In Silico HTS and Structure Based Optimization of Indazole-Derived ULK1 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00344

We present the outcome of an in silico high throughput screen (HTS) and optimization of a small molecule Unc-51-Like Kinase 1 (ULK1) inhibitor hit, SR-17398, with an indazole core. Docking studies guided design efforts that led to inhibitors with increase

Full text of DOI:10.1021/acsmedchemlett.7b00344

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX-XXX
In Silico HTS and Structure Based Optimization of Indazole-Derived
ULK1 Inhibitors
Spencer D. Wood,§ Wayne Grant,† Isabel Adrados,† Jun Yong Choi,§ James M. Alburger,§
Derek R. Duckett,† and William R. Roush*,§
†
§Department of Chemistry and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter,
Florida 33458, United States
S
* Supporting Information
ABSTRACT: We present the outcome of an in silico high
throughput screen (HTS) and optimization of a small molecule
Unc-51-Like Kinase 1 (ULK1) inhibitor hit, SR-17398, with an
indazole core. Docking studies guided design efforts that led to
inhibitors with increased activity vs ULK1 (IC50 < 50 nM). The
most advanced molecules in this inhibitor series (3a and 3g)
hold promise for further development into selective ULK1
molecular probes to interrogate the biology of ULK1 and to
assess whether selectively targeting autophagy is an effective
anticancer strategy.
KEYWORDS: In silico high-throughput screen, ULK1, inhibitor, indazole
acroautophagy (hereafter autophagy) is an important
unstructured serine−proline-rich domain.14−16 ULK1 is also
Mcellular process that maintains energy homeostasis activated by the GSK3-TIP60 signaling pathway upon growth
factor deprivation.17 Small molecule inhibition of ULK1
during periods of stress and starvation and plays a major role
in controlling protein and organelle quantity and quality.1
potentially provides an avenue for suppressing autophagy.
Recently, X-ray crystal structures of ULK1 were elucidated by
the Shokat group featuring ATP competitive inhibitors
cocrystallized with the kinase; there are also reports of other
early stage inhibitors in the literature.18−21
Autophagy is an ancient, cannibalistic (literally “self-eating”)
pathway, wherein cellular components including long-lived
proteins, bulk cytoplasmic material, and aged or damaged
organelles are encapsulated by a double membrane vesicle,
coined the autophagosome.2 These vesicles then fuse with the
lysosome, which degrades the delivered cargo to recoup
building blocks and adenosine triphosphate (ATP) necessary
for cell survival.3,4
Autophagy has been implicated in the pathology of various
diseases such as neurodegeneration and cancer.5 The observed
role of autophagy in cancer is complex and can be tumor-
suppressive or tumor-promoting depending on context. Recent
studies using genetically engineered mouse models have
implicated autophagy in KRAS- and BRAF- driven cancers by
demonstration of tumor suppression in response to inhibition
of autophagy.6−8 Moreover, autophagy is protective for cancers
experiencing a decrease in nutrient availability or damage
caused by cancer therapeutics.9,10 Accordingly, blocking
autophagy via small molecule inhibitors in autophagy-reliant
cancers could increase the efficacy of current chemotherapeu-
tics and may result in tumor suppression as a standalone
chemotherapy.11,12
Physical HTS campaigns are useful for generating chemical
starting points for drug discovery programs.22 Screening a large
library of characterized ligands against a biochemical target
provides insight into efficacious chemical scaffolds and
structure−activity relationship (SAR) patterns. This approach
has led to the generation of numerous therapeutic candidates
following SAR optimization of screening hits.23 Experimental
screens require expensive resources such as large chemical
libraries, miniaturized assays, automated instruments, costly
reagents, etc. By comparison, an in silico screen has far fewer
requirements. The resources needed to carry out an in silico
screen are minimal, including some that can be sourced freely.
