Selective CDK6 degradation mediated by cereblon, VHL, and novel IAP-recruiting PROTACs

Article abstract of DOI:10.1016/j.bmcl.2020.127106

Inhibitors of CDK4 and CDK6 have emerged as important FDA-approved treatment options for breast cancer patients. The properties and pharmacology of CDK4/6 inhibitor medicines have been extensively profiled, and investigations into the degradation of these

Full text of DOI:10.1016/j.bmcl.2020.127106

Journal Pre-proofs
Selective CDK6 degradation mediated by cereblon, VHL, and novel IAP-re‐
cruiting PROTACs
Niall A. Anderson, Jenni Cryan, Adil Ahmed, Han Dai, Grant A. McGonagle,
Christine Rozier, Andrew B. Benowitz
PII:
S0960-894X(20)30195-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127106
BMCL 127106
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
3 February 2020
9 March 2020
Please cite this article as: Anderson, N.A., Cryan, J., Ahmed, A., Dai, H., McGonagle, G.A., Rozier, C.,
Benowitz, A.B., Selective CDK6 degradation mediated by cereblon, VHL, and novel IAP-recruiting PROTACs,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127106
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Selective CDK6 degradation mediated by cereblon, VHL, and novel IAP-recruiting PROTACs
†
†
Niall A. Anderson , Jenni Cryan , Adil Ahmed, Han Dai, Grant A. McGonagle, Christine Rozier, Andrew
B. Benowitz*
Medicine Design, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire, SG1 2NY, United Kingdom
†
These authors contributed equally
*
Corresponding author: andrew.b.benowitz@gsk.com
Abstract
Inhibitors of CDK4 and CDK6 have emerged as important FDA-approved treatment options for breast
cancer patients. The properties and pharmacology of CDK4/6 inhibitor medicines have been
extensively profiled, and investigations into the degradation of these targets via a PROTAC strategy
have also been reported. PROTACs are a novel class of small-molecules that offer the potential for
differentiated pharmacology compared to traditional inhibitors by redirecting the cellular ubiquitin-
proteasome system to degrade target proteins of interest. We report here the preparation of
palbociclib-based PROTACs that incorporate binders for three different E3 ligases, including a novel
IAP-binder, which effectively degrade CDK4 and CDK6 in cells. In addition, we show that the
palbociclib-based PROTACs in this study that recruit different E3 ligases all exhibit preferential CDK6
vs. CDK4 degradation selectivity despite employing a selection of linkers between the target binder
and the E3 ligase binder.
Keywords
CDK4; CDK6; PROTAC; palbociclib
Cyclin-dependent kinase (CDK) 4 and CDK6 are cell-cycle regulatory kinases that can become
dysregulated in cancer.1-3 Along with cyclin D, they play key roles in regulating the transition point
between the G1- and S-phase during mitosis. In isolation, CDK4 and CDK6 lack intrinsic catalytic
kinase activity, but their kinase activity becomes functional when these proteins bind to members of
the cyclin D family. When activated, CDK4-cyclin D and CDK6-cyclin D complexes function by
initiating the phosphorylation of retinoblastoma protein (Rb). Rb is a tumor suppressor protein that
normally exerts a repressive effect on cell cycling until the cell is ready to enter mitosis.4, 5 However,
upon phosphorylation, Rb no longer suppresses cell cycle progression, and inappropriate Rb
6
phosphorylation by CDK4 and CDK6 complexes can be an important oncogenic driver. Inhibition of
CDK4 and CDK6 has proven to be a clinically validated strategy for the treatment of metastatic and
advanced ER+ HER- breast cancer, and palbociclib, ribociclib, and abemaciclib are FDA-approved
CDK4/6 inhibitor medicines currently available to patients (Figure 1).

Figure 1: FDA-approved CDK4/6 inhibitors.
H
H
O
N
N
N
N
O
N
N
N
N
N
O
N
N
N
N
NH
NH
1
: palbociclib
2: ribociclib
F
N
N
H
N
N
N
N
N
N
F
3
: abemaciclib
In addition to the described kinase-dependent functions of both CDK4 and CDK6, important kinase-
independent biology associated with CDK6 has been reported, including correlation of CDK6 levels
7
with tumor blood vessel density and with Vegf-A mRNA and protein levels in cancer cell lines. Since
inhibitors would not be expected to have any effect on these or other kinase-independent
scaffolding functions of CDK6, we were interested in exploring the degradation of this protein
through the use of a PROTAC (Proteolysis Targeting Chimera).8
PROTACs have emerged as an important new small-molecule modality to study the biology of
medicinally important targets, and as potential medicines in their own right.9-13 They function by
redirecting the cellular ubiquitin-proteasome system (UPS) to tag specifically selected proteins for
proteasomal degradation via E3 ligase complex-mediated ubiquitin transfer, thereby effecting a
chemically-induced knock-down of the target protein. Cereblon-recruiting PROTACs based on the
dual CDK4/6 inhibitor palbociclib have been reported,14-17 and intriguingly were demonstrated to
selectively degrade CDK6 over CDK4, despite palbociclib itself having approximately equal inhibitory
potency against both CDK4- and CDK6-cyclin D complexes.18
Previous studies have demonstrated that PROTAC composition plays a critical role in determining
degradation selectivity in the case of non-specific target protein binders.19, 20 Selectivity differences
are presumably influenced by the stability and accessible conformations of the requisite ternary
complex between the bound protein, the PROTAC molecule, and the recruited E3 ligase complex,21-23
and differential ternary complex formation has been invoked to explain CDK6 vs. CDK4 degradation
selectivity.14 With selective CDK6 degradation having been previously reported for cereblon-
recruiting palbociclib-based PROTACs, we sought to investigate what effect the choice of E3 ligase
would have on the degradation of these targets by von Hippel-Lindau (VHL)- and Inhibitor of
Apoptosis (IAP)-recruiting palbociclib-based PROTACs.
To probe the PROTAC-mediated CDK4 and CDK6 degradation effects of different E3 ligases, we
prepared a series of compounds (Figure 2) incorporating various linkers and known binders to VHL
1
5
15
(
9
PROTACs 4-6), and cereblon (PROTACs 10, 11, and 12 ), as well as a novel IAP binder (PROTACs 7-
). As has been previously described by others, we viewed the palbociclib piperazine moiety as a
suitable linker design vector for PROTAC construction.

Figure 2: PROTACs prepared to test CDK4 and CDK6 degradation. Each individual PROTAC consists
of the palbociclib-based target binder combined with a linker and an E3 ligase binder according to
the connectivity indicated. PROTACs 4-6 contain the VHL binder shown, PROTACs 7-9 contain the
novel IAP binder shown, and PROTACs 10-12 contain the cereblon binder shown.
H
O
O
N
N
N
N
E3 LIGASE
BINDER
LINKER
N
N
N
PALBOCICLIB-BASED TARGET BINDER
N
HN
O
S
H
N
NH
H
O
N
N
O
N
O
O
NH
HN
O
O
F
O
H
HN
N
O
N
F
OH
VHL (PROTACs 4-6)
IAP (PROTACs 7-9)
Cereblon (PROTACs 10-12)
O
N
H
O
O
PROTAC 4
PROTAC 7
PROTAC 1015
O
O
O
O
O
O
O
O
O
N
O
O
H
PROTAC 5
PROTAC 8
PROTAC 11
O
O
H
O
O
O
O
N
O
N
O
N
O
O
O
H
H
O
O
O
PROTAC 6
PROTAC 9
PROTAC 1215
Following preparation of PROTACs 4-12, we evaluated the degradation of CDK4 and CDK6 in Jurkat
cells after 24 h compound treatment in a dose-response manner using capillary electrophoresis
combined with antibody labelling to measure protein levels (Figure 3). As expected, palbociclib 1 did
not cause any significant degradation of CDK4 or CDK6 (Figure SI-1). However, we observed potent
degradation of CDK4 for seven of the nine PROTACs studied, and of CDK6 for eight of the nine
PROTACs studied, encompassing VHL, IAP, and cereblon E3 ligase binders. The exceptions to the
potent target degradation were PROTACs 6 and 9, which were found to weakly degrade CDK4 with
pDC values between 5-6 (Table 1). Additionally, PROTAC 9 was also found to be a weak degrader
5
0
of CDK6, with a pDC of 5.8. The relatively weak target degradation potency of PROTACs 6 and 9
5
0
may be related to the linker connecting the palbociclib-based target binder and the E3 ligase
binders, since the analogous VHL-recruiting PROTACs 4 and 5, as well as the analogous IAP-recruiting
PROTACs 7 and 8 were each found to be potent degraders of both CDK4 and CDK6. With the
exception of PROTACs 6 and 9, there were no meaningful differences found between the ability of

the PROTACs incorporating VHL, IAP, or cereblon ligase binders to cause the degradation of CDK4
and CDK6 as measured by their respective pDC values.
5
0
The most potent CDK6 degrader that we observed was the previously reported short linker cereblon-
1
5
recruiting PROTAC 10, with a CDK6 pDC of 9.1. This compound was also found to potently cause
5
0
the degradation of CDK4, with a pDC of 8.0. It is noteworthy that this degradation selectivity
5
0
preference for CDK6 over CDK4 was maintained for each compound studied ranging from a  pDC50
of 0.5 to 1.5 (pDC CDK6 – pDC CDK4), and that even the weak CDK4/6 degrader PROTAC 9 was
5
0
50
found to selectively degrade CDK6 over CDK4 with a  pDC of 0.5 (Table 1).
5
0
Figure 3. Dose-response protein level curves for CDK4 (blue symbols) and CDK6 (red symbols) in
Jurkat cells measured after 24 h treatment with PROTACs 4-12. Individual data points are the
average of three experiments. Error bars indicate standard deviation.
PROTAC 4
PROTAC 5
PROTAC 6
PROTAC 7
PROTAC 8
PROTAC 9
PROTAC 10
PROTAC 11
PROTAC 12
To explore whether the differences in the observed CDK4 and CDK6 degradation potency for these
compounds might be related to differences in CDK4 and CDK6 inhibitory potencies, we determined
the inhibitory potencies of PROTACs 4-12 as well as palbociclib 1 against CDK4-cyclin D1 and CDK6-
cyclin D3 (Table 1). Although the measured inhibitory potency of palbociclib 1 against CDK4-cyclin
D1 (pIC 7.5) and CDK6-cyclin D3 (pIC 7.0) in our assay was reduced as compared to the reported
5
0
50
1
8
inhibitory potencies for this compound (CDK4-cyclin D1 pIC 8.0, CDK6-cyclin D2 pIC 7.8), we
5
0
50
observed sub-micromolar inhibition of CDK4-cyclin D1 and CDK6-cyclin D3 for all compounds tested.
However, there was little correlation between CDK4/6-cyclin D inhibitory potency and CDK4/6
degradation potency (Figure 4), and it is notable that the weak CDK4/6 degrader PROTAC 9 was still
a potent inhibitor of both CDK4-cyclin D1 and CDK6-cyclin D3, and that the 200 nM IC CDK6-cyclin
5
0
D3 inhibitor PROTAC 4 was surprisingly found to be a 4 nM DC CDK6 degrader.
5
0

2
4
Table 1. CDK4/6 inhibition and degradation potency for palbociclib 1 and PROTACs 4-12.
Experimentally determined data are reported as the average of three experiments.
Compound:
1
4
5
6
7
8
9
1015
11
1215
CDK4-cyclin D1
pIC50
CDK6-cyclin D3
pIC50
7
.5
.0
7.5
7.4
7.5
8.3
8.2
7.8
8.5
8.2
7.7
7
6.7
7.6
8.4
0.8
6.8
6.2
7.7
1.5
6.9
5.6
6.7
1.1
7.9
7.7
8.3
0.6
7.8
7.1
7.8
0.7
7.4
5.3
5.8
0.5
8.1
8.0
9.1
1.1
7.8
7.3
8.3
1.0
7.3
6.9
7.9
1.0
CDK4 pDC50
CDK6 pDC50
<4.0
<4.0
-
CDK6-CDK4 
pDC50
Figure 4. Comparison of CDK4-cyclin D1 and CDK6-cyclin D3 inhibitor potency vs. CDK4 and CDK6
degradation potency.
To determine whether the observed CDK4 and CDK6 degradation was UPS-dependent, we pre-
treated Jurkat cells with 1 M of the known proteasome inhibitor epoxomicin, followed individually
1
5
by PROTACs 4, 7, and 10, representing compounds that recruit VHL, IAP, and cereblon,
respectively. Measuring CDK4 and CDK6 protein levels after 5 h treatment with each PROTAC
following epoxomicin treatment clearly demonstrated an inhibition of protein loss (Figure 5),
indicating that CDK4/6 protein level decreases caused by PROTAC treatment in the absence of
epoxomicin was due to proteasomal degradation of these targets. Interestingly, we also observed a
1
5
putative Hook effect after 5 h treatment with PROTAC 10 in the absence of epoxomicin, as well as
after 24 h treatment with PROTAC 5 (Figure 3). The appearance of a Hook effect is indicative of
2
5
competing binary and ternary complex formation, and provides additional evidence for a PROTAC-
mediated ubiquitin transfer and degradation mechanism.

Figure 5. Dose-response protein level curves for CDK4 (blue symbols) and CDK6 (red symbols) in
Jurkat cells measured after 5 h treatment without and with epoxomicin and with PROTACs 4, 7, and
1
0.15 No curves could be fitted to the data generated from treatment with epoxomicin and the
PROTACs. Individual data points are the average of three experiments, except for the data points for
PROTAC 10 with no epoxomicin treatment, which are the average of two experiments. Error bars
indicate standard deviation, where present.
PROTAC 4
PROTAC 7
PROTAC 1015
No epoxomicin
No epoxomicin
No epoxomicin
PROTAC 4
PROTAC 7
PROTAC 1015
With epoxomicin
With epoxomicin
With epoxomicin
In contrast to a recent report indicating that neither CDK4 nor CDK6 was degraded with VHL or IAP
1
7
PROTACs, we observed potent degradation of both CDK4 and CDK6 with VHL- and with novel IAP-
recruiting palbociclib-based PROTACs incorporating multiple linkers between the target binding and
the E3 ligase binding moieties. Surprisingly, in addition to confirming the previously reported CDK6
vs. CDK4 degradation selectivity with cereblon-recruiting palbociclib-based PROTACs, we observed
that the preference for CDK6 degradation over CDK4 degradation was maintained for VHL- and IAP-
recruiting palbociclib-based PROTACs with all linkers studied. This preference for selective CDK6 vs.
CDK4 degradation may be due to differential ternary complex effects related to the choice of
palbociclib as the target binding moiety, as recent reports have shown that cereblon-recruiting
ribociclib-based PROTACs induced preferential degradation of CDK4 over CDK6.14, 17 Additional
investigations studying differences in ternary complex formation and stability between palbociclib-
and ribociclib-based PROTACs may be required to more fully understand the nature of degradation
selectivity differences between CDK4 and CDK6 induced by these compounds.
Acknowledgements
The authors would like to acknowledge John Harling for helpful discussions.
Supplementary Material
Full synthetic routes, experimental procedures, and Figure SI-1 are available on-line in the
Supplementary Information.

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CDK4-cyclin D1 and CDK6-cyclin D3 inhibition data for all compounds tested was generated
2
6
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: