Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling

Article abstract of DOI:10.1016/j.chembiol.2019.11.014

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small-molecule AKT inhibitors have entered clinical trials, robust and durable therape

Full text of DOI:10.1016/j.chembiol.2019.11.014

Brief Communication
Discovery of an AKT Degrader with Prolonged
Inhibition of Downstream Signaling
Graphical Abstract
Authors
Inchul You, Emily C. Erickson,
Katherine A. Donovan,
Nicholas A. Eleuteri, Eric S. Fischer,
Nathanael S. Gray, Alex Toker
Correspondence
nathanael_gray@dfci.harvard.edu
(N.S.G.),
atoker@bidmc.harvard.edu (A.T.)
In Brief
You and Erickson et al. report the
development and characterization of INY-
03-041, a highly selective AKT degrader.
The authors demonstrate that INY-03-041
not only exhibits more potent anti-
proliferative effects than the catalytic
inhibitor GDC-0068, but also induces
sustained AKT destabilization and
inhibition of downstream signaling, even
after compound washout.
Highlights
d
d
d
An AKT-selective heterobifunctional degrader was
developed
AKT degradation induced stronger anti-proliferative effects
than AKT inhibition
AKT loss and inhibition of downstream signaling was
sustained, even after washout
You et al., 2020, Cell Chemical Biology 27, 1–8
January 16, 2020 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.11.014

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
Cell Chemical Biology
Brief Communication
Discovery of an AKT Degrader
with Prolonged Inhibition of Downstream Signaling
Inchul You,1,2,4 Emily C. Erickson,3,4 Katherine A. Donovan,1,2 Nicholas A. Eleuteri,1 Eric S. Fischer,1,2
3,5,
Nathanael S. Gray,1,2, and Alex Toker
*
*
1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA
3Department of Pathology, Medicine and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
MA 02215, USA
4These authors contributed equally
5Lead Contact
*Correspondence: nathanael_gray@dfci.harvard.edu (N.S.G.), atoker@bidmc.harvard.edu (A.T.)
https://doi.org/10.1016/j.chembiol.2019.11.014
SUMMARY
Neel, 1999; Fruman et al., 2017; Shayesteh et al., 1999).
Thus, AKT is an attractive therapeutic target and significant ef-
The PI3K/AKT signaling cascade is one of the most forts have been made to develop AKT-targeted therapies
(Brown and Banerji, 2017).
Current strategies to target AKT have focused on ATP-
commonly dysregulated pathways in cancer, with
over half of tumors exhibiting aberrant AKT activa-
tion. Although potent small-molecule AKT inhibitors
have entered clinical trials, robust and durable thera-
peutic responses have not been observed. As an
alternative strategy to target AKT, we report the
development of INY-03-041, a pan-AKT degrader
consisting of the ATP-competitive AKT inhibitor
competitive, allosteric, and covalent inhibitors. Several ATP-
competitive inhibitors, such as ipatasertib (GDC-0068) and
capivasertib (AZD5363), are currently under clinical investiga-
tion in phase II and III studies (Oliveira et al., 2019; Turner
et al., 2019). However, these inhibitors suffer from a lack of
selectivity among the AGC kinase family, and this may limit
their clinical efficacy or tolerability (Huck and Mochalkin,
2017). By contrast, allosteric inhibitors which target the pleck-
strin homology domain, such as MK-2206 and miransertib
GDC-0068 conjugated to lenalidomide, a recruiter
of the E3 ubiquitin ligase substrate adaptor Cereblon
(CRBN). INY-03-041 induced potent degradation of (ARQ-092), exhibit a high degree of specificity toward AKT,
but either lack significant efficacy in the clinic or require further
clinical evaluation (Do et al., 2015; Keppler-Noreuil et al., 2019).
all three AKT isoforms and displayed enhanced
anti-proliferative effects relative to GDC-0068.
Notably, INY-03-041 promoted sustained AKT degra-
dation and inhibition of downstream signaling effects
for up to 96 h, even after compound washout. Our
findings suggest that AKT degradation may confer
prolonged pharmacological effects compared with
inhibition, and highlight the potential advantages of
Covalent allosteric inhibitors which target AKT at Cys296 and
Cys310 have been reported, but have not advanced to clinical
trials (Uhlenbrock et al., 2019).
An alternative pharmacological approach to inhibiting AKT ac-
tivity is to directly reduce cellular AKT protein levels via targeted
protein degradation. Heterobifunctional degraders, also known
as proteolysis targeting chimeras, consist of a moiety that binds
AKT-targeted degradation.
to an E3 ubiquitin ligase chemically linked to a second moiety
that engages a target protein, thereby recruiting the E3 ligase
into close proximity to the target protein to induce its ubiquitina-
tion and subsequent proteasomal degradation (Winter et al.,
2015). Several advantages of degraders over inhibitors have
INTRODUCTION
The serine/threonine kinase AKT is a central component of the been reported, which include enhancing the selectivity of
phosphoinositide 3-kinase (PI3K) signaling cascade and is a multi-targeted inhibitors (CDK9) (Olson et al., 2018), abrogating
key regulator of critical cellular processes, including prolifera- non-kinase-dependent functions (FAK) (Cromm et al., 2018),
tion, survival, and metabolism (Manning and Toker, 2017). and overcoming resistance mutations (BTK) (Dobrovolsky
The AKT protein kinase family comprises three highly homolo- et al., 2018). Because the pharmacological effects of degraders
gous isoforms, AKT1, AKT2, and AKT3, that possess both depend on the re-synthesis rate of the target protein rather than
redundant functions and isoform-specific activities (Toker, sustained target occupancy, small-molecule degraders may
2012). Hyperactivation of AKT, due to gain-of-function muta- also have significantly prolonged effects in comparison with
tions or amplification of oncogenes (receptor tyrosine kinases reversible inhibitors. However, while the potential for degraders
and PI3K) or inactivation of tumor suppressor genes (PTEN, to achieve an extended pharmacological duration of action has
INPP4B, and PHLPP), is one of the most common molecular been noted, there have been no reported degraders to date
perturbations in cancer and promotes malignant phenotypes that highlight such a feature (Churcher, 2018). Given the long
associated with tumor initiation and progression (Cantley and half-life of AKT (Basso et al., 2002) and its importance in cancer
Cell Chemical Biology 27, 1–8, January 16, 2020 ª 2019 Elsevier Ltd.
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Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
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Figure 1. Design and Development of INY-03-041
(A) Co-crystal structure of GDC-0068 (green) bound to AKT1 (gray, PDB: 4EKL) revealing solvent-exposed isopropylamine (circled) where linker was attached.
(B) Chemical structure of INY-03-041.
(C) TREEspot visualization of the biochemical kinome selectivity profile of GDC-0068 and INY-03-041 (1 mM). AKT isoforms are highlighted in blue, while all other
inhibited kinases are highlighted in red (Table S1).
etiology and progression, AKT is an attractive protein to target tested in a commercially available fluorescence resonance en-
ergy transfer-based assay (Invitrogen, Z0-Lyte) for AKT1, AKT2,
for degradation.
Here, we report the development of INY-03-041, a pan-AKT and AKT3 inhibition (Table S1). INY-03-041 had similar inhibitory
degrader that induces potent degradation of all three AKT iso- activity against AKT1 (half maximal inhibitory concentration
forms for an extended period of time. Through cell-based as- [IC50] = 2.0 nM), AKT2 (IC50 = 6.8 nM), and AKT3 (IC50
=
says, we demonstrate that INY-03-041 exhibits more potent 3.5 nM) as GDC-0068 (IC50 values for AKT1, 2, and 3 = 5, 18,
and prolonged effects on downstream signaling than GDC- and 8 nM, respectively) (Blake et al., 2012), demonstrating that
0068, which may explain its enhanced anti-proliferative effects INY-03-041 retained comparable biochemical affinity with all
in comparison with the parent catalytic inhibitor. Our data three AKT isoforms as its parent inhibitor. In addition, we evalu-
demonstrate that AKT degradation has more durable pharmaco- ated the biochemical selectivity of INY-03-041 against a panel of
logical effects than AKT inhibition, which highlights a potentially 468 kinases at 1 mM (KINOMEscan) and observed that INY-03-
novel aspect of degrader pharmacology.
041 had a similar selectivity profile as GDC-0068 (Figure 1C).
Although INY-03-041 scored as strong binder of RET
a
RESULTS
(V804M) in the KINOMEscan assay, this was confirmed to be a
false positive, as its biochemical IC50 was determined to
be >10,000 nM (Invitrogen, LanthaScreen) (Table S1).
Design and Development of the Pan-AKT Degrader INY-
03-041
To develop an AKT-targeting heterobifunctional degrader, we INY-03-041 Is a Potent and Highly Selective Pan-AKT
designed compounds based on GDC-0068, the most advanced Degrader
AKT inhibitor in clinical trials (Oliveira et al., 2019). The co-crystal After verifying that INY-03-041 engaged AKT biochemically, we
structure of GDC-0068 bound to AKT1 (PDB: 4EKL) revealed that sought to characterize its degradation activity in cells. We first
the isopropylamine is solvent exposed, suggesting that the chose to evaluate the AKT degrader in the triple-negative breast
amine could serve as a suitable attachment site for linkers cancer cell line MDA-MB-468 due to their high expression of all
without adversely affecting affinity to AKT (Figure 1A). A ten-hy- three AKT isoforms. We found that INY-03-041 induced potent
drocarbon linker was used to conjugate GDC-0068 with lenalido- degradation of all three AKT isoforms in a dose-dependent
mide to generate INY-03-041 (Figure 1B).
manner after a 12-h treatment, with maximal degradation
To verify that conjugation of the linker and lenalidomide did not observed between 100 and 250 nM (Figure 2A). At concentrations
affect the ability of INY-03-041 to bind to AKT, INY-03-041 was of 500 nM and greater, we observed diminished AKT degradation,
2
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Figure 2. INY-03-041 Induces Potent Degradation of AKT Isoforms Dependent on CRBN, Neddylation, and the Proteasome
(A) Immunoblots for AKT1, AKT2, AKT3, pan-AKT, and Vinculin in MDA-MB-468 cells after 12-h treatment with DMSO, GDC-0068 (GDC), or INY-03-041 at the
concentrations indicated (n = 4).
(B) Immunoblots for AKT1, AKT2, AKT3, pan-AKT, and Vinculin in MDA-MB-468 cells after treatment with INY-03-041 (250 nM) at indicated times or DMSO (24 h)
(n = 4).
(C) Immunoblots for AKT1, AKT2, AKT3, pan-AKT, and Vinculin after 12-h co-treatment of MDA-MB-468 cells with DMSO, bortezomib (0.5 mM), MLN-4924 (1 mM),
lenalidomide (10 mM), or GDC-0068 (10 mM) and either INY-03-041 (250 nM) or DMSO (n = 4).
(D) Scatterplot depicts the change in relative protein abundance of INY-03-041 (250 nM, 4 h)-treated MOLT4 cells compared with DMSO vehicle control-treated
cells. Protein abundance measurements were made using tandem mass tag quantitative mass spectrometry and significant changes were assessed by
moderated t test as implemented in the limma package (Ritchie et al., 2015). The log2 fold change (log2 FC) is shown on the y axis and negative log10 p value
(-log10 p value) on the x axis for three independent biological replicates of each treatment.
See also Table S2.
consistent with the hook effect, in which independent engage- RING ligases, such as CRL4CRBN (Soucy et al., 2009), prevented
ment of AKT and CRBN by INY-03-041 prevents formation of a AKT destabilization, indicating that degradation was dependent
productive ternary complex (An and Fu, 2018). Treatment of on the ubiquitin-proteasome system (Figure 2C). Finally, we co-
MDA-MB-468 cells with 250 nM of INY-03-041 over time revealed treated INY-03-041 with excess quantities of either GDC-0068 or
partial degradation of all AKT isoforms within 4 h and progressive lenalidomide to compete for binding to AKT or CRBN, respec-
loss of AKT abundance out to 24 h (Figure 2B).
tively, both of which prevented AKT degradation, demonstrating
To ensure that INY-03-041-induced AKT degradation was that engagement to both AKT and CRBN is required for INY-03-
dependent on CRBN, we synthesized INY-03-112, a negative 041-induced AKT degradation (Figure 2C).
control compound with an N-methylated glutarimide that sub-
To broadly assess degrader selectivity, MOLT4 cells, a cell line
stantially weakens CRBN binding (Figure S1A) (Brand et al., that is amenable to proteomics and expresses all three AKT iso-
2018). INY-03-112 did not induce potent degradation of any forms, were treated with 250 nM of INY-03-041 for 4 h and an un-
AKT isoform (Figure S1B), demonstrating that INY-03-041- biased, multiplexed massspectrometry-basedproteomicanalysis
induced AKT degradation was CRBN dependent. Furthermore, was performed (Donovan et al., 2018). This analysis identified sig-
co-treatment of INY-03-041 with bortezomib, a proteasome in- nificant downregulation of all three AKT isoforms, as well as
hibitor, or MLN-4924, an NEDD8-activating enzyme inhibitor RNF166, a ring-finger protein known to be downregulated by lena-
that prevents neddylation required for the function of cullin lidomide treatment (Figure 2D) (Kronke et al., 2015). Although
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Figure 3. INY-03-041 Induces Enhanced Anti-proliferative Effects Compared with GDC-0068
GR values across concentrations in (A) ZR-75-1, (B) T47D, (C) LNCaP, (D) MCF-7, (E) MDA-MB-468, and (F) HCC1937 cells after 72-h treatment with GDC-0068
(blue), INY-03-041 (red), INY-03-112 (green), and lenalidomide (orange). Error bars represent the standard deviation of three technical replicates.
INY-03-041 exhibited potent in vitro inhibition of S6K1 (IC50
=
16 nM), with a 14-fold increased potency compared with
37.3 nM) and PKG1 (IC50 = 33.2 nM), both of which are known GDC-0068 (GR50 = 229 nM). The anti-proliferative effect of
off-targets of GDC-0068, no downregulation of either kinase was INY-03-041 was degradation dependent, as INY-03-112 was
observed in the proteomics (Table S2). Further immunoblot anal- significantly less potent (GR50 = 413 nM) than INY-03-041 and
ysis confirmed that INY-03-041 did not induce S6K1 degradation had a comparable GR50 value with GDC-0068 (Figure 3A; Table
(Figure S1C). Although the time course (Figure 2B) indicates that S4). Similar trends were seen in the other cell lines sensitive to
stronger AKT degradation would be observed at longer time AKT inhibition, with 8- to 14-fold lower GR50 values for INY-03-
points, it is likely that the results would be confounded by subse- 041 in comparison with GDC-0068 (Figures 3A–3D; Table S4).
quent transcriptional changes caused by AKT degradation.
In addition, lenalidomide, used as a control for RNF166, IKZF1,
As CRBN-targeting degraders often destabilize zinc finger and IKZF3 degradation, did not have strong anti-proliferative ef-
proteins, and because we observed IMiD-induced downregula- fects, suggesting that the enhanced anti-proliferative effects
tion of RNF166, we examined whether INY-03-041 affected were due to AKT degradation (Figures 3A–3D).
protein abundance levels of Ikaros (IKZF1) and Aiolos (IKZF3),
Although INY-03-041 displayed enhanced anti-proliferative ef-
well-established targets of lenalidomide (Kronke et al., 2014). fects compared with GDC-0068 in MDA-MB-468 and HCC1937
Immunoblot analysis revealed weak IKZF1 and IKZF3 degrada- cells, there were no apparent differences in GR50 values between
tion after 24 h of drug treatment, albeit at relatively high concen- INY-03-041 and INY-03-112, its non-CRBN binding control (Fig-
trations of 500 nM or greater, indicating that INY-03-041 is ures 3E and 3F; Table S4). Thus, the anti-proliferative effects of
primarily a selective degrader for AKT (Figure S1C).
INY-03-041 in these cell lines were likely due to off-target effects
unrelated to AKT degradation that manifest at elevated concen-
trations of INY-03-041 and INY-03-112. This is consistent with
previous studies reporting resistance of MDA-MB-468 and
INY-03-041 Exhibits Enhanced Anti-proliferative Effects
Compared with GDC-0068
As AKT has well-characterized functions in regulating cell prolif- HCC1937 to AKT inhibition (Lin et al., 2013), and indicates that
eration, we next compared the anti-proliferative effects of AKT AKT degradation has similar phenotypic effects as AKT inhibition
degradation and inhibition using growth rate inhibition (GR) to in these cell lines. Overall, the data show that INY-03-041 sup-
account for variation in division rates among cells, as this can presses proliferation more potently than GDC-0068, and high-
confound other drug response metrics, such as IC50 values (Haf- light the potential therapeutic value of targeted AKT degradation.
ner et al., 2017). In a panel of cell lines with PI3K pathway muta-
tions that have been reported to be sensitive (ZR-75-1, T47D, INY-03-041 Suppresses Downstream Signaling More
LNCaP, and MCF-7) and insensitive (MDA-MB-468 and Potently Than GDC-0068
HCC1937) to AKT inhibition (Table S3) (Lin et al., 2013), we found Given the enhanced anti-proliferative effects of INY-03-041
that INY-03-041 was most potent in ZR-75-1 cells (GR50
=
compared with GDC-0068, we sought to compare their effects
4
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Figure 4. INY-03-041 Exhibits More Potent and Durable Downstream Signaling Effects Than GDC-0068
(A) Immunoblots of pan-AKT, phospho-PRAS40 (T246), total PRAS40, phospho-GSK3b (S9), total GSK3b, phospho-S6 (S240/244), total S6, and Vinculin after
treating T47D or MDA-MB-468 cells for 24 h with DMSO, INY-03-041, or GDC-0068 at the concentrations indicated (n = 3).
(B) Immunoblots of pan-AKT, phospho-PRAS40 (T246), total PRAS40, and Vinculin after treatment of T47D or MDA-MB-468 cells with 250 nM of INY-03-041 or
GDC-0068 at the time points indicated (n = 3).
(C) Immunoblots of pan-AKT, phospho-PRAS40 (T246), total PRAS40, and Vinculin in T47D or MDA-MB-468 cells treated for 12 h with INY-03-041 or GDC-0068
(250 nM), followed by washout for indicated times (n = 4). Solid vertical white line indicates samples run on separate gels.
on downstream AKT signaling. In T47D cells, which were highly 0068 in MDA-MB-468, MOLT4, IGROV1, and PC3 cells (Figures
sensitive to INY-03-041 in terms of anti-proliferation, we 4A and S3A–S3C). Similar to that observed in T47D cells, INY-
confirmed that INY-03-041 induced potent AKT degradation, 03-041 significantly reduced phosphorylation of PRAS40,
with no detectable levels of all three AKT isoforms observed after GSK3b, and S6 at 250 nM, while weaker responses were seen
a 24-h treatment with 250 nM of INY-03-041 (Figure S2). More- with equivalent doses of GDC-0068 (Figures 4A and S3A–
over, INY-03-041 treatment resulted in robust and dose-depen- S3C). Although INY-03-041 suppresses downstream AKT
dent inhibition of phosphorylated PRAS40 (pPRAS40) and signaling more potently than GDC-0068 in a variety of cancer
GSK3b (pGSK3b), well-established direct substrates of AKT cell lines, longer time points were required for INY-03-041 to
(Cross et al., 1995; Wang et al., 2012), as well as S6 (pS6), a display maximal pharmacodynamic effects relative to GDC-
downstream marker of AKT activity (Lin et al., 2013) (Figure 4A). 0068 (Figures S4A–S4C), consistent with the rate of AKT degra-
Although 250 nM INY-03-041 significantly reduced pPRAS40, dation observed.
pGSK3b, and pS6 levels, doses up to 1 mM of GDC-0068 were
Notably, we also found that INY-03-041 promoted sustained
required to observe comparable effects (Figure 4A). To test destabilization of all three AKT isoforms for at least 96 h after
whether these effects were generalizable across distinct cell treatment with 250 nM of INY-03-041 in both T47D and MDA-
lines, we also compared the effects of INY-03-041 and GDC- MB-468 cells (Figure 4B). This durable AKT degradation resulted
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(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
in sustained inhibition of downstream signaling, as pPRAS40 peutic intervention in cancers with PI3K/AKT pathway alter-
levels were also significantly reduced for up to 96 h (Figure 4B). ations. Although our data suggest that AKT degraders can
By contrast, treatment with an equivalent dose of GDC-0068 not induce more potent and durable downstream effects than AKT
only resulted in less-pronounced inhibition of pPRAS40, but the inhibitors, further optimization, particularly of the pharmacoki-
duration of this effect was also shorter (Figure 4B).
netic properties of the large heterobifunctional degrader mole-
To further characterize the mechanism underlying the cules will be necessary. In addition, reported on-target toxicities
extended duration of AKT degradation induced by INY-03-041, with AKT inhibitors (Jansen et al., 2016) suggest that more potent
we performed compound washout experiments after 12 h of degraders may have a narrow therapeutic window, further
treatment with either 250 nM of INY-03-041 or GDC-0068. We necessitating the need to investigate the therapeutic viability of
observed no detectable rebound of AKT levels for up to 96 h after targeted AKT degradation.
washout in INY-03-041-treated cells (Figure 4C), suggesting that
Our data also support the value of INY-03-041 as a chemical
the re-synthesis rate of AKT is slow. Consistently, INY-03-041 probe for studying the acute biological effects of pan-AKT deple-
potently suppressed levels of pPRAS40 for up to 96 h after tion. Existing methods for inducing AKT1, AKT2, and AKT3
washout, while washout in GDC-0068-treated cells resulted in depletion in cells have relied on CRISPR knockout or repression,
rebound of pPRAS40, as would be expected of a reversible in- doxycycline-inducible small-hairpin RNAs, or transient transfec-
hibitor (Figure 4C). Taken together, our data suggest that INY- tion with small interfering RNA, all of which require relatively long
03-041-mediated AKT degradation resulted in more potent and incubation periods (Degtyarev et al., 2008; Koseoglu et al.,
durable pharmacological effects than AKT inhibition.
2007). This not only prevents assessment of acute biological
changes resulting from loss of AKT, but also may allow reprog-
ramming and compensation of cellular networks. By contrast,
INY-03-041 induces rapid degradation of all three AKT isoforms,
DISCUSSION
Heterobifunctional degraders have garnered attention recently thereby permitting the investigation of the phenotypes of acute
due to potential advantages over traditional small-molecule in- pan-AKT depletion. Therefore, INY-03-041 will be a useful chem-
hibitors, including their ability to target proteins deemed undrug- ical tool to explore AKT biology inaccessible with current
gable through the use of non-functional ligands (Farnaby et al., strategies.
2019; Silva et al., 2019) and to achieve selectivity with promiscu-
ous ligands (Nowak et al., 2018). However, one advantage of de-
graders that has not been fully investigated is their potential to
exert long-lasting pharmacological effects. Because degraders
deplete abundance of a targeted protein, short-term drug expo-
sure may deliver sustained pharmacological effects, uncoupling
pharmacodynamics from pharmacokinetics. This distinction
may be more apparent for proteins with slow re-synthesis rates,
as longer periods of time may be required for protein levels to
recover and re-establish physiological signaling.
SIGNIFICANCE
Although many small-molecule AKT inhibitors have been
developed, here we demonstrate an alternative approach
to targeting AKT by inducing its degradation, which resulted
in distinct pharmacological effects. INY-03-041, a heterobi-
functional degrader, promoted rapid and highly selective
degradation of all three AKT isoforms and had more potent
anti-proliferative effects than GDC-0068, the most clinically
advanced AKT inhibitor. More importantly, INY-03-041-
induced degradation of AKT and the ensuing suppression
of downstream signaling was sustained for several days.
Our work showcases the extended pharmacodynamic ef-
fects of degraders, and presents INY-03-041 as a tool to
study acute changes to the AKT signaling network after
rapid AKT degradation.
Consistent with the long-reported half-life for AKT (Basso
et al., 2002), INY-03-041 destabilized all three AKT isoforms
and diminished downstream signaling effects for up to 96 h,
even after compound washout. This durable and prolonged
pharmacodynamic effect, which was significantly greater than
that of the parent AKT inhibitor GDC-0068, may explain, at least
in part, the more potent anti-proliferative effects of INY-03-041.
To the best of our knowledge, this is the first example of a
small-molecule degrader that sustains knockdown and
downstream signaling effects for such an extended period of
time, and exemplifies a distinguishing feature of degrader
pharmacology.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
Although several ATP-competitive and allosteric AKT inhibi-
tors have entered clinical trials, dose-limiting toxicities or lack
of clinical efficacy have hindered their progress (Jansen et al.,
2016). Moreover, AKT has been reported to have kinase-inde-
pendent functions (Vivanco et al., 2014) that would reduce
efficacy of small-molecule inhibitors, and resistance to AKT in-
hibitors can arise in some contexts due to upregulation of
AKT3 (Stottrup et al., 2016). As degraders have been reported
to abrogate kinase-independent functions (Cromm et al., 2018)
and overcome inhibitor-induced compensatory upregulation of
target proteins (Cai et al., 2012; Han et al., 2019), targeted AKT
degradation may be a promising alternative approach for thera-
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHODS DETAILS
B Drug Treatment Experiments
B Immunoblotting
B Growth Rate Assay and Analysis
B In Vitro Kinase Assays
B TMT LC-MS Sample Preparation
B LC-MS Data Analysis
B Chemistry Synthetic Scheme
6
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B General Chemistry Methods
B Abbreviations Used
Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol. Chem.
277, 39858–39866.
Blake, J.F., Xu, R., Bencsik, J.R., Xiao, D., Kallan, N.C., Schlachter, S.,
Mitchell, I.S., Spencer, K.L., Banka, A.L., Wallace, E.M., et al. (2012).
Discovery and preclinical pharmacology of a selective ATP-competitive Akt in-
hibitor (GDC-0068) for the treatment of human tumors. J. Med. Chem. 55,
8110–8127.
B Chemical Synthesis of INY-03-041 and INY-03-112
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Anti-proliferation Assay
B LC-MS Data Analysis
d DATA AND CODE AVAILABILITY
Brand, M., Jiang, B., Bauer, S., Donovan, K.A., Liang, Y., Wang, E.S., Nowak,
R.P., Yuan, J.C., Zhang, T., Kwiatkowski, N., et al. (2018). Homolog-selective
degradation as a strategy to probe the function of CDK6 in AML. Cell Chem
Biol. 26, 300–306.e9.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.11.014.
Brown, J.S., and Banerji, U. (2017). Maximising the potential of AKT inhibitors
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Cai, J., Cai, L.Q., Hong, Y., and Zhu, Y.S. (2012). Functional characterisation of
a natural androgen receptor missense mutation (N771H) causing human
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ACKNOWLEDGMENTS
We would like to thank Eric Wang and Milka Kostic for helpful comments on the
manuscript. We would also like to thank Caitlin Mills and Kartik Subramanian
(Laboratory for Systems Pharmacology at Harvard Medical School) for their
help with proliferation data collection and analysis. This work was supported
by the Training Grant in Chemical Biology (NIH 7 T32 GM095450-07, to I.Y.),
the Training Grant in Pharmacological Sciences (NIH 1 T32 GM132089-01,
to I.Y.), the National Science Foundation Graduate Research Fellowship Pro-
gram (DGE1745303, to E.C.E.), the NIH 1R01 CA218278-01A1 (to E.S.F. and
N.S.G.) and the Ludwig Center at Harvard (to E.C.E. and A.T.).
Cantley, L.C., and Neel, B.G. (1999). New insights into tumor suppression:
PTEN suppresses tumor formation by restraining the phosphoinositide 3-ki-
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Churcher, I. (2018). Protac-induced protein degradation in drug discovery:
breaking the rules or just making new ones? J. Med. Chem. 61, 444–452.
Cromm, P.M., Samarasinghe, K.T.G., Hines, J., and Crews, C.M. (2018).
Addressing kinase-independent functions of Fak via PROTAC-mediated
degradation. J. Am. Chem. Soc. 140, 17019–17026.
Cross, D.A., Alessi, D.R., Cohen, P., Andjelkovich, M., and Hemmings, B.A.
(1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein
kinase B. Nature 378, 785–789.
AUTHOR CONTRIBUTIONS
A.T. and N.S.G. conceived the study. I.Y. designed and synthesized the com-
pounds. E.C.E. and I.Y. designed and conducted the biological profiling of the
degraders. K.A.D. and N.A.E. performed the proteomics. I.Y and E.C.E wrote
the manuscript, with guidance from E.S.F., N.S.G., and A.T. All authors gave
feedback on the manuscript.
Degtyarev, M., De Maziere, A., Orr, C., Lin, J., Lee, B.B., Tien, J.Y., Prior, W.W.,
van Dijk, S., Wu, H., Gray, D.C., et al. (2008). Akt inhibition promotes autophagy
and sensitizes PTEN-null tumors to lysosomotropic agents. J. Cell Biol. 183,
101–116.
Do, K., Speranza, G., Bishop, R., Khin, S., Rubinstein, L., Kinders, R.J., Datiles,
M., Eugeni, M., Lam, M.H., Doyle, L.A., et al. (2015). Biomarker-driven phase 2
study of MK-2206 and selumetinib (AZD6244, ARRY-142886) in patients with
colorectal cancer. Invest. New Drugs 33, 720–728.
DECLARATION OF INTERESTS
A.T. is a consultant for Oncologie, Inc. N.S.G. is a scientific founder and mem-
ber of the Scientific Advisory Board (SAB) of C4 Therapeutics, Syros, Soltego,
B2S, Gatekeeper, and Petra Pharmaceuticals, and has received research
funding from Novartis, Astellas, Taiho, and Deerfield. E.S.F. is a founder
and/or member of the SAB, and equity holder of C4 Therapeutics and Civetta
Therapeutics, and a consultant to Novartis, AbbVie, and Pfizer. The Fischer lab
receives research funding from Novartis, Deerfield, and Astellas. I.Y., E.C.E.,
K.A.D., E.S.F., A.T., and N.S.G. are inventors on a patent application related
to the AKT degraders described in this manuscript.
Dobrovolsky, D., Wang, E.S., Morrow, S., Leahy, C., Faust, T., Nowak, R.P.,
Donovan, K.A., Yang, G., Li, Z., Fischer, E.S., et al. (2018). Bruton’s tyrosine
kinase degradation as a therapeutic strategy for cancer. Blood 133, 952–961.
Donovan, K.A., An, J., Nowak, R.P., Yuan, J.C., Fink, E.C., Berry, B.C., Ebert,
B.L., and Fischer, E.S. (2018). Thalidomide promotes degradation of SALL4, a
transcription factor implicated in Duane Radial Ray syndrome. Elife 7, https://
doi.org/10.7554/eLife.38430.
Farnaby, W., Koegl, M., Roy, M.J., Whitworth, C., Diers, E., Trainor, N.,
Zollman, D., Steurer, S., Karolyi-Oezguer, J., Riedmueller, C., et al. (2019).
BAF complex vulnerabilities in cancer demonstrated via structure-based
PROTAC design. Nat. Chem. Biol. 15, 672–680.
Received: September 12, 2019
Revised: October 28, 2019
Accepted: November 25, 2019
Published: December 16, 2019
Fruman, D.A., Chiu, H., Hopkins, B.D., Bagrodia, S., Cantley, L.C., and
Abraham, R.T. (2017). The PI3K pathway in human disease. Cell 170, 605–635.
Hafner, M., Heiser, L.M., Williams, E.H., Niepel, M., Wang, N.J., Korkola, J.E.,
Gray, J.W., and Sorger, P.K. (2017). Quantification of sensitivity and resistance
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(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Rabbit monoclonal anti AKT1
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cat# 2938;
RRID: AB_915788
Rabbit monoclonal anti AKT2
Mouse monoclonal anti AKT3
Rabbit monoclonal anti AKT (pan)
Mouse monoclonal anti AKT (pan)
Cat# 3063;
RRID: AB_2225186
Cat# 8018;
RRID: AB_10859371
Cat# 4691;
RRID: AB_915783
Cat# 2920;
RRID: AB_1147620
Rabbit monoclonal anti Phospho-PRAS40
(T246)
Cat# 2997;
RRID: AB_2258110
Rabbit monoclonal anti PRAS40
Cat# 2691;
RRID: AB_2225033
Rabbit monoclonal anti GSK3b
Cat# 9315;
RRID: AB_490890
Rabbit polyclonal anti Phospho-GSK3b (S9)
Rabbit monoclonal anti S6 Ribosomal Protein
Cat# 9336;
RRID: AB_331405
Cat# 2217;
RRID: AB_331355
Rabbit polyclonal anti Phospho-S6 Ribosomal
Protein (S240/244)
Cat# 2215;
RRID: AB_331682
Rabbit monoclonal anti Phospho-S6 Ribosomal
Protein (S240/244)
Cat# 5364;
RRID: AB_1069423
Rabbit monoclonal anti Vinculin
Rabbit monoclonal anti p70S6 Kinase
Rabbit monoclonal anti Ikaros
Rabbit monoclonal anti Aiolos
Cat# 13901;
RRID: AB_2728768
Cat# 2708;
RRID: AB_390722
Cat# 14859;
RRID: AB_2744523
Cat# 15103;
RRID: AB_2744524
Rabbit monoclonal anti
Cat# 4970;
b-actin
RRID: AB_2223172
Chemicals, Peptides, and Recombinant Proteins
DMSO
Fisher
Cat# BP231-100
Cat# 10008822
Cat# S7109
Cat# S2808
Cat# 901558
Cat# 15140122
Cat# 350000CL
Cat# 11965118
Cat# L34974
N/A
Bortezomib (PS-341)
Pevonedistat (MLN4924)
GDC-0068
Cayman Chemical
Selleckchem
Selleckchem
Lenalidomide
Sigma Aldrich
Penicillin Streptomycin
RPMI Medium
Thermo Fisher Scientific
Wisent Bioproducts
Thermo Fisher Scientific
Thermo Fisher Scientific
This study
DMEM Medium
LIVE/DEAD Far Red
INY-03-041
INY-03-112
This study
N/A
(Continued on next page)
Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020 e1

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
Continued
REAGENT or RESOURCE
Critical Commercial Assays
Z’ Lyte (AKT1)
SOURCE
IDENTIFIER
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Assay ID 247
Assay ID 250
Assay ID 253
Assay ID 757
Assay ID 810
Assay ID 703
Assay ID 801
Assay ID 1115
Assay ID 1847
Z’ Lyte (AKT2)
Z’ Lyte (AKT3)
Z’ Lyte (PKG1)
Z’ Lyte (S6K1)
Z’ Lyte (PKN1)
Z’ Lyte (bMSK2)
Z’ Lyte (Haspin)
Lanthascreen (RET (V804M))
Deposited Data
INY-03-041 Proteomics in MOLT4 cells
Experimental Models: Cell Lines
MOLT4
This study
Pride: PXD015207
ATCC
ATCC
ATCC
CRL-1582;
RRID: CVCL_0013
Jurkat
TIB-152;
RRID: CVCL_0367
ZR-75-1
CRL-1500;
RRID: CVCL_0588
LNCaP
T47D
Steven P. Balk’s Lab
ATCC
N/A
HTB-133;
RRID: CVCL_0553
MCF7
ATCC
ATCC
ATCC
HTB-22;
RRID:CVCL_0031
MDA-MB-468
HCC1937
IGROV1
HTB-132;
RRID: CVCL_0419
CRL-2336;
RRID: CVCL_0290
Panagiotis A.
Konstantinopoulos’s
Lab
N/A
PC3
Jarrod A. Marto’s Lab
N/A
Software and Algorithms
GraphPad Prism
GraphPad Software, Inc.
PerkinElmer Informatics
www.graphpad.com/
Columbus image storage and analysis
http://www.perkinelmer.com/product/
image-data-storage-and-analysis-
system-columbus
Adobe Illustrator
Adobe Creative Cloud
Thermo Fisher Scientific
https://www.adobe.com/creativecloud.html
Proteome Discoverer 2.2
https://www.thermofisher.com/order/
catalog/product/OPTON-30795
R Framework
Python 3.7.3
Team RCR: A Language and Environment
for Statistical Computing
http://www.R-project.org/
Python Software Foundation
https://www.python.org/
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Alex
Toker (atoker@bidmc.harvard.edu). Requested compounds will be provided following completion of an MTA.
e2 Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
EXPERIMENTAL MODEL AND SUBJECT DETAILS
MOLT4 (male, CVCL_0013), Jurkat (male, CVCL_0065), ZR-75-1 (female, CVCL_0588), LNCaP (male, VCL_0395), T47D (female,
CVCL_0553), MCF-7 (female, CVCL_0031) , MDA-MB-468 (female, CVCL_0419) , and HCC1937 (female, CVCL_0290) cells were
cultured in RPMI media (Wisent Bioproducts) supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher Scientific)
and 100U/mL Penicillin-Streptomycin (Gibco) at 37ꢀC in the presence of 5% CO2. IGROV1 (female, CVCL_1304) and PC3 (male,
CVCL_0035) cells were cultured in DMEM media (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher
Scientific) and 100U/mL Penicillin-Streptomycin (Gibco) at 37ꢀC in the presence of 5% CO2.
METHODS DETAILS
Drug Treatment Experiments
Cells were plated at 250,000 cells per mL (MDA-MB-468, MOLT4, IGROV1, PC3, and Jurkat) or 200,000 cells per mL (T47D) in 2 mL
per well of RPMI or DMEM media with 10% serum in 6-well treated tissue culture plates (Greiner, Cat # TCG-657160) or 60 mm
treated tissue culture plates (Corning, Cat # 430166) and incubated overnight. The next day, cells were treated with the indicated
compounds at the appropriate concentration and protein lysates were harvested at the times specified.
Immunoblotting
Cells were washed once in 1x PBS then lysed in RIPA buffer (150 mM Tris-HCl, 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 1%
(v/v) NP-40, pH 7.5) containing 0.1% (w/v) sodium dodecyl sulfate, 1 mM sodium pyrophosphate, 20 mM sodium fluoride, 50 nM
calyculin, and 0.5% (v/v) protease inhibitor cocktail (Sigma-Aldrichꢀ) for 15 minutes. Cell extracts were precleared by centrifugation
at 14,000 rpm for 10 minutes at 4ꢀC. The Bio-Rad DC protein assay was used to assess protein concentration, and sample concen-
tration was normalized using SDS sample buffer. Lysates were resolved on acrylamide gels by SDS-polyacrylamide gel electropho-
resis and electrophoretically transferred to nitrocellulose membrane (BioRad) at 100 volts for 90 minutes. Membranes were blocked
in 5% (w/v) nonfat dry milk in tris-buffered saline (TBS) buffer for 1 hour then incubated with specific primary antibodies diluted in 5%
(w/v) nonfat dry milk in TBS-T (TBS with 0.05% Tween-20) at 4ꢀC overnight, shaking. The next day, membranes were washed with
TBS-T then incubated for 1 hour at room temperature with fluorophore-conjugated secondary antibodies (LI-COR Biosciences). The
membrane was washed again with TBS-T then imaged with a LI-COR Odyssey CLx Imaging System (LI-COR Biosciences).
Growth Rate Assay and Analysis
Cell lines were plated at densities ranging from 500 to 2000 cells per well in a 384-well plate using a Matrix WellMate Reagent
Dispenser (Thermo Fisher) and allowed 24 hours to adhere to plate prior to treatment. A D300 Digital Dispenser (Hewlett-Packard)
was used to treat cells with dilution series of compounds as indicated. At the time of treatment and after 72 hours of treatment, cells
were stained and fixed for subsequent analysis. Cells were stained with LIVE/DEAD Far Red Dead Cell Stain (LDR) (Thermo Fisher
Scientific) at 1:2000 for 1 hour at 37ꢀC. Cells were fixed for 30 minutes at room temperature in 4% formaldehyde (Sigma Aldrich) then
permeabilized with 0.5% Triton X-100 in PBS. Cells were blocked for 1 hour using Odyssey Blocking Buffer (LI-COR Biosciences) and
stained overnight at 4ꢀC with 2 mg/ml Hoechst 33342 (Sigma Aldrich). An Operetta microscope was used to image fixed cells, and
data was stored and analyzed using Columbus software (PerkinElmer). Nuclei were segmented by Hoechst signal using the
Columbus system (Perkin Elmer). The average LDR and Hoechst intensities were determined within the nuclear area. Dead cells
were classified by LDR signal. Experiments were performed in technical triplicate.
In Vitro Kinase Assays
Z’-LYTE assays were conducted for AKT1, AKT2, AKT3, PKG1, S6K1, PKN1, ßMSK2, and Haspin at Life Technologies in a 10-point
dose response using Km ATP concentrations. LanthaScreen assays were conducted for RET (V804M) in a 10-point dose response at
Life Technologies.
TMT LC-MS Sample Preparation
MOLT4 cells were treated with DMSO, 250 nM INY-03-041-01 for 4 hours in biological triplicates. Cells were harvested by centrifu-
gation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, 1x Roche pro-
tease inhibitor and 1x Roche PhosphoStop was added to the cell pellets and cells were homogenized by 20 passes through a 21
gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 0.5 – 4 mg mL-1. The homogenized sample
was clarified by centrifugation at 20,000 x g for 10 minutes at 4ꢀC. A Bradford assay was used to determine the final protein concen-
tration in the cell lysate. 200 mg protein for each sample were reduced and alkylated as previously described (An et al., 2017). Proteins
were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one
volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged at 14,000 x g for 5 minutes
Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020 e3

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol,
centrifuged at 14,000 x g for 5 min, and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was
resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS pH 8 for digestion
with LysC (1:50; enzyme:protein) for 12 hours at RT. The LysC digestion was diluted to 0.5 M Urea, 200 mM EPPS pH 8 and then
digested with trypsin (1:50; enzyme:protein) for 6 hours at 37ꢀC. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were
dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide
sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio
of 1:4 peptide:TMT label. The 11-plex labeling reactions were performed for 1.5 hours at RT and the reaction quenched by the addi-
tion of 0.3% hydroxylamine for 15 minutes at RT. The sample channels were combined at a 1:1:1:1:1:1:1:1:1:1:1 ratio, desalted using
C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined
using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was
then resuspended in 1% formic acid and acidified (pH 2ꢁ3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters).
Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide
xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mo-
bile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were
then pooled in a non-continuous manner into 24 fractions and every fraction was used for subsequent mass spectrometry analysis.
Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled
with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on a 50 cm and 75 mm inner diameter
Easyspray column (ES803a, Thermo Fisher Scientific). Peptides were separated using a 190 minute gradient of 6 – 27% acetonitrile in
1.0% formic acid with a flow rate of 300 nL/min.
Each analysis used an MS3-based TMT method as described previously (McAlister et al., 2014). The data were acquired using a
mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120
seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized
collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in
the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000
and with a maximum synchronous precursor selection (SPS) precursors set to 10.
LC-MS Data Analysis
Proteome Discoverer 2.2 (Thermo Fisher) was used for .RAW file processing and controlling peptide and protein level false discovery
rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot
human database (September 2016) with both the forward and reverse sequences. Database search criteria are as follows: tryptic with
two missed cleavages, a precursor mass tolerance of 10 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of cysteine
(57.02146 Da), static TMT labeling of lysine residues and N-termini of peptides (229.16293 Da), variable phosphorylation of serine,
threonine and tyrosine (79.966 Da), and variable oxidation of methionine (15.99491 Da). TMT reporter ion intensities were
measured using a 0.003 Da window around the theoretical m/z for each reporter ion in the MS3 scan. Peptide spectral matches
with poor quality MS3 spectra were excluded from quantitation (summed signal-to-noise across 10 channels < 200 and precursor
isolation specificity < 0.5). Only proteins containing at least two unique peptides identified in the experiment were included in final
quantitation.
Chemistry Synthetic Scheme
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Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
General Chemistry Methods
Reagents and solvents were purchased from commercial suppliers and were used without further purification unless otherwise
noted. Reactions were monitored using a Waters Acquity UPLC/MS system (Waters PDA el Detector, QDa Detector, Sample man-
ager - FL, Binary Solvent Manager) using Acquity UPLCꢀ BEH C18 column (2.1 x 50 mm, 1.7 mm particle size): solvent gradient = 85%
A at 0 min, 1% A at 1.7 min; solvent A = 0.1% formic acid in Water; solvent B = 0.1% formic acid in Acetonitrile; flow rate: 0.6 mL/min.
Products were purified by flash column chromatography using CombiFlashꢀRf with Teledyne Isco RediSepꢀ normal-phase silica
flash columns (4 g, 12 g, 24 g, 40 g or 80 g) and preparative HPLC using Waters SunFireTM Prep C18 column (19 x 100 mm,
5 mm particle size) using a gradient of 15-75% methanol in water containing 0.05% trifluoroacetic acid (TFA) over 48 min (60 min
run time) at a flow of 40 mL/min. 1H NMR spectra were recorded on 500 MHz Bruker Avance III spectrometer, and chemical shifts
are reported in million (ppm, d) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities
are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Purities of assayed compounds were
in all cases greater than 95%, as determined by reverse-phase HPLC analysis.
Abbreviations Used
DCM: Dichloromethane
DIEA: N,N-Diisopropylethylamine
DMF: Dimethylformamide
TEA: Triethylamine
DMP: Dess–Martin periodinane
STAB: Sodium triacetoxyborohydride
DCE: 1,2-Dichloroethane
MeOH: Methanol
Chemical Synthesis of INY-03-041 and INY-03-112
Chemical Synthesis of 3
Tert-butyl((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-
oxopropyl)carbamate (2):. To a solution of known intermediate 1 (Blake et al., 2012) (727 mg, 2.37 mmol), (S)-3-((tert-butoxycar-
bonyl)amino)-2-(4-chlorophenyl)propanoic acid (711 mg, 2.37 mmol) and HATU (902 mg, 2.37 mmol) in DMF (12 mL) was
added DIEA (2.1 mL) and stirred for 1 hour at r.t. The reaction mixture was diluted ethyl acetate (30 mL) and washed with water
(2 x 15 mL) and brine (2 x 15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo
to afford 2 as a yellow oil (1.22 g, 100% yield). The crude was used directly for the next reaction. LC-MS: m/z 516.27 [M+1].
(S)-3-amino-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)
propan-1-one (3):. To a solution of intermediate 2 (1.22 g, 2.37 mmol) in dioxane (7 mL) was added 4 M HCl in dioxane (5 mL). The
reaction was stirred at r.t. for 4 hours. The reaction mixture was concentrated in vacuo and purified by reverse-phase HPLC (95-35%
methanol in water) to obtain 3 as a TFA salt. The resultant TFA salt was dissolved in a mixture of 4:1 chloroform: isopropanol (50 mL)
and washed with sat. aq sodium bicarbonate (50 mL) to give 3 as a white foam (868 mg, 88% yield). LC-MS: m/z 416.25 [M+1]. 1H
Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020 e5

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
NMR (500 MHz, DMSO-d6) d 8.43 (s, 1H), 7.43 – 7.38 (m, 2H), 7.35 – 7.28 (m, 2H), 4.84 (t, J = 7.3, 6.2 Hz, 1H), 4.07 (dd, J = 8.3, 5.3 Hz,
1H), 3.73 – 3.66 (m, 1H), 3.64 – 3.56 (m, 3H), 3.52 – 3.35 (m, 4H), 3.22 – 3.16 (m, 1H), 3.09 (dd, J = 12.6, 8.3 Hz, 1H), 2.72 – 2.67 (m, 1H),
2.01 – 1.87 (m, 2H), 1.04 (dd, J = 6.5, 3.9 Hz, 3H).
Synthesis of INY-03-041 and INY-03-112
3-(4-(10-hydroxydec-1-yn-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (5a). Known intermediate 4a (Zhou et al., 2018) (500 mg,
1.55 mmol), dec-9-yn-1-ol (478 mg, 3.10 mmol), Pd(PPh3)2Cl2 (113 mg, 0.16 mmol) and CuI (61 mg, 0.32 mmol) were dissolved in TEA
(4 mL) and DMF (8 mL). The reaction mixture was flushed with nitrogen (x3) and stirred at 70ꢀC for 3 hours. The reaction mixture was
cooled to r.t., diluted with ethyl acetate (50 mL) and filtered through celite. The organic layer was washed with brine (3 x 20 mL), dried
over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by column chromatography on
1
silica gel (9:1 DCM:MeOH) to afford 5a as a pale yellow solid (503 mg, 82% yield). LC-MS: m/z 397.24 [M+1]. H NMR (500 MHz,
DMSO-d6) d 11.00 (s, 1H), 7.71 (dd, J = 7.6, 1.0 Hz, 1H), 7.64 (dd, J = 7.7, 1.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 5.76 (s, 2H), 5.15
(dd, J = 13.3, 5.2 Hz, 1H), 4.45 (d, J = 17.6 Hz, 1H), 4.31 (d, J = 17.6 Hz, 1H), 3.37 (t, J = 6.5 Hz, 2H), 2.92 (ddd, J = 17.3, 13.6,
5.5 Hz, 1H), 2.64 – 2.57 (m, 1H), 2.50 – 2.40 (m, 2H), 2.06 – 1.99 (m, 1H), 1.62 – 1.54 (m, 2H), 1.47 – 1.37 (m, 3H), 1.36 – 1.23
(m, 5H), 1.18 (t, J = 7.3 Hz, 1H).
3-(4-(10-hydroxydecyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (6a). To a solution of intermediate 5a (100 mg, 0.25 mmol) in
MeOH (10 mL) was added Pd/C (10 mg). H2 (g) was introduced to the reaction mixture, and the reaction was stirred at r.t. for 4 hours.
The reaction mixture was filtered over celite and concentrated in vacuo to afford 6a as a white solid (91 mg, 91% yield). LC-MS: m/z
401.30 [M+1]. 1H NMR (500 MHz, DMSO-d6) d 10.98 (s, 1H), 7.60 – 7.54 (m, 1H), 7.50 – 7.41 (m, 2H), 5.13 (dd, J = 13.3, 5.1 Hz, 1H),
4.45 (d, J = 17.2 Hz, 1H), 4.34 – 4.28 (m, 2H), 3.36 (td, J = 6.5, 4.9 Hz, 2H), 2.92 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.66 – 2.58 (m, 3H),
2.43 (qd, J = 13.3, 4.5 Hz, 1H), 2.01 (dtd, J = 12.7, 5.2, 2.2 Hz, 1H), 1.66 – 1.54 (m, 2H), 1.43 – 1.35 (m, 2H), 1.33 – 1.21 (m, 12H).
3-(4-(10-(((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-
oxopropyl)amino)decyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (INY-03-41). To a solution of intermediate 6a (83 mg, 0.21 mmol)
in DCM (4 mL) was added DMP (132 mg, 0.31 mmol). The reaction was stirred at r.t. for 2 hours. The reaction mixture was filtered and
concentrated in vacuo to obtain 10-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)decanal (84 mg, quantitative yield). LC-MS: m/z
399.27 [M+1]. The crude material was then dissolved in DCE (2 mL), followed by addition of intermediate 3 (131 mg, 0.31 mmol). The
reaction was stirred at r.t. for 30 minutes, after which STAB (89 mg, 0.42 mmol) was added. The reaction mixture was stirred for an
additional 1 hour. The reaction was quenched by sat. aq sodium bicarbonate (10 mL) and extracted with DCM (3 x 20 mL). The
organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude residue was pu-
rified by reverse-phase HPLC (75-15% methanol in water) to obtain INY-03-041 (TFA salt). The resultant TFA salt was dissolved in 4:1
chloroform:isopropanol (10 mL) and washed with sat. aq sodium bicarbonate. The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo to afford title compound as an off-white solid (42 mg, 25% yield). LC-MS: m/z 798.42
[M+1]. 1H NMR (500 MHz, DMSO-d6) d 8.43 (s, 1H), 7.56 (dd, J = 5.4, 3.2 Hz, 1H), 7.45 – 7.43 (m, 2H), 7.41 (d, J = 8.2 Hz, 2H),
7.34 (d, J = 8.2 Hz, 2H), 5.38 (d, 1H), 5.13 (dd, J = 13.3, 5.1 Hz, 1H), 4.87 – 4.80 (m, 1H), 4.45 (d, J = 17.1 Hz, 1H), 4.29 (dd, J =
15.4, 7.6 Hz, 2H), 3.70 – 3.56 (m, 5H), 3.51 – 3.42 (m, 3H), 3.41 – 3.36 (m, 1H), 3.24 – 3.16 (m, 2H), 2.92 (ddd, J = 17.9, 13.8,
5.4 Hz, 1H), 2.77 – 2.71 (m, 1H), 2.63 (t, J = 7.5 Hz, 3H), 2.60 – 2.55 (m, 2H), 2.42 (tt, J = 13.3, 6.6 Hz, 1H), 2.04 – 1.93 (m, 2H),
1.93 – 1.87 (m, 1H), 1.62 – 1.55 (m, 2H), 1.41 – 1.35 (m, 2H), 1.32 – 1.26 (m, 4H), 1.22 (s, 8H), 1.03 (d, J = 6.8 Hz, 3H).
3-(4-(10-hydroxydec-1-yn-1-yl)-1-oxoisoindolin-2-yl)-1-methylpiperidine-2,6-dione (5b). 5b was synthesized with similar proced-
ures as 5a using intermediate 4b (90 mg, 0.27 mmol) as the starting material. 5b was obtained as a light orange solid (49 mg,
44% yield). LC-MS: m/z 411.51 [M+1]. 1H NMR (500 MHz, DMSO-d6) d 7.71 (dd, J = 7.6, 1.0 Hz, 1H), 7.63 (dd, J = 6.8, 1.2 Hz,
1H), 7.52 (t, J = 7.6 Hz, 1H), 5.21 (dd, J = 13.5, 5.1 Hz, 1H), 4.44 (d, J = 17.6 Hz, 1H), 4.30 (d, J = 17.6 Hz, 1H), 3.36 (t, J = 6.5 Hz,
2H), 3.04 – 2.95 (m, 4H), 2.79 – 2.72 (m, 1H), 2.47 (t, J = 7.1 Hz, 2H), 2.45 – 2.39 (m, 1H), 2.03 (dtd, J = 12.7, 5.2, 2.2 Hz, 1H),
1.61 – 1.52 (m, 2H), 1.47 – 1.34 (m, 4H), 1.34 – 1.24 (m, 6H).
3-(4-(10-hydroxydecyl)-1-oxoisoindolin-2-yl)-1-methylpiperidine-2,6-dione (6b). 6b was synthesized with similar procedures as 6a
using intermediate 5b (42 mg, 0.1 mmol) as the starting material. 6b was obtained as an off-white solid (33 mg, 79% yield). LC-MS: m/
z 415.57 [M+1]. 1H NMR (500 MHz, DMSO-d6) d 7.60 – 7.53 (m, 1H), 7.49 – 7.43 (m, 2H), 5.20 (dd, J = 13.4, 5.1 Hz, 1H), 4.45 (d, J =
17.1 Hz, 1H), 4.32 – 4.27 (m, 2H), 3.36 (td, J = 6.6, 5.1 Hz, 2H), 3.01 (s, 3H), 3.00 – 2.95 (m, 1H), 2.79 – 2.74 (m, 1H), 2.66 – 2.61 (m, 2H),
2.48 – 2.39 (m, 1H), 2.08 – 1.98 (m, 1H), 1.63 – 1.56 (m, 2H), 1.43 – 1.34 (m, 2H), 1.32 – 1.22 (m, 12H).
3-(4-(10-(((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-
oxopropyl)amino)decyl)-1-oxoisoindolin-2-yl)-1-methylpiperidine-2,6-dione (INY-03-112). INY-03-112 was synthesized with
similar procedures as INY-03-041 using intermediate 6b (27 mg, 0.07 mmol) as the starting material. INY-03-112 was obtained
as an off-white solid (9 mg, 16% yield). LC-MS: m/z 812.47 [M+1]. 1H NMR (500 MHz, DMSO-d6) d 8.43 (s, 1H), 7.57 (dd, J = 5.8,
2.8 Hz, 1H), 7.49 – 7.43 (m, 4H), 7.37 – 7.32 (m, 2H), 5.40 (d, J = 5.5 Hz, 1H), 5.21 (dd, J = 13.4, 5.1 Hz, 1H), 4.83 (q, J = 6.3 Hz,
1H), 4.45 (d, J = 17.1 Hz, 1H), 4.38 (dd, J = 8.9, 5.1 Hz, 1H), 4.29 (d, J = 17.1 Hz, 1H), 3.76 – 3.41 (m, 8H), 3.41 – 3.34 (m, 3H),
3.10 (t, J = 10.0 Hz, 1H), 3.01 (s, 3H), 2.99 – 2.95 (m, 1H), 2.83 – 2.73 (m, 3H), 2.63 (t, J = 7.7 Hz, 2H), 2.43 (qd, J = 13.2, 4.5 Hz,
1H), 2.05 – 1.87 (m, 3H), 1.59 (t, J = 7.5 Hz, 2H), 1.50 (s, 2H), 1.33 – 1.27 (m, 4H), 1.23 (s, 8H), 1.02 (d, J = 6.9 Hz, 3H).
e6 Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020

Please cite this article in press as: You et al., Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling, Cell Chemical Biology
(2019), https://doi.org/10.1016/j.chembiol.2019.11.014
QUANTIFICATION AND STATISTICAL ANALYSIS
Anti-proliferation Assay
Growth rate inhibition values were determined as described previously (Hafner et al., 2017) using python 3.7.3. Data was visualized
using a non-linear regression curve fit in GraphPad Prism 8. N=3 biological replicates were used for each treatment condition and
each data point represents the mean +/- SEM, as also indicated by the figure legend.
LC-MS Data Analysis
Reporter ion intensities were normalized and scaled using in house scripts and the R framework (Team, 2013). Statistical analysis was
carried out using a moderated t-test in the limma package within the R framework (Ritchie et al., 2015).
DATA AND CODE AVAILABILITY
The accession number for the proteomic data reported in this paper is Pride: PXD015207.
Cell Chemical Biology 27, 1–8.e1–e7, January 16, 2020 e7