Selective degradation of CDK6 by a palbociclib based PROTAC

Article abstract of DOI:10.1016/j.bmcl.2019.03.035

Development of selective kinase inhibitors that target the ATP binding site continues to be a challenge largely due to similar binding pockets. Palbociclib is a cyclin-dependent kinase inhibitor that targets the ATP binding site of CDK4 and CDK6 with similar potency. The enzymatic function associated with the kinase can be effectively probed using kinase inhibitors however the kinase-independent functions cannot. Herein, we report a palbociclib based PROTAC that selectively degrades CDK6 while sparing the homolog CDK4. We used competition studies to characterize the binding and mechanism of CDK6 degradation.

Full text of DOI:10.1016/j.bmcl.2019.03.035

Accepted Manuscript
Selective Degradation of CDK6 by a Palbociclib Based PROTAC
Sandeep Rana, Mourad Bendjennat, Smit Kour, Hannah M. King, Smitha
Kizhake, Muhammad Zahid, Amarnath Natarajan
PII:
S0960-894X(19)30174-X
https://doi.org/10.1016/j.bmcl.2019.03.035
BMCL 26349
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2019
21 March 2019
25 March 2019
Please cite this article as: Rana, S., Bendjennat, M., Kour, S., King, H.M., Kizhake, S., Zahid, M., Natarajan, A.,
Selective Degradation of CDK6 by a Palbociclib Based PROTAC, Bioorganic & Medicinal Chemistry Letters
(2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.035
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Selective Degradation of CDK6 by a Palbociclib Based PROTAC
Sandeep Rana,*,#,a Mourad Bendjennat,#,a Smit Kour,a Hannah M. King,a Smitha
Kizhake,a Muhammad Zahid,b Amarnath Natarajan,*,a,c,d
aEppley Institute for Research in Cancer and Allied Diseases, bDepartment of
c
Environmental, Agricultural and Occupational Health, College of Public Health Fred &
d
Pamela Buffett Cancer Center, Department of Pharmaceutical Sciences, University of
Nebraska Medical Center, Omaha, Nebraska 68022, USA
Abstract: Development of selective kinase inhibitors that target the ATP binding site
continues to be a challenge largely due to similar binding pockets. Palbociclib is a
cyclin-dependent kinase inhibitor that targets the ATP binding site of CDK4 and CDK6
with similar potency. The enzymatic function associated with the kinase can be
effectively probed using kinase inhibitors however the kinase independent functions
cannot. Herein, we report a palbociclib based PROTAC that selectively degrades CDK6
while sparing the homolog CDK4. We used competition studies to characterize the
binding and mechanism of CDK6 degradation.
Cyclin dependent kinases are a family of serine-threonine kinases that exert a
variety of functions, which include regulation of cell cycle (CDK1, 2, 4, 6 and 11) and
gene expression (CDK7, and 9) 1-9. CDK4/6 is a therapeutic target for cancer and
palbociclib is the first CDK4/6 selective inhibitor that was approved by the FDA in 2015
for cancer therapy5. CDK4/6 is catalytically inactive, and upon binding to cyclin D,
CDK4/6 is activated resulting in phosphorylation of RB family of proteins. This leads to
the release of RB mediated inhibition of E2F transcription factors. E2F activates many
cell cycle genes including cyclin E that binds to and activates CDK2, which in turn
hyperphosphorylates RB proteins. This feedback loop ensures the irreversible
progression of the cell cycle from G1 to S phase10. Knock out studies have shown that
cyclin D1 and CDK4/6 may be dispensable in normal cells; however, they are critical for
tumor growth11.

Palbociclib inhibits the kinase activity of CDK4-cyclin D1 and CDK6-cyclin D3
complexes. Palbociclib is an ATP competitive kinase inhibitor that binds to the hinge
region of CDK4/6 and inhibits phosphorylation of downstream substrates. In addition to
kinase-dependent cell cycle regulation function of CDK6, a recent report suggests
CDK6 plays a role in transcriptional regulation through a kinase-independent
mechanism12. The available CDK4/6 inhibitors can be used to probe the role of kinase
dependent functions of CDK4/6. However, the lack of selectivity and their inability to
target the non-kinase domain makes them unsuitable to probe the above-mentioned
kinase independent function of CDK6.
To address this, we employed the emerging proteolysis targeting chimera
(PROTAC) based strategy to develop CDK6 selective degrader that will target both
kinase-dependent and kinase-independent CDK6 function. PROTAC is
a
heterobifunctional molecule wherein one fragment interacts with the protein of interest
and the other binds to a component of an E3-ubiquitin ligase and the two are connected
via a linker. PROTAC facilitates the formation of a ternary complex by binding to both
the target protein and either a component of E3 ubiquitin ligase or the E2 ligase. The
resulting ternary complex facilitates poly-ubiquitination of the target protein, which is
subsequently degraded by the proteasome8, 13-20. Recent studies with BET degraders
demonstrated improved inhibition of cancer cell growth and the induction of apoptosis
when compared to the corresponding BET inhibitors15, 16, 18, 21, 22
.
Although the kinase fold of CDK4 and CDK6 are identical, the distribution of
surface exposed lysine residues, which is required for ubiquitination by an E3 ligase in
CDK4 and CDK6 are different (Supplementary Figure S1). We hypothesized that a
PROTAC strategy might yield a selective CDK6 degrader. X-ray crystal structure (pdb:
5L2I) of palbociclib (1) bound to CDK6 showed that nitrogen atoms in the amino-
pyrimidine core of palbocilcib interacts with the hinge region residues of CDK6 and the
piperazine ring is solvent exposed23. Structure-activity relationship (SAR) studies
demonstrated that modifications on the piperazine ring did not result in loss of CDK4/6
binding affinity24. Thus, we speculated that the nitrogen atom of the piperazine ring is
ideally positioned to conjugate the linker to generate bifunctional PROTAC molecules
(Figure 1).

Figure 1: Binding of Palbociclib to CDK6 (PDB code 5L2I). The terminal piperazine ring
is solvent exposed and was used to design to heterobifunctional PROTACs.
We synthesized a set of five PROTAC molecules by conjugating palbociclib (1) to
phthalimide based cereblon E3 ligase ligands (pomalidomide) via flexible linkers with
varying lengths and composition (Figure 2).

Figure 2: Design of palbociclib-based PROTACs.
The synthetic route to access PROTACs (2 – 6) is summarized in Scheme 1.
Briefly, a reaction of palbociclib (1) with t-butyl 2-bromoacetate in N-Methyl-2-
pyrrolidone (NMP) solvent resulted in intermediate 7. Condensation of 4-
fluoroisobenzofuran-1,3-dione (8)16 with commercially available 3-aminopiperidine-2,6-
dione hydrochloride afforded previously reported intermediate 925. Displacement of the
fluoro group with different alkyl amines yielded intermediate 1025, 1126 and 1915 which
were treated with 7 to generate PROTACs 2, 3 and 6 in two steps respectively.
Condensation of commercially available 4-nitroisobenzofuran-1,3-dione (12) with 3-
aminopiperidine-2,6-dione hydrochloride resulted in the formation of 2-(2,6-
dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione which was subsequently reduced under
H2 over 5% Pd/C to yield 13. Treatment of 6-bromohexanoic acid with thionyl chloride
resulted in 6-bromohexanoyl chloride which then refluxed with 13 followed by a
Finkelstein reaction to yield 14. Alkylation of palbociclib (1) with 14 resulted in PROTAC
4. A reaction of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (15) with p-toluenesulfonyl
chloride yielded 2-(2-(2-chloroethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (16).
Alkylation of the hydroxyl group of 17 with 2-(2-(2-chloroethoxy)ethoxy)ethyl 4-
methylbenzene sulfonate (16) followed by a Finkelstein reaction yielded 18. Alkylation of
1 with intermediate 18 yielded PROTAC 5.

Scheme 1: (i) t-butyl 2-bromoacetate, NMP, 70 °C, 3h; (ii) 3-aminopiperidine-2,6-dione
hydrochloride, KOAc, Acetic acid, reflux, 24h; (iii) t-butyl (4-aminobutyl)carbamate,
DMA, DIEA, 80 °C, 2h; (iv) 7, TFA, DCM, 0 °C, 3h; (v) HATU, HOBT, DIEA, DMF, 2h;
(vi) t-butyl (8-aminooctyl)carbamate, DMA, DIEA, 80 °C, 2h; (vii) 3-aminopiperidine-2,6-
dione hydrochloride, KOAc, Acetic acid, reflux, 24h; (viii) H2, 5% Pd/C, DMF, 16h; (ix)
(a) 6-bromohexanoic acid, thionyl chloride, 0 °C, 2h, (b) 13, NEt3, THF, 80 °C, 16h; (x)
NaI, acetone, reflux, 24h; (xi) 1, NEt3, NMP, 70 °C, 12h; (xii) p-toluenesulfonyl chloride,

KOH, DCM; (xiii) 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol, tosyl chloride, K2CO3,DMF, 70
°C, 3h; (xiv) NaI, acetone, reflux, 24h; (xv) t-butyl (3-(2-(2-(3-aminopropoxy)ethoxy)
ethoxy)propyl)carbamate, DMA, DIEA, 80 °C, 2h.
To identify a CDK6 selective degrader we subjected MiaPaCa2 cells to 500 nM
of PROTACs 2 – 6 and assessed degradation of CDK family of kinases by Western blot
analysis (Figure 3). Interestingly, shorter linker length (6-11 atoms) PROTAC 2 – 5
exhibited partial degradation of CDK6 whereas PROTAC 6 with longer linker (17 atoms)
selectively degraded CDK6 (Figure 3A) while sparing other members of the CDK (2, 4,
5, 7 and 9) family and Rb (a non-CDK). Degrader 2, 4, 5 induced partial degradation of
CDK4 but robust degradation of CDK6. Palbociclib inhibits both CDK4 and CDK6,
however, Longer linker length PROTAC 6 selectively degrade CDK6. This could be
because of the significant modifications to palbociclib in PROTAC 6 that resulted in the
loss of CDK4 binding. To assess if the lack of CDK4 degradation is due to lack of
binding of 6 to CDK4 we subjected PROTAC 6 to in vitro cell-free kinase assay (Figure
3B)

Figure 3: Effects of palbociclib-based degraders in MiaPaCa2 cells. (A) Western blot
analyses of a panel of kinases with lysates generated from MiaPaCa2 cells treated with
0.5 M of degrader analogs for 4h (CDK4, 6 and RB) 24h (CDK2, 5, 7 and 9 for 24h).
(B) Cell-free assay showing inhibition of CDK4 and CDK6 with PROTAC 6. (C) Dose-
response studies with different degraders in MiaPaCa2 cells when treated for 24h.
PROTAC 6 inhibited both CDK4 and CDK6 in vitro with similar potency. This
suggests that a stable ternary complex could not be formed with CDK4 thus preventing
its degradation. However, the other possibility that cannot be rule out is the rapid
deubiquitination of CDK4 as a contributing factor for the absence of CDK4 degradation.
Next, we performed a dose-dependent study with PROTACs 2 - 6 to estimate relative
potency. Consistent with the single dose screens, we observed potent and selective
degradation of CDK6 only with PROTAC 6. Together, these studies identified PROTAC
6 as a potent and selective degrader of CDK6.
We next evaluated PROTAC 6 in a dose-response study at 4 and 24 hours in
HPNE and MiaPaCa2 cell lines (Figure 4). At both time points, we observed nearly
quantitative degradation of CDK6 in both cell lines at 100 nM while CDK4 levels
remained unchanged. At the 4h time point in both cell lines we observed reduced
degradation of CDK6 at 5 and 10M. This is because at higher concentrations of the
PROTAC the corresponding binary complexes CDK6-PROTAC 6 and CRBN-PROTAC
6 are formed, which prevents the formation of the ternary complex that is required for
degradation. This observation is consistent with previously reported PROTAC studies
and is commonly known as the “hook effect” (Figure 4A and 4B). Next, we subjected
PROTAC 6 (500 nM) to a time-dependent study and probed for CDK6 in HPNE and
MaiPaCa2 cells. We observed a time-dependent decrease in CDK6 levels as early as 4
hours and complete degradation by 8h and remained so until the 24h time point.

Figure 4: Effect of PROTAC 6 in HPNE and MiaPaCa2 cell lines. Dose-response
studies with different concentrations of PROTAC 6 at 4 and 24h in (A) HPNE and (B)

MiaPaCa2 cells. Time-course studies with degrader 6 at different intervals in (C) HPNE
and (D) MiaPaCa2 cells
To evaluate the mechanism of action of PROTAC 6, we conducted a series of
competition experiments. First, we conducted a competition experiment with PROTAC 6
and the CDK4/6 ligand, palbociclib. Briefly, MiaPaCa2 cells were treated with 10 M of
palbociclib, PROTAC 6 (1M) individually or in combination and incubated for 24 hours.
The lysates were then probed for CDK4 and CDK6 using Western blot analysis. We
observed a reduction of CDK6 levels in PROTAC 6 treated sample and no such effect
was observed in both palbociclib or palbociclib + PROTAC 6 treated lysates. Since
palbociclib saturated CDK6 binding sites in the combination treatment, PROTAC 6
failed to degrade CDK6 (Figure 5A). Since PROTAC strategy requires formation of
ternary complex, next, we probed competition with CRBN ligand. MiaPaCa2 cells were
treated with increasing concentrations of pomalidomide (0.1 – 10M) alone and in
combination with PROTAC 6 (1M) for 24 hours, and the lysates were subjected to
Western blot analyses (Figure 5B). Pomalidomide by itself did not affect CDK6 and at 5
and 10M pomalidomide was able to block PROTAC 6 mediated degradation of CDK6.
These competitions studies demonstrate the need for simultaneous engagement of
CDK6 and CRBN by PROTAC 6 for productive degradation.
It has been suggested that PROTAC based strategy involves ubiquitination of
protein of interest followed by its proteasomal degradation. To probe this, we subjected
MiaPaCa2 cells to proteasome inhibitor MG132 for 1 hour followed by PROTAC 6 for 8
hours. As anticipated, CDK6 degradation was abrogated in the presence of MG132,
which is consistent with previously reported studies with other PROTACs (Figure 5C)
25. Collectively, these studies elucidate the mechanism of action i.e., the formation of the
ternary complex (CDK6•PROTAC 6•CRBN) and a proteasomal based degradation of
CDK6 by PROTAC 6.

Figure 5: Mechanism of action of degrader 6. (A) Western blot analyses showing
inhibition of CDK6 degradation upon simultaneous treatment of palbociclib (10 M) and
PROTAC 6 for 24h. (B) Western blot analysis showing inhibition of CDK6 degradation
upon simultaneous treatment of increasing concentrations of pomalidomide and
PROTAC 6 for 24h. (C) Western blot analysis showing inhibition of CDK6 degradation
when pre-incubation with 10 M of proteasome inhibitor MG132 for 1h followed by
treatment of PROTAC 6 for 8h.
Our results in pancreatic cell lines are consistent with a recent report from the
Gray and Winter lab that showed a palbociclib-based CDK6 PROTAC with a different

linker degraded CDK6 in acute myeloid leukemia cell lines27,28 (while this manuscript
was in preparation). The need for CDK6 selective PROTACs reported by the
Winter/Gray lab and PROTAC 6 reported here are highlighted by two recent reports.
Kollmann et al. characterized a kinase-independent role for CDK6, which cannot be
probed using the current CDK4/6 inhibitors12. Yang et al. showed that abemaciclib
treatment mediated resistance is due to elevated CDK6 levels29. CDK6 selective
PROTACs will serve as effective compounds that can be used to dissect the kinase
independent function of CDK6 and as viable hits for optimization as therapeutics to
overcome inhibitor resistance.
In conclusion, we report the development of a palbociclib-based PROTAC (6) as
a selective CDK6 degrader. Although Palbociclib (1) inhibits CDK4 and CDK6 with
equal potency, PROTAC 6 selectively degrades CDK6 while sparing other members of
the CDK family. A PROTAC strategy efficiently converted a non-selective CDK4/6
inhibitor to a selective CDK6 degrader. Competition studies confirmed the need for the
formation of a ternary complex as a prerequisite for efficient CDK6 degradation.
Acknowledgements: This work was supported in part by NIH grants CA197999, and
CA036727. We would like to thank Ed Ezell for NMR, and the Natarajan lab members
for helpful discussions.
AUTHOR INFORMATION
Corresponding Author
S.R.: phone, +402 559 3794; fax, +402 559 8270; E-mail, sandeep.rana@unmc.edu.
A.N.: phone, +402 559 3793; fax, +402 559 8270; E-mail, anatarajan@unmc.edu.
Author Contributions
# Equal contribution
Supplementary data
Supplementary data associated with this article can be found, in the online version.
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