Suitable computational facilities are the largest physical asset
needed. An additional benefit is that an in silico screen can be
performed on any target as long as suitable crystal structures
(or homology model) with three-dimensional coordinates of
the protein target are available.24 We chose to perform an in
silico HTS to identify ULK1 inhibitors due to the simplicity and
cost-effective nature of this approach. There are several
programs capable of executing in silico HTS campaigns available
Unc-51-like kinase 1 (ULK1) is a 112 kDa ubiquitously
expressed protein and is required for efficient stress-induced
autophagy under most conditions.13 ULK1 is negatively
regulated by mTOR under normal nutrient conditions and
activated during periods of amino acid or glucose deprivation
by AMPK through phosphorylation at multiple sites in the
Received: August 19, 2017
Accepted: November 10, 2017
© XXXX American Chemical Society
A
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Figure 1. (a) Workflow for in silico HTS and structure of SR-17398 selected from analysis of the best candidates. (b,c) Docking images of SR-17398
and optimized scaffold 3g in the ULK1 crystal structure.
both free of charge and for purchase.25 Approaches utilizing in
silico HTS campaigns have generated hits for numerous drug
discovery projects.26
incorporating the following parameters: OPLS2005 force field,
pH 7, and generation of tautomers. This process produced a
digital file containing ∼1.2 million tautomers of the original
structures. The prepared compounds were subsequently
docked in a standard precision (SP) protocol, as this method
has been observed to proceed with a balance of speed and
accuracy.28 The output of this docking campaign was analyzed
by inspection of the top 500 hits selected according to the
Glide docking score.
Screening hits were initially prioritized by their H-bonding
interactions with the hinge-binding region of the ULK1 ATP
pocket. Next, the top hits were grouped into common cores
based on repeat scaffolds observed in the top tier.
While performing this evaluation, hits containing promiscu-
ous binding groups or PAINS were eliminated.29 In this way,
we identified a variety of cores and purchased a small set of
representative compounds from ChemNavigator. The pur-
chased compounds were selected by using substitution patterns
and functional groups observed in screening hits with the
highest Glide scores. The molecules purchased were >90%
similar, if not identical, to the molecules identified in the
docking studies. Structures of a subset of the hits obtained from
this screen are provided in Figure S1.
We employed Schrodinger’s Maestro software in our
̈
studies.27 Our protein target was a publicly available crystal
structure of ULK1 with a bound ATP competitive inhibitor
published by the Shokat group (PDB ID: 4WNP).19 The
enzyme coordinates were obtained from the protein structure
database (http://www.rcsb.org/pdb/). The protein was first
prepared for docking studies via the Protein Prep application.
Then, using the cocrystallized inhibitor as the center
coordinate, we generated a grid with the Schrodinger Glide,
̈
Receptor Grid Generation task.27 The grid dimensions were 25
× 25 × 25 Å, encompassing the critical hinge-binding region
residues Cys95, Tyr94, Glu93, and Met92. The grid also
encompassed proximal solvent exposed and binding pocket
areas, which could provide interactions with amino acids that
are specific to ULK1.
Using this structure, we performed an in silico HTS campaign
employing the molecular structures contained in the ∼650,000
Scripps HTS library (Figure 1a). The Scripps Molecular Library
Screening Center hosts this library and is maintained by the
Lead Identification Department at Scripps Florida. This library
comprises primarily commercially available compounds but also
includes small molecules developed in-house. We prepared the
digital screening ligand library using the LigPrep workflow
The purchased hits were evaluated in a biochemical assay for
ULK1.30 This assay used full-length ULK1 and full-length
human Atg13 tagged with Flag. DMSO solutions (10 mM) of
B
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a
Scheme 1. Synthesis of Representative ULK1 Inhibitors
a
Reagents and conditions: (a) Boc2O, Et3N, DMAP, THF, 0 °C; (b) Pd/C, H2, MeOH, 22 °C; (c) 3-(Boc-amino)cyclohexanecarboxylic acid,
HATU, Et3N, CH2Cl2, 22 °C; (d) 10% TFA in CH2Cl2, 22 °C; (e) 1-naphthylamine, DIPEA, CH2Cl2, 0 to 22 °C; (f) Lawesson’s reagent, toluene,
110 °C; (g) hydrazine, EtOH, 78 °C; (h) Br2, AcOH, 80 °C; (i) 3,4-dihydro-2H-pyran, p-TsOH, EtOAc, 77 °C; (j) 5-aminoisoquinoline, Pd2(dba)3,
[(t-Bu)3PH]BF4, NaOtBu, 1,4-dioxane; (k) 4 M HCl in dioxane, 22 °C.
