure 2). Since E3 ligase presentation to
the target is important for ubiquitination of an available
lysine residue, we speculated that a differently oriented E3
ligase is required for adequate ubiquitination and degradation
of BCR-ABL. Given the recent success of induced protein
degradation through the recruitment of the Cereblon
(CRBN) E3 ligase, we hypothesized that a switch to the
CRBN E3 ligase may allow us to access BCR-ABL for
degradation as well as provide the first head-to-head compar-
ison between the two E3 ligases.[10,11]
When dasatinib was conjugated to pomalidomide to
recruit CRBN, the dasatinib-CRBN (DAS-CRBN)
PROTAC not only retained its ability to induce degradation
of c-ABL (> 85% at 1 mm) but also induced BCR-ABL
degradation (> 60% at 1 mm), demonstrating the first
PROTAC-induced degradation of an oncogenic tyrosine
kinase (Figure 3). This result was consistent across the several
different linkers used previously in the series of VHL-based
PROTACs (Supporting Information, Figures S1–S4). Even
more strikingly, when the VHL recruiting ligand in the
bosutinib PROTAC series was exchanged for the CRBN
ligand, we observed c-ABL (> 90%) and BCR-ABL
(> 80%) degradation at 2.5 mm (Figure 3A).The accessibility
of BCR-ABL and c-ABL for degradation with the BOS-
CRBN series stands in contrast with the BOS-VHL series
Figure 4. Cell viability with DAS-6-2-2-6-CRBN. This PROTAC is >103-
fold more effective against the BCR-ABL driven cell line K562 over non-
BCR-ABL driven cell lines as determined by CellTiter-Glo Luminescent
Cell Viability assay after a 48 h treatment. Error bars displayed are the
standard error of the median (n=3). Data was normalized to DMSO-
treated controls.
Furthermore, the PROTAC compound is more than 103-fold
less active against the non-BCR-ABL driven cell lines,
HEK293T and SK-BR-3 breast carcinoma. Thus, this
PROTAC compound retains selective activity against the
BCR-ABL driven cell line K562 and will be an excellent tool
for future investigations into kinase-independent roles of
BCR-ABL in LSCs.
In summary, we report the design and synthesis of
bifunctional small molecules based on two potent TKIs
(bosutinib and dasatinib) that mediate the degradation of c-
ABL and BCR-ABL by hijacking either CRBN or VHL E3
ubiquitin ligase. Furthermore, these novel PROTACs are
selective against the BCR-ABL driven cell line K562. In the
course of their development, we have found that changing the
inhibitor warhead and the recruited E3 ligase influences
which protein targets are susceptible to PROTAC-induced
degradation (Figure 5). By varying the recruited E3 ligase, we
have found that the substrate spectrum of PROTACs can be
significantly altered. This last observation hints at the
potential to narrow the selectivity of a promiscuous inhibitor
by creating a more selective degrader via the coupling to
different E3 ligase recruiting ligands. In conclusion, for those
seeking to utilize PROTAC technology for post-translational
protein removal, a starting point for synthesis should focus on
exchanging the identity of the target ligand and the recruited
Figure 3. CRBN-based PROTACs. A) BOS-6-2-2–6-CRBN and B) DAS-6-
2-2-6-CRBN were incubated with K562 cells for 24 h. The concentra-
tions of the parent inhibitors are 1 mm. As determined by immunoblot,
degradation of BCR-ABL and c-ABL can be observed in the DAS-CRBN
and BOS-CRBN series.
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[3] A. Quintµs-Cardama, J. Cortes, Blood 2008, 113, 1619 – 1630.
[4] B. Druker, S. Tamura, E. Buchdunger, Nat. Med. 1996, 2, 561 –
566.
[5] M. Okada, S. Adachi, T. Imai, K. Watanabe, S. Toyokuni, M.
Ueno, A. S. Zervos, G. Kroemer, T. Nakahata, Blood 2003, 103,
2299 – 2307.
[6] X. An, A. K. Tiwari, Y. Sun, P. -R. Ding, C. R. Ashby, Jr., Z.-S.
Chen, Leuk. Res. 2010, 34, 1255 – 1268.
[7] J. Wertheim, K. Forsythe, B. J. Druker, D. Hammer, D.
Boettiger, W. Pear, Blood 2002, 99, 4122 – 4130.
[8] C. V. Ichim, Stem Cells Transl. Med. 2014, 3, 405 – 415.
[9] A. Hamilton, G. V. Helgason, M. Schemionek, B. Zhang, S.
Myssina, E. K. Allan, F. E. Nicolini, C. Müller-Tidow, R. Bhatia,
V. G. Brunton et al., Blood 2011, 119, 1501 – 1510.
[10] J. Lu, Y. Qian, M. Altieri, H. Dong, J. Wang, K. Raina, J. Hines,
J. D. Winkler, A. P. Crew, K. Coleman et al., Chem. Biol. 2015,
22, 755 – 763.
[11] G. E. Winter, D. L. Buckley, J. Paulk, J. M. Roberts, A. Souza, S.
Dhe-Paganon, J. E. Bradner, Science 2015, 348, 1376 – 1381.
[12] M. Zengerle, K.-H. Chan, A. Ciulli, ACS Chem. Biol. 2015, 10,
1770 – 1777.
[13] D. P. Bondeson, A. Mares, I. E. D. Smith, E. Ko, S. Campos,
A. H. Miah, K. E. Mulholland, N. Routly, D. L. Buckley, J. L.
Gustafson et al., Nat. Chem. Biol. 2015, 11, 611 – 617.
[14] K. M. Sakamoto, K. B. Kim, A. Kumagai, F. Mercurio, C. M.
Crews, R. J. Deshaies, Proc. Natl. Acad. Sci. USA 2001, 98, 8554 –
8559.
[15] Y. Itoh, M. Ishikawa, R. Kitaguchi, S. Sato, M. Naito, Y.
Hashimoto, Bioorg. Med. Chem. 2011, 19, 3229 – 3241.
[16] D. L. Buckley, J. L. Gustafson, I. Van Molle, A. G. Roth, H. S.
Tae, P. C. Gareiss, W. L. Jorgensen, A. Ciulli, C. M. Crews,
Angew. Chem. Int. Ed. 2012, 51, 11463 – 11467; Angew. Chem.
2012, 124, 11630 – 11634.
[17] D. L. Buckley, I. Van Molle, P. C. Gareiss, H. S. Tae, J. Michel,
D. J. Noblin, W. L. Jorgensen, A. Ciulli, C. M. Crews, J. Am.
Chem. Soc. 2012, 134, 4465 – 4468.
Figure 5. Summary. Varying the inhibitor warhead and the recruited E3
ligase permits targets to be accessed for degradation. IMA-based
PROTACs did not induce the degradation of c-ABL or BCR-ABL, despite
target engagement.
E3 ligase in order to quickly survey the degradation potential
of small-molecule PROTACs.
Acknowledgements
This work was partially supported by the Leukemia &
Lymphoma Society and the NIH (T32GM067543,
R35CA197589,
R01AI084140,
and
MTSP
TG-
T32GM007205). D.H. is supported by the EMBO Long-
Term Fellowship ALTF-1102-2014.
[18] B. Nagar, W. Bornmann, P. Pellicena, Cancer Res. 2002, 62,
4236 – 4243.
[19] J. S. Tokarski, J. Newitt, C. Y. J. Chang, J. D. Cheng, M.
Wittekind, S. E. Kiefer, K. Kish, F. Y. F. Lee, R. Borzillerri,
L. J. Lombardo et al., Cancer Res. 2006, 66, 5790 – 5797.
[20] N. M. Levinson, S. G. Boxer, PLoS One 2012, 7, e29828.
[21] E. F. Douglass, C. J. Miller, G. Sparer, H. Shapiro, D. A. Spiegel,
J. Am. Chem. Soc. 2013, 135, 6092 – 6099.
Keywords: cancer · drug design · E3 ubiquitin ligases ·
inhibitors · protein degradation
How to cite: Angew. Chem. Int. Ed. 2016, 55, 807–810
Angew. Chem. 2016, 128, 818–821
[1] O. Hantschel, G. Superti-Furga, Nat. Rev. Mol. Cell Biol. 2004, 5,
33 – 44.
[2] O. Hantschel, W. Warsch, E. Eckelhart, I. Kaupe, F. Grebien, K.-
U. Wagner, G. Superti-Furga, V. Sexl, Nat. Chem. Biol. 2012, 8,
285 – 293.
Received: August 14, 2015
Revised: October 7, 2015
Published online: November 23, 2015
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