Structure-Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00402

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure-activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

Full text of DOI:10.1021/acsmedchemlett.9b00402

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Letter
Structure-Activity Relationship Study of Covalent Pan-
Phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors
Theresa D. Manz, Sindhu C. Sivakumaren, Adam Yasgar, Matthew D. Hall, Mindy
I. Davis, Hyuk-Soo Seo, Joseph D Card, Scott B Ficarro, Hyeseok Shim, Jarrod A.
Marto, Sirano Dhe-Paganon, Atsuo T. Sasaki, Matthew B. Boxer, Anton Simeonov,
Lewis C. Cantley, Min Shen, Tinghu Zhang, Fleur M. Ferguson, and Nathanael S Gray
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00402 • Publication Date (Web): 03 Nov 2019
Downloaded from pubs.acs.org on November 4, 2019
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Structure-Activity Relationship Study of Covalent Pan-
Phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors
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†,‡,┴
†,‡
Δ
Δ
Δ
Theresa D. Manz,
Sindhu C. Sivakumaren, Adam Yasgar, Matthew D. Hall, Mindy I. Davis,
†,‡
ψ,ϴ,φ
ψ,ϴ,φ
§
ψ,ϴ,φ
Hyuk-Soo Seo, Joseph D. Card,
Scott B. Ficarro,
Hyeseok Shim, Jarrod A. Marto,
†
,‡
ᴨ
Δ
Δ
§
Sirano Dhe-Paganon, Atsuo T. Sasaki, Matthew B. Boxer, Anton Simeonov, Lewis C. Cantley,
Δ
†,‡,*
†,‡,*
†,‡,*
Min Shen, Tinghu Zhang,
Fleur M. Ferguson,
and Nathanael S. Gray
†
‡
Department of Cancer Biology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, MA 02215, USA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA
Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbruecken, Germany
┴
ΔNational Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
ᴨDivision of Hematology and Oncology, University of Cincinnati, 3125 Eden Ave., Cincinnati, OH 45267-0508, USA
ψDepartment of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Ma 02215, USA
ϴDepartment of Oncologic Pathology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, MA 02215, USA
φDepartment of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215, USA
§Meyer Cancer Center, Weill Cornell Medicine and New York Presbyterian Hospital, New York, NY 10065, USA
ABSTRACT: Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such
as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular
on-target activity. In this study, we present a new class of covalent
pan-PI5P4K inhibitors with potent biochemical and cellular activity.
Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor
previously developed in our lab. Here, we report further structure-
guided optimization and structure-activity relationship (SAR) study
of this scaffold, resulting in compound 30, which retained
biochemical and cellular potency, while demonstrating
a
significantly improved selectivity profile. Furthermore, we confirm
that the inhibitors show efficient binding affinity in the context of
HEK 293T cells using isothermal CETSA methods. Taken together,
compound 30 represents a highly selective pan-PI5P4K covalent
lead molecule.
KEYWORDS: PI5P4K, lipid kinase inhibitors, structure-activity relationship, covalent inhibitors, phosphoinositide, drug discovery
N
The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks),
consisting of the three isoforms, PI5P4Kα, β, and γ, are lipid
kinases that catalyze phosphorylation of phosphatidylinositol 5-
I
N
HO
HO
OH
HN
OMe
N
O
phosphate (PI5P) on its 4-position to form phosphatidylinositol-
1
I-OMe Tyrphostin AG 538
a131
4,5-bisphosphate (PI-4,5-P
2
). In the cellular membrane, PI-4,5-
IC50 = 1 M (PI5P4K)
IC50 = 1.9 M (PI5P4K)
P
2
is also produced by another signaling pathway, in which
phosphatidylinositol 4-phosphate (PI4P) is phosphorylated by
phosphatidylinositol 4-phosphate 5-kinases (PI4P5Ks).
Although the majority of PI-4,5-P is produced via the PI4P5K
CF3
O
NH2
N
O
N
N
N
O
N
N
S
N
2
N
N
CF3
N
S
N
H
NH
pathway, the PI5P4Ks have been recognized as key regulators
of many cell functions including metabolism, stress response,
N
HN
S
S
O
2-8
autophagy, and immunological processes. Dysregulation of
SAR088
IC50 = 3.8 M (PI5P4K)
NIH-12848
NCT-504
IC50 = 15.8 M (PI5P4K)
the PI5P4K signaling pathway has been further linked to
IC50 = 1 M (PI5P4K)
3
5
diseases such as diabetes, neurodegenerative disorders, and
Figure 1. Reported PI5P4K inhibitors.
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cancers9-11. These findings indicate that the inhibition of
PI5P4K kinase activity with a small molecule inhibitor might
have a therapeutic potential across various diseases.
To understand the SAR of THZ-P1-2 analogs, we employed
biochemical assays for PI5P4Kα (ADP Glo) and PI5P4Kβ
Scheme 1. Synthesis of THZ-P1-2 and Compounds 6-11a
1
2
3
4
5
6
7
8
9
To date, several scaffolds have been developed as PI5P4K
5
,12-15
N
N
a or b
N
N
c
N
N
d, e
inhibitors.
For example, I-OMe Tyrphostin AG 538 was
Cl
Cl
Cl
R
H2N
N
R
1
2
reported to inhibit PI5P4Kα with an IC50 of 1 µM, while NIH-
12848 was found to be a PI5P4Kγ selective compound
Figure 1). However, though some of the reported compounds
H
1
2a-g
3a-g
1
5
O
N
N
O
N
N
(
f or g
N
N
H
R
O
N
N
H
R
might seem able to covalently label kinases, such as a131, these
inhibitors are reversible inhibitors with micromolar
biochemical potency against PI5P4K, which limits their use in
the investigation of PI5P4K function in the cellular context
H
H
N
H2N
N
R
R'
4a-g
H
5a,b, 7-11
h
THZ-P1-2, 6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R =
R' =
N
(Supporting Figure 6). In general, irreversible binding through
N
N
N
N
H
N
SO2Ph
a unique cysteine represents a viable strategy to improve
16
potency and selectivity. We recently reported the discovery of
N
NH
THZ-P1-2 as a pan-PI5P4K covalent small molecule inhibitor
by targeting conserved cysteines across all three PI5P4K
isoforms, that are situated outside of the ATP-binding pocket.
Here we report the structure-activity relationship (SAR) for this
series of compounds.
N
N
H
H
SO2Ph
a
Reagents and conditions: (a) (HO)
boronic acid esters, NaHCO , Pd(PPh
2
B-R or respective pinacol
Cl , ACN/H O, 90-100 °C,
3
3
)
2
2
2
2
1
.5-19 h, 53-83% yield; (b) 4-methylaniline, TEA, BuOH, 140 °C,
h or 3/4-phenylaniline, TEA, EtOH, 120 °C, 3 h, 99%-quant.
yield; (c) m-phenylenediamine, DIEA, NMP, 140-160 °C, 1.5 h -
2 d, 37-95% yield; (d) 4-nitrobenzoyl chloride, pyridine or
TEA/DCM, rt, 5 h or 4-((tert-butoxycarbonyl)amino)benzoic acid,
HATU, Hunig’s Base, DMF, rt, 1 h, then TFA/DCM, rt, 2 h, 37%
yield; (e) SnCl
2 2 2
·2H O, EtOAc/MeOH, 80 °C, 4.5 h - 2 d or H ,
1
0% Pd/C, EtOAc/MeOH or MeOH, rt, 16 h, 4-87% yield over 2
steps; (f) 4-bromocrotonyl chloride, DIEA, DCM or ACN, 0 °C,
then dimethylamine, THF, rt, 1 h, 24-71% yield; (g) 4-
dimethylaminocrotonic acid, DIEA, HATU, DCM, or oxalylic
chloride, DMF rt, 1-12 h, 6-30% yield; (h) 1.0 M NaOH/1,4-
dioxane, rt, 4-6 h, 8-60% yield.
Table 1. IC50 Evaluation of THZ-P1-2 and Compounds 6-11
O
N
N
O
N
N
R
H
H
N
N
Figure 2. Cocrystal structure of PI5P4Kα and THZ-P1-2 (dark
grey). The pocket bound by the inhibitor is highlighted in pale-
green. H-bonds are shown as dashed bonds. For clarity, only
Asn198, Val199, Phe134 and Phe200 are shown as capped sticks.
PDB-ID: 6OSP
H
IC50 [µM]a
Cpd
R
PI5P4Kα
PI5P4Kβ
THZ-P1-2
0.95 ± 0.34
5.9 ± 5.4
Our recently reported irreversible inhibitor of PI5P4Ks,
THZ-P1-2, displays sub-micromolar biochemical potency
N
H
17
against PI5P4Kα kinase activity. Although the THZ-P1-
6
7
2.1 ± 0.0
10.9 ± 4.0
15.8 ± 7.5
N
N
H
2
/PI5P4Kα co-crystal structure (PDB-ID: 6OSP) did not
resolve the loop that contains the covalently targeted cysteine
(Cys293), the structure clearly revealed key non-covalent
interactions, including two hydrogen bonds between the
aminopyrimidine, as well as the benzamide carbonyl and
Asn198, a hydrogen bond forming between the pyrimidine and
N
3.5 ± 0.24
N
8
9
1.3 ± 0.36
6.3 ± 2.6
N
H
Val199,
and
T-shaped
π-stacking
between
the
phenylenediamine group and Phe134 in the p-loop (Figure 2).
In addition, Phe134 and Phe200 form a narrow passage
encaging the phenylenediamine of THZ-P1-2 (Supporting
Figure 7). Even though the electron-density for the war head tail
was insufficiently resolved, the acrylamide moiety seemed to
be positioned towards the solvent exposed region, which allows
proximity to the targetable cysteines for each isoform: Cys 293
(α), 307 or 318 (β), and 313 (γ) (Supporting Figure 8).
Hydrophobic interactions between the indole and the
hydrophobic pocket under the p-loop contribute to the overall
affinity.
1.3 ± 0.16
19.3 ± 0.0
6.2 ± 1.3
N
H
1
1
0
1
N
H
24.5 ± 6.1
17.4 ± 4.8
30.4 ± 32.8
a
Average ± SD of three experiments, each using an 11-point
titration.
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Transcreener), which have produced reliable IC50 values for variety of different indole ring systems, or bulkier mono- or
Page 4 of 8
(
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9
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1
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1
1
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2
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5
5
5
5
5
5
6
THZ-P1-2. The THZ-P1-2 analogs, which contain either a
bicyclic
Scheme 2. Synthesis of Compounds 14-19a
H
R
H2N
R
N
R
N
N
a
N
N
d
N
N
g, i
N
N
N
H
H
H2N
N
O
N
Cl
N
N
H
N
0,1
H
0,1
13a,b
0,1
H
N
O
N
O
NH
N
2a
SO2Ph
12a,b
SO2Ph
SO2Ph
14, 15
O
O
R
R
O
R
N
N
b or c
N
N
e or f
N
N
g or h, i
N
N
H2N
N
N
N
H
H2N
A
H
N
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
N
N
H
N
H
H
H
N
N
N
N
N
N
N
N
H
SO2Ph
SO2Ph
SO2Ph
2b
3b, 12c,d
13c-f
16-19
R = H, CH3
A = phenyl, cyclohexyl
a
Reagents and conditions: (a) anilines, DIEA, NMP, 150 °C, 6-8 h, then TFA/DCM, rt, 1 h, 67-84% yield; (b) anilines, DIEA, NMP, 150 °C,
h – overnight, 46-60% yield; (c) nitroaniline, Cs CO , X-Phos, Pd (dba) , 1,4-dioxane, 104 °C, 3 h, then SnCl , MeOH, 80 °C, 8 h, 61%
·2H O, EtOAc/MeOH, 80 °C, 1.5-2 d, 23-39% yield over 2 steps; (e)
carboxylic acid chlorides, DIEA or TEA, DCM, 0 °C, 5 min or rt, 1.5 h, then H , 10% Pd/C, MeOH or EtOAC/MeOH, rt, 12 h – overnight,
6-51% over 2 steps; (f) carboxylic acids, HATU, DIEA, DCM, rt, 0.5-3 h, then HCl, MeOH or EtOAc, rt, 0.5 h – overnight, 54-85% over
steps; (g) 4-bromocrotonyl chloride, DIEA, DCM or ACN, 0 °C, 5 min - 0.5 h, then dimethylamine (2.0 M in THF), rt, 0.5-2 h, 30-91%
8
2
3
2
3
2
yield; (d) carboxylic acid chlorides, pyridine, rt, overnight, then SnCl
2
2
2
3
2
yield; (h) 4-dimethylaminocrotonic acid hydrochloride, DIEA, HATU, DCM, rt, 12 h, 30% yield; (i) 1.0 M NaOH/1,4-dioxane, 10 °C - rt,
-6 h, 10-46% yield.