assayed compounds were prepared for IC50 determination. Of
these compounds, only one (SR-17398) displayed ULK1
inhibitory activity <30 μM (the IC50 for SR-17398 was 22.4
μM). IC50 values are reported with a standard deviation < 5
nM based on repeat experiments carried out with control
compounds. We used this compound as a starting point for
subsequent structure based design and SAR studies.
structures capable of maintaining two H-bonding interactions,
one with Lys46 and the second with Asn134 or a proximal
residue. The best candidates based in these criteria were
selected for synthesis. Lastly, we designed compounds
predicted to interact with Tyr94. The aryl ring system of this
residue overhangs the active site. We envisioned projecting an
aryl unit from the 3-amino position of the indazole that could
engage in a π-stacking or edge-to-face π-interaction with Tyr
94. Aryl substituents capable of H-bonding interaction with
Tyr94 were also considered. We used a combination of
biochemical data along with docking poses to assist in
validating hypotheses about structural changes capable of
improving the ULK1 inhibitory activity in this compound
series.
The screening hit SR-17398 was synthesized via a coupling
reaction of 5-aminoindazole and 1-N-Boc-3-carboxyl-cyclo-
hexane followed by deprotection of the Boc group (Scheme
1a). This allowed us to verify its structure and activity. Multiple
unsubstituted 3-aminoindazole derivatives (Table 1) were
synthesized following the sequence in Scheme 1a, featuring
1a as a representative example. This compound was synthesized
by first Boc protection of 1 followed by reduction of the 5-nitro
substituent to yield 2. The same amide coupling and
deprotection sequence described above was used to complete
the synthesis of 1a from the 5-aminoindazole intermediate (2),
as well as for all other compounds in Table 1.
We critically evaluated the docking pose of SR-17398 in the
ULK1 crystal structure. The two nitrogen atoms contained in
the indazole ring system make two H-bonding interactions with
the amide backbone of the hinge region, specifically with Glu93
and Cys95 (Figure 1b). The amide carbonyl at the 5-position of
the indazole participates in a H-bonding interaction with Lys46
mediated by a water molecule in the active site. Lastly, the
primary amine attached to the cyclohexyl ring makes a H-
bonding interaction with Asn143. This residue is located in an
oxygen-rich portion of the active site made up of an amino acid
backbone orienting amide carbonyls toward the binding pocket.
The first objective was to increase the activity of SR-17398
by adding functional groups capable of engaging in additional
binding interactions with ULK1. The first modification was the
addition of an amino group at the 3-position of the indazole
core, as in structure 1a. Our docking models predicted this
amine would make a new H-bonding interaction with the
amide carbonyl of Cys95 in the ATP binding domain. A second
aim was to modify the 3-aminocyclohexane unit by eliminating
stereocenters. We also considered using other amines capable
of maintaining the interactions that we identified as important
for binding in the oxygen-rich portion of the active site. To
achieve this goal, we examined the docking pose of various
The analogs presented in Tables 2 and 3 were synthesized
using one of two processes. First, compounds 2c−h and all the
compounds in Table 3 were synthesized by building an
indazole core from a substituted benzene derivative. This
C
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process is illustrated in Scheme 1b using the intermediates for
the synthesis of 3a as an example. The first reaction involves
amide formation between 1-naphthylamine and 5-fluoro-3-
nitrobenzoyl chloride (5). The amide was converted into
thioamide 6 using Lawesson’s reagent. Subsequent treatment of
6 with hydrazine forms the 5-nitroindazole ring system 7.
Compounds 2j and 2k were synthesized from 7 by using a
Buchwald−Hartwig coupling reaction.31 The synthesis of 2k is
found in Scheme 1 for the cis representative. The sequence
begins with bromination of 5-nitroindazole (6) followed by
THP protection to generate 3-bromoindazole 7. This
intermediate was then coupled with 5-aminoisoquinoline
using Buchwald−Hartwig coupling conditions and subse-
quently THP deprotected to yield indazole derivative 8. The
5-nitroindazole intermediates of type 5 and 8 were then
subjected to acylation reactions as described in Scheme 1. The
synthesis of 2a, 2b, and 2i utilized modified procedures that are
described in the Supporting Information.
Table 1. Structure−Activity Relationship for ULK1
Inhibitors with Various Amide Units
compd
SR #
R1
IC50 ULK1 (μM)
a
la
SR-18938
SR-18939
SR-19557
SR-19558
SR-19559
SR-19778
SR-19779
SR-19780
SR-19874
3-aminocyclohexane
3-aminophenyl
0.368
18.10
13.96
1.49
1b
1c
1d
1e
1f
trans-4-aminocyclohexane
cis-4-aminocyclohexane
propylamine
>33
4-piperidine
0.560
0.670
0.840
0.330
a
1g
1h
1i
3-piperidine
a
2-piperidine
(1S,3R)-3-aminocyclohexane
a
Compounds tested as a mixture of stereoisomers.
Analogs of screening hit SR-17398 were evaluated for ULK1
inhibitory activity using the previously described biochemical
assay.30 As predicted based on the modeling efforts, addition of
an amino group at the 3-position of the indazole ring system
led to a significant increase in ULK1 inhibition potency (368
nM, 1a, Table 1). Next, we attempted to alter the cyclohexane
ring of 1a. Our goal was to eliminate the stereocenters while
also retaining the H-bonding interactions believed to be
important for activity. Analog 1b with a 3-aminophenyl group
in place of the 3-aminocyclohexane had markedly reduced
inhibitory activity (18.1 μM). 3-Aminopropyl functionalization
also resulted in a loss of activity (1e, >33 μM). 4-
Aminocyclohexane derivatives (cis and trans, 1c and 1d)
proved to be much less active than the 3-amino variant.
However, 2-, 3-, and 4-piperidine derivatives retained
moderate activity against ULK1 (1f, 1g, 1h). The 4-pipderidine
analog 1f was the most potent in this series (560 nM) apart
from 1a. As noted in Table 1, many of these derivatives were
synthesized as a mixture of stereoisomers, but in the case of 1a,
we purposefully examined the effect of stereochemistry on
ULK1 activity. Inhibitor 1i has (1R,3S) configuration in the
cyclohexane ring and proved to have about equivalent potency
when compared to the mixture of isomers 1a (330 nM).
Derivatives functionalized with various aryl units at the 3-
amino position of the indazole ring were examined next (Table
2). Appending a benzamide to this position led to retention of
activity (2a, 242 nM), while using reductive amination to
incorporate a 4-methoxybenzyl unit retained comparable
activity to 1a (2b, 477 nM). Appending an aromatic six-
membered ring directly to this position with varying methoxy
or trifluoromethyl substituent groups resulted in a retention of
submicromolar inhibition (2e, 2f, 2g); the ortho-methoxy
derivative displayed the most potent activity (2f, 110 nM). 4-
Bromo and 3-methoxycarbonyl substitution did not provide
additional activity (2d, 2h). Introduction of 1-naphthyl unit as
in 3a (Table 3) resulted in a significant increase in activity,
generating an inhibitor with 11 nM ULK1 inhibition potency.
When using a quinoline derivative with the nitrogen at the 4 or
8 position (analogs 2i, 2j), a reduction in activity occurred
compared to 3a. However, a 5-isoquinoline derivative yielded
comparable activity to the 1-naphthyl functionalized system
(2k, 40 nM). We hypothesize that potent activity is retained in
2k (compared to that in 2i, 2j) because the quinolone nitrogen
of 2k is not conjugated to the 3-amino group in the indazole
core.
Table 2. Structure−Activity Relationship for ULK1
Inhibitors with Various Aryl Groups at the 3-Amino Position
compd
SR #
R1
benzamide
IC50 ULK1 (μM)
2a
2b
2c
2d
2e
2f
SR-18937
SR-19398
SR-19399
SR-19401
SR-19403
SR-19873
SR-20077
SR-20078
SR-20080
SR-20291
SR-20296
0.242
0.477
2.73
4-methoxybenzyl
phenyl
4-bromophenyl
2-methoxyphenyl
4-trifluoromethylphenyl
3-trifluoromethylphenyl
methyl-3-benzoate
4-quinoline
0.416
0.110
0.840
0.288
0.183
0.239
0.589
0.040
2g
2h
2i
2j
8-quinoline
2k
5-isoquinoline
Table 3. Structure−Activity Relationship for ULK1
Inhibitors with a 3-Aminonaphthyl Group and Various
Amides
compd
SR #
R1
IC50 ULK1 (μM)
a
3a
3b
3c
3d
3e
3f
SR-19871
SR-20079
SR-20290
SR-20292
SR-20293
SR-20294
SR-20295
SR-20297
SR-20298
3-aminocyclohexane
0.011
4.23
3-dimethylaminocyclohexane
4-piperidine
0.315
3.05
(1S,3R)-3-aminocyclohexane
3-hydroxycyclohexane
4-tetrahydropyran
2.22
1.83
3g
3h
3i
(1R,3S)-3-aminocyclohexane
0.045
0.024
4.94
a
trans-3-aminocyclohexane
2-azetidine
a
Compounds tested as a mixture of stereoisomers.
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Author Contributions
SAR data for analogs deriving from potent inhibitor 3a are
presented in Table 3. The cis-(1S,3R) configuration of the
cyclohexyl amine led to a significant reduction in activity (3d, 3
μM), while the cis-(1R,3S) enantiomer (3g, 45 nM) was about
four-fold less active compared to the mixture of stereoisomers
(3a). We evaluated a racemic mixture of the two trans isomers
(3h; 24 nM), which proved to be only two-fold less active than
the stereoisomeric mixture 3a. These data suggest that the most
potent isomer is in the trans configuration. Replacement of the
primary amine with a hydroxyl group or a dimethylamine
resulted in a significant reduction in activity (3b, 3d).
Tetrahydropyran or azetidine substituents led to a significant
loss of activity (3f, 3i). Lastly, use of a 4-piperdine in place of
the cyclohexane unit (3c) resulted in a modest drop in activity
to 315 nM.
Assessment of in vitro drug metabolism was obtained on
some of our most potent analogs (2e, 3a, 3c, and 3g). These
compounds proved to have excellent stability in human, rat, and
mouse microsomes, and they also exhibited negligible CYP
inhibition (Supplementary Figure 2). These results are
encouraging for our goal of using these (or further optimized)
compounds in animal models of cancer.
In summary, using an in silico HTS campaign utilizing a
published X-ray structure of ULK1 and the electronic
coordinates of an in-house chemical library, we identified SR-
17398 as a moderately active ULK1 inhibitor. Further
optimization of SR-17398 using structure-guided rational
drug design then led to the generation of significantly more
potent ULK1 inhibitors. Utilizing two specific modifications:
(1) addition of an amino group at the 3-position of the indazole
and (2) substitution of the 3-amino unit with an aromatic 10-
membered ring system [either naphthyl (3a) or 5-isoquinolyl
(2k)]. Docking models suggest how these substituents
potentially interact with active site residues (Figure 1c). The
3-amino group provides a third H-bonding interaction with the
hinge region, while the 10-membered aromatic system can
engage in π-interactions with Tyr94. SAR efforts for the 3-
aminocyclohexane substituent have confirmed that it is essential
for ULK1 inhibition.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This research was supported by NIH grant R01 GM113972
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by grants from the National
Institutes of Health.
ABBREVIATIONS
■
HTS, high throughput screening; ULK1, Unc-51-Like Kinase 1;
ATP, adenosine triphosphate; SAR, structure−activity relation-
ship
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* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
* (W.R.R.) E-mail: roush@scripps.ede. Phone: 1-561-228-
2540.
ORCID
William R. Roush: 0000-0001-9785-5897
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DOI: 10.1021/acsmedchemlett.7b00344
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX