Phosphorylation-Inducing Chimeric Small Molecules

Article abstract of DOI:10.1021/jacs.0c05537

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases - AMPK and PKC - to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton's tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Full text of DOI:10.1021/jacs.0c05537

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Phosphorylation-Inducing Chimeric Small Molecules
Sachini U. Siriwardena,⊥ Dhanushka N. P. Munkanatta Godage,⊥ Veronika M. Shoba,⊥ Sophia Lai,⊥
Mengchao Shi, Peng Wu, Santosh K. Chaudhary, Stuart L. Schreiber, and Amit Choudhary*
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ABSTRACT: Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules
that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given
protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small
molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small
molecules (PHICS), which enable two example kinasesAMPK and PKCto phosphorylate target proteins that are not otherwise
substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit
several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of
phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-
phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target
protein Bruton’s tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic
research and medicine.
he appendage of a phosphoryl group to many proteins
adaptor proteins,12,13 to the best of our knowledge, these
1
Tprofoundly influences their structures and functions. studies provide first examples of induction of phosphorylation
by rational rewiring of kinase specificity using small molecules.
To generate PHICS for AMPK and PKC, we designed small-
molecule kinase binders with functional groups for linker
attachment. For AMPK, we modified its allosteric activator,14
PF-06409577, by replacing the cyclobutyl ring with the more
synthetically amenable aminoethyl handle (Figure S1), while
for PKC 9-(4-aminomethylbenzyloxy)-substituted benzolac-
tam activator was used.15 These chemical modifications did
not perturb the ability of the binders to activate AMPK or PKC
as assessed by the ADP-Glo assay,16 which measures the
amount of ADP produced from the kinase reaction (Figure
S2). Next, we generated PHICS by conjugating these binders
to (S)-JQ1, a BRD4 binder, with linkers of varying length
(Figure S3 and S4). The AMPK PHICS were synthesized
using a modular, click-chemistry-based convergent synthesis17
(Figure S3), while the PKC PHICS were constructed using
two consecutive amidation steps (Figure S4).
Unsurprisingly, small molecules that block protein phosphor-
ylation via kinase inhibition have had a transformative impact
in basic science and medicine.2 We hypothesize that small
molecules that induce phosphorylation of any given protein-of-
interest on-demand will also be useful in myriad scenarios. For
example, such molecules can be used to trigger cell-signaling
events or neo-phosphorylations that are not observed in native
cellular environment.3 Neo-phosphorylation can also alter
protein structure and function,4 evoke an immune response,5,6
or affect the protein’s interaction with other biomolecules,
particularly with RNA/DNA that have negatively charged
phosphodiester backbones.7,8
To design such phosphorylation-inducing molecules, we
drew inspiration from chemical inducers of dimerization9,10
and ubiquitination-inducing small molecules (e.g., PRO-
TACs).11 The latter increase the effective molarity of the
ubiquitin ligase around the target protein, triggering ubiquiti-
nation even when the target protein is otherwise not a
substrate of the given ligase. Herein, we describe a new class of
bifunctional molecules that we term as phosphorylation-
inducing chimeric small molecules (PHICS) formed by linking
a kinase activator with a small-molecule binder of the target
protein. Using PHICS, we demonstrate rewiring of substrate
specificity of two kinases, AMP-activated protein kinase
(AMPK) and protein kinase C (PKC), to induce phosphor-
ylation (native and neo) of bromodomain-containing protein 4
(BRD4), which is not known to be a substrate of AMPK or
PKC. Furthermore, an AMPK-based PHICS was able to
induce a signaling-relevant phosphorylation (S180) on
Bruton’s tyrosine kinase (BTK) in a non-native cellular
environment. While kinase specificity has been rewired using
We assessed both ternary complex formation and levels of
BRD4 phosphorylation (Figure S5 and S6) induced by these
PHICS to identify PHICS1 and PHICS2 as the most optimal
for AMPK and PKC, respectively (Figure 1A). We used (R)-
JQ1, the inactive enantiomer of the BRD4 binder,18 to
synthesize iPHICS1 and iPHICS2 to serve as negative
controls. Using the ADP-Glo assay, we confirmed that
Received: May 20, 2020
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© XXXX American Chemical Society
A

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Figure 1. PHICS induces proximity and phosphorylation in vitro. (A) Structures of PHICS and ternary complex formation between BRD4 and
AMPK (B) or BRD4 and PKC (C) observed by AlphaScreen assay. (D−F) Detection of BRD4 phosphorylation by immunoblotting using
phospho-AMPK substrate motif antibody (D), or phospho-PKC substrate motif antibody (E), or phospho-Ser484/488 antibody (F). (G,H) ADP-
Glo assay for BRD4 phosphorylation with PHICS1 (G) or PHICS2 (H) compared to their respective iPHICS.
iPHICS1 and iPHICS2 still activated AMPK and PKC (Figure
S7A−C), as the kinase-binding moiety remains unaltered. The
ternary complex formation was assessed by an AlphaScreen
assay (Amplified Luminescent Proximity Homogenous
assay)19,20 using BRD4 and AMPK (α1β1γ1 isoform) or
PKC (α-isoform). PHICS1 and PHICS2, but not iPHICS1
and iPHICS2, displayed the bell-shaped curve consistent with
ternary-complex equilibria (Figure 1B and C),21 where at high
concentrations of the bifunctional molecule, the kinase−
PHICS and BRD4−PHICS species dominate the equilibrium
eliciting the “hook effect.”22 Using phospho-AMPK- or
phospho-PKC-substrate motif antibodies, we observed BRD4
phosphorylation only when PHICS, BRD4, and kinase were
present (Figure 1D and E).23,24 BRD4 phosphorylation was
observed irrespective of the nature of the tag (i.e., GST vs. His
tag) (Figure S7D,E). The levels of BRD4 phosphorylation also
increased in an AMPK-/PKC-dependent manner with PHICS
but not with iPHICS (Figure S7F,G). We also observed the
hook effect in BRD4 phosphorylation with an increasing
PHICS concentration (Figure S8) with the highest levels of
phosphorylation achieved at 1 μM of PHICS1, corresponding
to the concentration of maximum signal in the AlphaScreen
assay for ternary-complex formation (Figure 1B). Using
pSer484/488 antibody, we observed PHICS1 induced
phosphorylation of those sites on BRD4 by AMPK (Figure
1F), which are phosphorylated in the native environment by
casein kinase II (CK2).25
T229 have not been reported before.26 To confirm that AMPK
does not have an intrinsic preference for phosphorylation of
these BRD4 sites and that they are not part of unknown
substrate motif, we tested peptides derived from the BRD4
sequence bearing those residues. In an ADP-Glo assay, these
peptides showed a 250-fold lower preference for phosphor-
ylation than AMPK’s natural ACC substrate, SAMS peptide
(Figure S11),27 further confirming that the PHICS-induced
proximity of AMPK and BRD4 is essential for rewiring the
AMPK specificity. Finally, we note that the sites T186, S324,
and S325 but not T169 and T221 are in the most preferred
AMPK consensus substrate recognition motif, wherein a basic
residue is preferred in the −3 position (RXXpS/pT), but
AMPK can also phosphorylate proteins lacking this motif.28−30
Since PHICS were designed based on reversible binders, we
hypothesized that they could exhibit turnover with each
PHICS molecule phosphorylating multiple BRD4 molecules.
Using the ADP-Glo assay and iPHICS1 and iPHICS2 as
negative controls (Figure 1G and H), we calculated ADP
production in the presence of the PHICS1 (324 22 nM, 2 h)
and PHICS2 (740 31 nM, 1 h) and found it to be higher
than the limiting AMPK (20 nM) or PKC (50 nM)
concentrations, respectively, suggesting that PHICS exhibit
turnover. Another hallmark of bifunctional molecules is the
isoform specificity that arises from not only a differential
binding affinity of PHICS to various isoforms but also from
intrinsically different interactions between the enzyme and the
target isoform upon ternary complex formation.31 With an
AMPK isoform (α1β2γ1) that is not activated by PF-06409577
(Figure S12),14 we did not observe the induction of BRD4
phosphorylation by PHICS1 (Figure 2A). Additionally,
PHICS2 also exhibited isoform specificity, with the highest
BRD4 phosphorylation occurring with PKCα, modest
Next, we confirmed that PHICS can induce neo-phosphor-
ylation on the truncated BRD4 (49−460 aa) using mass
spectrometry. For AMPK PHICS, the statistically significant
phosphorylation sites were T169, T186, T221, S324, and S325,
whereas those for PKC were T229, S324, and S338 (Figure S9
and S10). Phosphorylation at sites T169, T186, T221, and
B
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HEK293T cells allowing us to assess the ability of PHICS to
induce BTK phosphorylation in a non-native cellular environ-
ment.
We linked AMPK binder with a noncovalent analogue of
Ibrutinib via various linkers37 (Figure S14) and found eight
carbon alkyl linker (PHICS3, Figure 3A) to be optimum for
the in vitro phosphorylation (Figure S15), which was
monitored using phospho-BTK (Ser180) antibody. To
demonstrate the ternary complex formation inside the cells,
we transfected HEK293T with BTK-Flag and performed co-
immunoprecipitation of AMPK with BTK in the presence of
PHICS3 (Figure 3B). Furthermore, we were able to detect
PHICS-mediated BTK phosphorylation at Ser180 (Figure 3C
and S16A) and we validated this site of phosphorylation using
S180A variant, where the phosphorylation was not detected
(Figure 3D). We note that potent AMPK activator, PF-
06409577, alone did not induce the same levels of BTK
phosphorylation as PHICS3, even at 4-fold higher concen-
tration (Figure S16B). Finally, we were able to dosably control
PHICS-induced S180 phosphorylation with fast kinetics
(Figure S16C and D).
Figure 2. PHICS exhibits isoform and paralog specificity. (A) Effect
of AMPK isoforms on PHICS1-mediated BRD4 phosphorylation. (B)
Effect of PKC isoforms on PHICS2-mediated BRD4 phosphorylation.
(C,D) Phosphorylation of BRD4 paralogs by AMPK (C) or PKC
(D).
To confirm that the observed BTK phosphorylation in the
non-native environment was mediated by PHICS, we leveraged
several chemical genetics approaches. First, the pretreatment of
cells with covalent BTK binder, Ibrutinib, demolished the
ability of PHICS3 to induce BTK phosphorylation (Figure 4A
and S17A). Second, we mutated the residues purported to be
involved in the binding of PHICS to BTK (Thr474, Lys430,
and Asp539). Notably, Thr474 forms hydrogen bond with the
4-amino group of Ibrutinib (Figure 4B)38 and we observed
drastically lowered BTK phosphorylation for T474A variant
(Figure 4C). Alterations in the ATP binding pocket by D539N
and K430R mutations also markedly reduced the PHICS3
induced phosphorylation (Figure S17B and C). Finally, we
designed an inactive analogue of PHICS3, by placing a bulky
pivaloyl (Piv) group on the 4-aminopyrazolo[3,4-d]pyrimidine
that should sterically prevent the binding to BTK. Indeed, the
pivaloyl-bearing PHICS (Piv-PHICS3, Figure 3A) was unable
to induce a significant BTK phosphorylation (Figure 4D).
Taken together, these studies confirm that the Ser180
phosphorylation of BTK arises owing to proximity-effects
induced by PHICS.
phosphorylation with PKCβI and II, and only minor
phosphorylation with the PKCγ and δ isoforms (Figure
2B).32 Paralog specificity was also observed for the target
proteins BRD (2/3/4), with BRD4 showing the highest level
of phosphorylation (Figure 2C, 2D and S13).
We were unable to observe PHICS-mediated BRD4
phosphorylation in cells perhaps owing to different localization
of the kinases and BRD4while the former are mostly
cytosolic, the latter primarily resides in the nucleus. We chose
Bruton’s Tyrosine Kinase (BTK), a protein widely expressed in
B cells33,34 for several reasons. First, BTK is a cytoplasmic
protein and thus available for interactions with cytoplasmic
AMPK. Second, while BTK can interact with PKC, it is not
known to interact with AMPK.35 Third, high-quality chemical
probes of BTK and their co-crystal structures are available,
allowing rational design of PHICS and inactive controls by
engineering BTK or PHICS.36 Fourth, BTK possesses a
phosphorylation site (S180) that not only lies in substrate-like
motif of AMPK but plays an important role in negative
regulation of BTK.35 Finally, BTK is undetectable in
Figure 3. PHICS induces proximity and phosphorylation in cellulo. (A) Structures of PHICS3 for phosphorylation of BTK via AMPK and the
inactive analogue, Piv-PHICS3. (B) Detection of ternary complex formation in HEK293T cells by co-immunoprecipitation of AMPK and BTK-
Flag in the presence of PHICS3. WCL: Whole Cell Lysate (C,D) Western blot analysis of BTK phosphorylation by PHICS3 in HEK293T cells
expressing wild-type BTK-Flag (C) or S180A variant (D). See Figures S3 and S14 for structures of AMPK activator and BTK inhibitor.
C
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Figure 4. Cellular validation of PHICS. (A) Competition experiment with covalent BTK inhibitor, Ibrutinib. (B) Key interactions of Ibrutinib with
BTK (PDB ID: 5P9I). (C) Western blot analysis of PHICS3-induced phosphorylation of wild-type BTK-Flag and T474A variant. (D) Western blot
analysis of BTK phosphorylation with PHICS3 and its inactive analogue, Piv-PHICS3.
Authors
Herein, we successfully rewired both AMPK and PKC
kinases using chimeric small molecules to induce novel
phosphorylation events, including neo-phosphorylations of
BRD4 and a signaling-relevant phosphorylation of BTK.
PHICS exhibited the hallmarks of a typical bifunctional
molecule, including the hook effect, turnover, dependence on
proximity (linker length), isoform specificity, and dose- and
temporal-control of phosphorylation. The turnover likely arises
from the reversible binding of the PHICS to the kinase and
target protein, which is also observed for PROTACs. Kinase
specificity has been previously rewired using adaptor
proteins,12,13 but we focused on small molecules since they
are cell-permeable and non-immunogenic. Furthermore, they
afford facile dose and temporal controls, exhibit fast kinetics
and turnover, and possess modular design allowing rapid
assembly. While these studies have focused on serine/
threonine-kinases, we are investigating the PHICS-mediated
rewiring of tyrosine kinases. Our future studies will deploy
PHICS to induce signaling-relevant phosphorylation as well as
neo-phosphorylation of oncogenic proteins that could evoke
an immune response against a tumor or the PHICS-mediated
deposition of a negative charge on the DNA-binding domains
of transcription factors (which are often deemed chemically
undruggable)39 that could adversely impact their ability to
bind to DNA. Overall, PHICS expands the toolkit of chimeric
small molecules that can be used to induce various post-
translational modifications.
Sachini U. Siriwardena − Chemical Biology and Therapeutics
Science, Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115, United
States
Dhanushka N. P. Munkanatta Godage − Chemical Biology
and Therapeutics Science, Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; Department of
Medicine, Harvard Medical School, Boston, Massachusetts
02115, United States
Veronika M. Shoba − Chemical Biology and Therapeutics
Science, Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115, United
States; orcid.org/0000-0003-0402-7414
Sophia Lai − Chemical Biology and Therapeutics Science, Broad
Institute of MIT and Harvard, Cambridge, Massachusetts
02142, United States; Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts 02138,
United States
Mengchao Shi − Chemical Biology and Therapeutics Science,
Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115, United
States; Divisions of Renal Medicine and Engineering, Brigham
and Women’s Hospital, Boston, Massachusetts 02115, United
States
Peng Wu − Chemical Biology and Therapeutics Science, Broad
Institute of MIT and Harvard, Cambridge, Massachusetts
02142, United States; Department of Medicine, Harvard
Medical School, Boston, Massachusetts 02115, United States;
Divisions of Renal Medicine and Engineering, Brigham and
Women’s Hospital, Boston, Massachusetts 02115, United
States; orcid.org/0000-0002-0186-1086
Santosh K. Chaudhary − Chemical Biology and Therapeutics
Science, Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115, United
States
ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c05537.
Supplementary Figures S1−S17; Materials and Meth-
ods; Characterization of compounds: 1H and 13C spectra
(Figures S18−S70); Supplementary References (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Stuart L. Schreiber − Chemical Biology and Therapeutics
Science, Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Chemistry
and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, United States; orcid.org/0000-0003-
1922-7558
Amit Choudhary − Chemical Biology and Therapeutics Science,
Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States; Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115, United
States; Divisions of Renal Medicine and Engineering, Brigham
and Women’s Hospital, Boston, Massachusetts 02115, United
States; orcid.org/0000-0002-8437-0150;
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c05537
Email: achoudhary@bwh.harvard.edu
D
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(13) Bhattacharyya, R. P.; Remenyi, A.; Yeh, B. J.; Lim, W. A.
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Author Contributions
⊥S.U.S., D.N.P.M.G., V.M.S., and S.L. contributed equally to
this work and are listed arbitrarily.
Notes
(14) Cameron, K. O.; Kung, D. W.; Kalgutkar, A. S.; Kurumbail, R.
G.; Miller, R.; Salatto, C. T.; Ward, J.; Withka, J. M.; Bhattacharya, S.
K.; Boehm, M.; Borzilleri, K. A.; Brown, J. A.; Calabrese, M.; Caspers,
N. L.; Cokorinos, E.; Conn, E. L.; Dowling, M. S.; Edmonds, D. J.;
Eng, H.; Fernando, D. P.; Frisbie, R.; Hepworth, D.; Landro, J.; Mao,
Y.; Rajamohan, F.; Reyes, A. R.; Rose, C. R.; Ryder, T.; Shavnya, A.;
Smith, A. C.; Tu, M.; Wolford, A. C.; Xiao, J. Discovery and
Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)-
phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct
Activator of Adenosine Monophosphate-activated Protein Kinase
(AMPK), for the Potential Treatment of Diabetic Nephropathy. J.
Med. Chem. 2016, 59 (17), 8068−81.
(15) Mach, U. R.; Lewin, N. E.; Blumberg, P. M.; Kozikowski, A. P.
Synthesis and pharmacological evaluation of 8- and 9-substituted
benzolactam-V8 derivatives as potent ligands for protein kinase C, a
therapeutic target for Alzheimer’s disease. ChemMedChem 2006, 1
(3), 307−314.
(16) Zegzouti, H.; Zdanovskaia, M.; Hsiao, K.; Goueli, S. A. ADP-
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The authors declare the following competing financial
interest(s): Broad Institute has filed a patent application
pertaining to the work described herein. S.L.S. serves on the
Board of Directors of the Genomics Institute of the Novartis
Research Foundation (GNF); is a shareholder and serves on
the Board of Directors of Jnana Therapeutics; is a shareholder
of Forma Therapeutics; is a shareholder and advises Decibel
Therapeutics and Eikonizo Therapeutics; serves on the
Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma
Foundation, Exo Therapeutics, and F-Prime Capital Partners;
and is a Novartis Faculty Scholar.
ACKNOWLEDGMENTS
■
V.M.S. and S.L. were supported by the Damon Runyon
Postdoctoral Fellowship Award and the NSF GRFP (DGE-
1745303), respectively. This work was supported by the
Merkin Institute of Transformative Technologies in Healthcare
and DARPA (N66001-17-2-4055). We thank Prof. C. M.
Chiang (UT Southwestern Medical Center) for providing the
BRD4 phospho-Ser484/488 antibody and Dr. P. K. Tiwari
(Broad Institute) for assistance with synthesis. This work is
dedicated to Prof. Laura L. Kiessling on the occasion of her
60th birthday.
(17) Wurz, R. P.; Dellamaggiore, K.; Dou, H.; Javier, N.; Lo, M. C.;
McCarter, J. D.; Mohl, D.; Sastri, C.; Lipford, J. R.; Cee, V. J. A “Click
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for Inducing Protein Degradation. J. Med. Chem. 2018, 61 (2), 453−
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(18) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
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Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.;
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