3
aromatic six-membered rings, were first designed to investigate
the tolerability of the hydrophobic pocket that is occupied by
the indole group in THZ-P1-2. The synthetic strategy for these
analogs revolved around the 4,6-dichloropyrimidine, which
underwent either a Suzuki coupling reaction with aromatic
a distinct 3-aminopiperidine group installed at 6-position of the
pyrimidine, followed by a urea functionality, which was utilized
to conjugate with 3-aminobenzeneamine (Scheme 3). All these
compounds contain a free amino group, which was eventually
converted to a 4-dimethylaminocrotonamide as a cysteine react-
N
boronic acids/esters, or an S 2 nucleophilic substitution
Table 2. IC50 Evaluation of Compounds 14-19,22,25,30
reaction with aromatic amines. The second chloride group was
then substituted with m-phenylenediamine, which was extended
through an amide bond with 4-aminobenzoic acid, containing a
free amino group for 4-dimethylaminocrotonamide
transformation, as the final step (Scheme 1).
IC50 [µM]a
Cpd
R
PI5P4K
α
β
H
N
THZ-P1-2 showed sub-micromolar potency (IC50 of 950 nM)
against PI5P4Kα, as well as an IC50 of 5.9 µM against PI5P4Kβ.
Pyrrolo[2,3-b]pyridine installed in compound 6 led to a slight
decrease in potency on both isoforms, while a 2-methylindazole
of compound 7 dampened the activity even further to 3.5 µM
and 15.8 µM for PI5P4Kα and PI5P4Kβ, respectively (Table 1).
Introducing an NH-linker, connecting the pyrimidine with a
phenyl group carrying either a 4-methyl group or even a bulky
-phenyl substituent (compounds 9 and 8, respectively) were
well tolerated and maintained IC50 values of around 1.3 µM and
.2-6.3 µM for the α- and β-isoform of the PI5P4Ks,
respectively. In contrast, a 3-phenylaniline or direct conjugation
of pyrimidine with a 3-phenylbenzene group resulted in a more
than 10-fold loss in PI5P4Kα activity (compounds 10 and 11),
underlining that these changes in the trajectory of binding seem
to cause a steric clash with some residues in the hydrophobic
pocket under the p-loop.
N
N
N
5.2
29.8
H
N
1
1
4
5
O
O
N
H
± 2.8
± 12.1
O
O
NH
N
N
5.3
33.6
N
H
N
H
N
NH
N
H
± 3.9
± 8.2
O
O
N
N
N
6
.5
20.5
O
N
H
N
H
16
17
N
N
N
N
H
N
N
N
± 0.90
± 11.9
H
4
N
H
N
2
.1
8.9
N
H
N
H
6
O
O
N
H
± 0.78
± 2.9
H
N
H
N
1.99
35.6
N
N
1
8
O
N
± 0.36
± 2.4
H
N
N
H
O
N
N
2
.0
31.8
Next, to understand the SAR for the groups that bind the
hinge region, as well as the solvent-exposed region, we
designed a second batch of THZ-P1-2 analogs, shown in
Table 2. To access these compounds, the second chloride
moiety at 6-position of the pyrimidine was substituted with
different aromatic amines through S
then further conjugated with either 3- or 4-nitro benzoic acid,
-aminocyclohexane-1-carboxylic acid or 4-aminopiperidine
O
N
H
N
1
2
2
9
2
5
H
N
N
N
H
N
N
H
± 1.17
± 4.1
N
N
H
N
H
N
N
N
inactive
inactive
H
O
O
O
N
N
H
N
2 reactions, which were
O
N
N
2.6
10.1
N
N
H
N
N
H
N
N
± 0.76
± 10.2
4
H
through amide bond formation to yield compounds 14-19 and
25 (Schemes 2 and 4). We also synthesized compound 22 with
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O
N
N
a
Average ± SD of three experiments, each using an 11-point
1
.34
9.9
O
N
N
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
30
H
H
titration.
N
N
± 0.156
± 2.8
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. A. KINOMEscan results for THZ-P1-2 and compound 30. Selectivity profile was determined for 469 kinases as percent DMSO
control at 1 µM inhibitor concentration (excluding mutant, atypical and pathogen). B. Table comparing percent DMSO control for top 5
off-targets of THZ-P1-2 to compound 30.
ive warhead.
respectively) showed almost identical activity with IC50 values
of 2.0-2.5 µM on PI5P4Kα. Interestingly, PI5P4Kβ seems to be
more sensitive to changes in this motif, since the IC50 of
compound 19 drops by 5-fold, compared to THZ-P1-2.
Similarly, the regiochemical variation of compound 6 to
compound 17, attaching the crotonamide residue in either 3- or
4-position of the benzamide, or to compound 18, carrying either
a m- or p-phenylenediamine, displayed again only minor effects
on the inhibitory activity on PI5P4Kα. While changing the
warhead attachment-site on the benzamide did not have any
effect on PI5P4Kβ activity either (compounds 6 and 17),
changes in the phenylenediamine moiety led to a 3.5-fold higher
IC50 (compound 18), underlining a higher sensitivity of the β-
isoform to alterations in this region. However, an extended
linker with an insertion of a methylene group, shifting the
benzamide carbonyl further away from the hinge region, in
compound 14 became more resistant to both, PI5P4Kα and
PI5P4Kβ.
We first found that introducing a methyl group to either
carbon-4 (compound 15) or carbon-6 (compound 16) led to up
to 5-fold higher IC50s compared to THZ-P1-2 and compound 6.
An explanation could be that the conformational changes in
these compounds are potentially causing steric clashes with the
relatively small passage (Supporting Figure 7) or are
jeopardizing the π-stacking interaction with Phe134. Going
even further, using a bulky, non-aromatic piperidine ring,
resulted in the overall inactive compound 22.
Scheme 3. Synthesis of Compound 22a
N
N
a, b
N
N
c, d
HN
Cl
N
H
N
N
N
N
SO2Ph
SO2Ph
2b
20
N
N
H
H2N
N
N
e, f
N
H
O
O
N
N
Scheme 4. Synthesis of Compound 25a
SO2Ph
21
N
N
N
N
a
O
N
N
b, c
H
N
H
N
N
N
N
Cl
HO
N
H
H
O
N
N
N
N
H
N
N
SO2Ph
SO2Ph
22
2b
23
a
O
N
N
Reagents: (a) tert-butyl 3-aminopiperidine-1-carboxylate, DIEA,
NMP, 110 °C, overnight, 50% yield; (b) TFA/DCM, rt, overnight,
quant. yield; (c) 3-nitroaniline, triphosgene, TEA, DCM, 0 °C, 2 h,
then rt, overnight, 83% yield; (d) SnCl , EtOAc/MeOH, reflux,
2
overnight, 74% yield; (e) 4-bromocrotonyl chloride, DIEA, DCM,
0 °C, 5 min, then dimethylamine (2.0 M in THF), rt, 1 h, 68% yield;
d, e
N
N
H
H2N
N
N
SO2Ph
24
O
N
N
O
N
N
H
N
N
H
N
N
H
(
f) 1.0 M NaOH/1,4-dioxane, 10 °C - rt, 4 h, 30% yield.
25
A scan of different crotonamide warhead-containing linkers
a
Reagents: (a) 2-(3-aminophenyl)acetic acid, DIEA, NMP, 140 °C,
2 h, 6% yield; (b) tert-butyl piperidin-4-ylcarbamate, HATU,
pointing towards the solvent-exposed area, including either
piperidine, cyclohexane or phenyl rings with different
substitution patterns demonstrated high tolerability to both the
composition and the conformation of the linker with respect to
activity against PI5P4Kα. For example, both piperidine and
cyclohexane containing compounds (compounds 25 and 19,
1
DIEA, DMF, rt, 0.5 h, 51% yield; (c) HCl, EtOAc, rt, 0.5 h, 90%
yield; (d) 4-dimethylaminocrotonic acid hydrochloride, DIEA,
HATU, DCM, rt, 0.5 h, 50% yield; (e) 1.0 M NaOH/1,4-dioxane,
rt, 4 h, 56% yield.
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Inspired by the SAR information we obtained above, as well
as the co-crystal structure of our inhibitor THZ-P1-2 with
PI5P4Kα , we cyclized the pyrimidine ring from carbon-4 and
-5 to yield a bicyclic pyrrolo[2,3-d]pyrimidine and introduced a
this could potentially improve the overall selectivity of the
scaffold, while maintaining on-target activity. The compound
was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
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3
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3
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4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
1
7
4
-methylphenyl, a favored residue (e.g. compound 9), attached
to the N-atom of the pyrrolo ring system. We hypothesized that
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
m
Figure 4. A..Melting-curves (T ) of PI5P4Kα and PI5P4Kβ. Treatment with THZ-P1-2 at 10 µM for 1 h, in comparison to DMSO.
B. Representative western blot of isothermal CETSA at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors at 1 or 10 µM.
C. Quantified relative band intensity (%) of isothermal CETSA experiment (Figure 4B). Mean of three independent experiments. D.
Dose-response curve of isothermal CETSA for PI5P4Kα at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors. Mean of
three independent experiments (Representative western blot, see Supporting Figure 5A). E. Dose-response curve of isothermal
CETSA for PI5P4Kβ at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors. Mean of three independent experiments
(Representative western blot, see Supporting Figure 5B).
Scheme 5. Synthesis of Compound 30a
coupled with m-phenylenediamine, extended with benzoic acid
via an amide bond and the free amino group was finally
converted to a crotonamide as already described in Scheme 1
N
N
a
N
N
b
Cl
NH
Cl
N
O
(Scheme 5), leading to active compound 30 with IC50 values of
26
27
1
.3 µM and 9.9 µM for PI5P4Kα and PI5P4Kβ, respectively.
As previously reported, the selectivity profiling of THZ-P1-
N
N
N
N
c, d
e, f
217 with KINOMEscan has shown that this compound exhibits
cross activity with the type I lipid kinase PIKFYVE, as well as
several protein kinases including BRK, TYK2 and Abl. We next
questioned whether the bicyclic pyrrolo[2,3-d]pyrimidine
scaffold on compound 30 could dial out these off-targets,
making it a more selective PI5P4K inhibitor. Therefore, another
KINOMEscan profiling assay for compound 30 was
undertaken. At 1 µM, compound 30 lost almost all off-targets
seen with THZ-P1-2, including the PI5P4K-related PIKFYVE
kinase (Figure 3). An ADP-Glo biochemical assay
H2N
N
N
N
N
N
H
H
H
H2N
O
28
29
N
N
O
N
N
N
H
H
N
N
H
30
a
Reagents: (a) 4-iodotoluidine, trans-1,2-cyclohexanediamine,
PO , CuI, toluene, 110 °C, 1 d, 48% yield; (b) m-
phenylenediamine, DIEA, NMP, 150 °C, 4 d, quant. yield; (c)
-nitrobenzoyl chloride, pyridine, rt, overnight; (d)
SnCl ·2H O, EtOAc/MeOH, 80 °C, 1 d, 40% yield over 2 steps;
e) 4-bromocrotonyl chloride, DIEA, ACN, 0 °C; (f)
K
3
4
4
(
commercially available from Carna Biosciences, Inc.) done
with PIKFYVE confirmed these findings as an IC50 value of
0.1 nM for THZ-P1-2, whereas compound 30 did not show
2
2
(
4
dimethylamine (2.0 M in THF), rt, 1 h, 4% yield over 2 steps.
any activity in this assay format, which was also true for other
off-targets (Supporting Table 2). Since the two compounds
solely differ in their head group moieties, facing the
hydrophobic pocket in the binding site, potential reasons for the
synthesized through the coupling of 4-chloro-7H-pyrrolo[2,3-
d]pyrimidine with 4-iodotoluene in
a copper-catalyzed
nucleophilic substitution reaction. The free chloride was then
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ACS Medicinal Chemistry Letters
selectivity improvement could be the slightly bulkier head
methods, we were able to confirm that the inhibitors show
efficient binding affinity in the context of HEK 293T cells.
These findings suggest that compound 30 is a selective,
covalent pan-PI5P4K inhibitor, which may overcome
limitations of cellular activity of reported PI5P4K inhibitors.
Further efforts to optimize cellular potency of compound 30
and its pharmacokinetics for in vivo studies are ongoing.
1
2
3
4
5
6
7
8
9
group in compound 30, as well as the missing hydrogen bond
offering NH group of the indole found in THZ-P1-2. It should
be noted, that PI5P4Kα is not assessed in the KINOMEscan
panel. Although PI5P4Kβ and PI5P4Kγ were included in the
kinases tested, we could not see significant binding of both
compounds tested, despite their biochemical potency and
cellular target engagement. Hence, the kinome-wide profiling
data (Figure 3A) was used to investigate potential off-targets.
ASSOCIATED CONTENT
Supporting Information
To assess the cellular activity of the THZ-P1-2 analogs, we
used a cellular thermal shift assay (CETSA), in which we
treated HEK 293T cells with our most potent pan-PI5P4K
inhibitors. We first determined the optimal incubation
temperature for the assay, by comparing melting curves of cells
treated with either DMSO or 10 µM THZ-P1-2 at temperatures
ranging from 40-67 °C (Figure 4A). While we observed loss of
soluble PI5P4Kα and PI5P4Kβ between 49 and 52 °C with the
DMSO control, THZ-P1-2 stabilized the enzymes to
temperatures up to 58 to 61 °C, showing that THZ-P1-2 exhibits
good pan-PI5P4K binding in the cellular context. We
determined 52 °C to be the optimal assay temperature to
investigate cellular target engagement of our compounds, since
incubation at this temperature resulted in still optimal
stabilization of soluble PI5P4Kα/β with THZ-P1-2, while the
DMSO treated cells show already an immense loss of soluble
protein. This temperature was used for further CETSA
experiments. Next, an isothermal CETSA was performed to
compare cellular target engagement of our top inhibitors,
showing good target binding activity of all selected compounds
in HEK 293T cells (Figure 4B and 4C). Dose-response curves
were obtained using the same isothermal CETSA conditions.
Surprisingly, compounds 8 and 30 generally showed slightly
less cellular activity compared to THZ-P1-2, while compound
1
1
1
1
1
1
1
1
1
1
2
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2
2
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2
2
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3
3
3
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6
0
1
2
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0
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0
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0
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0
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6
7
8
9
0
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental and characterization data for all new
compounds, additional biological data and protocols
(PDF).
AUTHOR INFORMATION
Corresponding Author
* Tinghu Zhang: Tinghu_Zhang@dfci.harvard.edu
* Fleur M. Ferguson: FleurM_Ferguson@dfci.harvard.edu
*
Nathanael S. Gray: Nathanael_gray@dfci.harvard.edu
Author Contributions
T.D.M., T.Z., F.M.F. and N.S.G. conceived the project and
designed the research strategy. T.D.M., T.Z. and F.M.F. designed
and synthesized the compounds. S.C.S. performed compound
testing and helped with planning experimental strategies. M.B.B.
synthesized additional compounds for the biochemical assay
validation. A.Y., M.I.D., A.S. and M.D.H. developed and
validated the biochemical assays. A.Y. performed the biochemical
assays under supervision of M.D.H. and M.S.. H.-S. S. and S.D.-
P. generated the co-crystal structure. A.T.S. provided reagents and
methods for the biochemical assays. S.C.S., J.D.C., S.B.F., and
J.A.M. performed the intact mass spectrometry analysis. T.D.M.
planed and performed the cellular assays. H.S. and L.C.C. were
involved in the planning and provided expertise and feedback for
the project. The manuscript was written through T.D.M and
contributions of all authors. All authors have given approval to the
final version of the manuscript. T.Z., F.M.F. and N.S.G.
supervised the research.
9
seems to be slightly more active in cells (Figure 4D, 4E and
Supporting Figure 5A and 5B).
Ultimately, comparing our lead compounds, THZ-P1-2 and
compound 30, with their reversible analogs (THZ-P1-2-R and
compound 30-R, respectively) lead to an overall decrease in
biochemical activity of 3-4-fold, except for compound 30 and
its PI5P4Kα activity, which did not change significantly
(Supporting Figure 9A). The same trend was seen in an
isothermal CETSA experiment, although compound 30 now
seems to show similar potency changes on both, PI5P4Kα and
β (Supporting Figure 9B and C). However, the observed
potency changes between covalent and non-covalent analogs
were again more pronounced with THZ-P1-2. To make sure that
compound 30 is still a covalent PI5P4K inhibitor, an intact mass
spectrometry experiment was undertaken, which showed
indeed covalent labeling of PI5P4Kα,β and γ by compound 30
Notes
The authors declare the following competing financial interest(s):
N.S.G. is a founder, SAB member and equity holder in
Gatekeeper, Syros, Petra, C4, B2S and Soltego. The Gray lab
receives or has received research funding from Novartis, Takeda,
Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield
and Sanofi. N.S.G. and T.Z. are inventors on a patent application
covering chemical matter in this publication, owned by Dana-
Farber Cancer Institute. L.C.C. is a founder and member of the
Board of Directors (BOD) of Agios Pharmaceuticals and is a
founder and receives research support from Petra
(Supporting Figure 10). These results indicate, that compound
3
0 might be a slower covalent binder than THZ-P1-2, which
Pharmaceuticals. These companies are developing novel therapies
for cancer. J.A.M. serves on the SAB of 908 Devices.
could explain not only the biochemical results, but also the
weaker cellular activity of compound 30.
In conclusion, the discovery of THZ-P1-2 and its analogs
present a novel class of covalent inhibitors for PI5P4Ks. The
SAR study reported here provides a rational for further
development, by systematically exploring the tolerability of
each binding region for chemical modification. The selectivity
profiling indicated a highly selective PI5P4K inhibitor is
achievable through the modification of the core 4,6-pyrimidine
scaffold, shown by compound 30. Furthermore, investigating
the cellular target engagement, using isothermal CETSA
ACKNOWLEDGMENT
The authors would like to thank M. Kostic for her help and
expertise with writing the manuscript.
Funding Sources
The authors would like to thank the National Institutes of Health
for their generous financial support through grants R01
CA197329 (to N.S.G., subcontracted to S.D.P.), R01 NS089815
and R03 MH096575 (to A.T.S.), R01 CA222218 and R03
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CA250020 (to J.A.M.),as well as R35 CA197588, R01
(7) Bulley, S. J.; Droubi, A.; Clarke, J. H.; Anderson, K. E.;
Stephens, L. R.; Hawkins, P.T.; Irvine, R. F. In cells,
phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P to
impact mTORC2 and Akt signaling. PNAS 2016, 113 (38), 10571-
0576.
8) Keune, W.-J.; Jones, D. R.; Divecha, N. PtdIns5P and Pin1 in
oxidative stress signaling. Advances in Biological Regulation 2013, 53
2), 179-189.
9) Jude, J. G.; Spencer, G. J.; Somerville, T. D. D.; Jones, D. R.;
Divecha, N.; Somervaille, T. C. P. A targeted knockdown screen of
genes coding for phosphoinositide modulators identifies PIP4K2A as
required for acute myeloid leukemia cell proliferation and survival.
Oncogene 2015, 34 (10), 1253-1262.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
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4
4
4
4
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4
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5
5
5
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5
5
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GM041890 and U54 CA210184 (to L.C.C.). This work was
further supported by the Breast Cancer Research Foundation (to
L.C.C.) and the NIH intramural research program (NCATS).
B
2
1
(
ABBREVIATIONS
ACN, acetonitrile; BRK, breast tumor-related kinase; CETSA,
cellular thermal shift assay; Cpd, compound; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; HATU, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
(
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b]pyridinium 3-oxide hexafluorophosphate; NMP, N-methyl-2-
(10) Luoh, S.-W.; Venkatesan, N.; Tripathi, R. Overexpression of
the amplified Pip4k2β gene from 17q11-12 in breast cancer cells
confers proliferation advantage. Oncogene 2004, 23, 1354-1363.
pyrrolidone; Pd
PI-4,5-P
2
(dba)
3
, Tris(dibenzylideneacetone)dipalladium(0);
PI4P,
2
,
phosphatidylinositol-4,5-bisphosphate;
(11) Emerling, B. M.; Hurov, J. B.; Poulogiannis, G.; Tsukazawa,
phosphatidylinositol 4-phosphate; PI4P5K, phosphatidylinositol 4-
phosphate 5-kinase; PI5P, phosphatidylinositol 5-phosphate;
PI5P4K, phosphatidylinositol 5-phosphate 4-kinase; PIKFYVE,
phosphatidylinositol 3-phosphate 5-kinase; rt, room temperature;
SAR, structure-activity relationship; SD, standard deviation; TEA,
triethylamine; TFA, trifluoro acetic acid; THF, tetrahydrofuran;
TYK2, tyrosine kinase 2; X-Phos, 2-Dicyclohexylphosphino-
K. S.; Choo-Wing, R.; Wulf, G. M.; Bell, E. L.; Shim, H.-S.; Lamia, K.
A.; Rameh, L. E.; Bellinger, G.; Sasaki, A. T.; Asara, J. M.; Yuan, X.;
Bullock, A.; DeNicola, G. M.; Song, J.; Brown, V.; Signoretti, S.;
Cantley, L. C. Depletion of a Putatively Druggable Class of
Phosphatidylinositol Kinases Inhibits Growth of p53-Null Tumors.
Cell 2013, 155 (4), 844-857.
(12) Davis, M. I.; Sasaki, A. T.; Shen, M.; Emerling, B. M.; Thorne,
2
',4',6'-triisoropylbiphenyl.
N.; Michael, S.; Pragani, R.; Boxer, M.; Sumita, K.; Takeuchi, K.;
Auld, D. S.; Li, Z.; Cantley, L. C.; Simeonov, A. A Homogeneous,
High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-
Kinase with a Novel, Rapid Substrate Preparation. PLoS ONE 2013, 8
(1), e54127.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment