Small molecule SUMOylation activators are novel neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2017.12.028

Neuronal loss characterizes many of the most intractable nervous system diseases that deprive our ageing population of their quality of life. Neuroprotective pharmacological modalities are urgently needed to address this burgeoning population. Small ubiqu

Full text of DOI:10.1016/j.bmcl.2017.12.028

Accepted Manuscript
Small molecule SUMOylation activators are novel neuroprotective agents
Katherine Krajnak, Russell Dahl
PII:
S0960-894X(17)31188-5
https://doi.org/10.1016/j.bmcl.2017.12.028
BMCL 25484
DOI:
Reference:
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
17 October 2017
11 December 2017
13 December 2017
Please cite this article as: Krajnak, K., Dahl, R., Small molecule SUMOylation activators are novel neuroprotective
agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.028
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Small molecule SUMOylation activators are novel neuroprotective agents
a
Katherine Krajnak , Russell Dahl *
b,
a
Van Andel Research Institute, Grand Rapids, MI 49503, USA
b
Neurodon LLC, Schererville, IN 46375, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Neuronal loss characterizes many of the most intractable nervous system diseases that deprive
our ageing population of their quality of life. Neuroprotective pharmacological modalities are
urgently needed to address this burgeoning population. Small ubiquitin-like modifier (SUMO)
conjugation has been established as an endogenous neuroprotective response, and we have
discovered several classes of small molecules that enhance SUMO conjugation. Herein we
describe the hit to lead campaign that enabled the discovery of 3 diverse classes of drug-like
SUMOylation activators. Optimized compounds were ultimately validated in cell-based models
of neuronal loss and provide a foundation for establishing systemically active SUMO activators
to treat degenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and stroke.
Received in revised form
Accepted
Available online
Keywords:
SUMO
Small Ubiquitin-like Modifier
Drug discovery
2
015 Elsevier Ltd. All rights reserved.
Small molecules
Medicinal chemistry
Neurodegeneration
Neuron death plays a central role in the pathogenesis of
multiple diseases that currently have no available therapeutics.
1
Consequently, neuroprotection is a major goal of neuroscience
drug discovery research, specifically relevant to diseases
involving neuronal insult and loss. Finding small molecules that
can prevent or alleviate neuron loss is critical to addressing these
significant neuropathies. While neuroprotection has the potential
to be an effective treatment for stroke, CNS trauma, Alzheimer’s
disease (AD), and Parkinson’s disease (PD), there are no
approved therapies that slow or halt neuron loss. The collective
economic and social burden of these diseases is enormous.
Currently, CNS trauma and related maladies including stroke
Figure 1. SUMOylation is an enzymatic process consisting of E1 (SAE), E2
(Ubc9), and, sometimes, an E3 ligase. Our activators increase the amount of
SUMO conjugation, which has been shown to be endogenously protective.
5
sometimes, various E3 ligases (many). A major function of this
conjugation is to modulate protein-protein interactions. A global
increase in SUMOylation levels mark the response to neuronal
insult caused by CNS trauma and neurodegenerative
6
pathogenesis. SUMO conjugation has been established as an
2
have estimated costs of $106 billion annually in the US. AD is
endogenous neuroprotective strategy in multiple models of
cellular stress that mimic a neurodegenerative environment. Cells
exposed to stress conditions such as hypothermia and hypoxia
the most frequent form of dementia in ageing affecting over 5
million Americans with annual economic burdens in the US
exceeding $180 billion annually. PD affects about 1% of the
7
show increased SUMO conjugation. Also, SUMOylation is
activated in hibernation as a neuroprotective response to
3
population aged 60 and over, and costs over $6 billion a year.
8
decreased bloodflow and nutrient availability. Massive increases
All of these disorders are marked by neuronal insult and
subsequent death. Thus, there is an urgent need for therapies with
neuroprotective effects. Protein conjugation with small ubiquitin-
like modifier (SUMO) proteins has been established by numerous
studies 4as an endogenous neuroprotective mechanism in
neurons, and the small molecule SUMOylation enhancers that
are presented herein serve to validate a novel strategy to treat
these important disorders and deliver valuable candidate
molecules for further drug development.
in SUMO and SUMOylation machinery are seen in animal
models of ischemia. Remarkably, overexpression of SUMO
9
components leads to a reduction in cell loss from stressors such
as oxygen and glucose deprivation (OGD) and ischemic
conditions, while knock down of SUMO in a cellular model
7
made cells more susceptible to OGD-induced cell death.
Increased SUMO conjugation has also been seen concurrently
with increased ubiquitination in neuronal injury as focal cerebral
ischemia increased both protein ubiquitination and SUMOylation
SUMO is a small protein that is covalently attached to target
proteins via an enzymatic cascade that is analogous to the
ubiquitin pathway (Figure 1). This includes an E1 enzyme
1
0
in various protein aggregates in the CNS. Finally, the
combination of ubiquitination and SUMOylation has been shown
to play a protective effect in the brains of transgenic mice
(
SUMO activating enzyme, SAE), an E2 enzyme (Ubc9), and,
11
following ischemic damage.

While inhibition of the various SUMO pathway components
to reduce protein SUMOylation has been targeted as a strategy
for various diseases including cancer, viral infection, and cystic
Due to our requirement for nascent SAR present in the
screening hits, we were able to implement a parallel synthesis
strategy of enriched, focused libraries to rapidly optimize the
SUMOylation activity of the 3 hit classes. Using the bioactivity
information present from the primary screen, we envisioned
synthesizing 50 to 100 analogues of each scaffold to answer
important SAR questions and generate lead compounds to
progress to cell-based neuroprotection studies. Chemistries were
chosen to minimize optimization time with final products rapidly
isolated using preparative HPLC-MS.
1
2,13
fibrosis,
relatively few reports of pharmacological activation
of SUMO conjugation are currently available. One such report
describes development of a high-throughput assay to find
activators of SUMO conjugation via the inhibition of microRNAs
1
4
182 and 183.
Reported activators were shown to be
neuroprotective in an in vitro model of ischemia. Another recent
report describes a SUMOylation activator for the substrate
15
2
+
protein Sarco/Endoplasmic Reticulum Ca -ATPase (SERCA).
This increase in SUMO conjugation leads to the subsequent
activation of SERCA and improved muscle contraction for
applications in heart failure.
Chemical Series 1: Quinolines. The optimization plan,
synthesis, and resultant SAR for our quinoline series of SUMO
activators are depicted in Scheme 1. We synthesized 3 sub-series
in this class, including sulfonamides, reverse-sulfonamides, and
amides using commercially available building blocks and
standard reaction conditions. Briefly, acid chlorides or sulfonyl
chlorides were reacted with amines, 1:1, in N,N-
dimethylformamide (DMF) with 1.5 equivalents of triethylamine
(
TEA) at room temperature for 2-4 hours. As can be seen in the
table, while a variety of functionality is tolerated at the para
position of the sulfonyl aromatic portion, it should be noted that
substitution at the ortho and meta positions resulted in a
significant loss of activity. The bis-sulfonamide 1 gave the best
activation and was progressed to cellular neuroprotection studies.
Effects of substitution on the quinoline moiety were detrimental,
and all actives were devoid of substitution at this moiety. In
addition, replacing the existing sulfonamide with a reverse-
sulfonamide or an amide, which has a subtle difference in
geometry with the planar amide as opposed to the tetrahedral
Figure 2. Components of the high-throughput HTRF assay to discover
activators of SUMOylation. Increase in the FRET signal over baseline
indicated potential activators which were validated in orthogonal assays that
directly measured protein sumoylation.
We discovered several classes of SUMO activators via a
high-throughput homogeneous time-resolved fluorescence
(
HTRF) assay. SUMO is covalently attached to target proteins
through a concerted process that is catalyzed by three enzymes,
known generally as the activation enzyme (E1), conjugation
1
6-21
enzyme (E2), and ligase (E3).
Our HTRF assay contained
Optimization Plan:
R2
SUMO-E1 (SAE), SUMO-E2 (UBC9), GST-SUMO and His-
RanGap, a known SUMO substrate, as well as the respective
R1
O
NH
R1
DMF/TEA
S
2
2
fluorescent antibodies (Figure 2). The assay was run in 1536-
well plate format and the selected activators were chosen based
on their ability to increase the FRET signal over a baseline level.
All active series were subsequently validated in 2 orthogonal
assays to confirm their ability to increase SUMOylation in HEK-
O
N
N
O
S
NH2
O
Cl
R2
5
quinoline
amines
10 sulfonyl
chlorides
50 sulfonamides
R1
2
93 cells. The assays included YFP-SUMO1 accumulation and
DMF/TEA
O
S
R1
HN
O
SUMOylation of the known SUMO substrate Sp1 above
endogenous levels. Following hit confirmation and validation, we
selected 3 hit series based on several criteria. First, we wanted
classes that showed nascent SAR in the primary screen rather
than single hit classes (i.e. “one-hit wonders”). As can be seen in
Figure 3, the classes chosen for follow up all had multiple
analogues showing activity. Second, we used drug-like
physicochemical filters as a means to rank order our compound
classes and select leads showing acceptable physical properties in
regards to lipophilicity (LogP < 5), polar surface area (PSA <
N
N
O
NH2
analines
S
O
Cl
5 reverse-
sulfonamides
5
R1
DMF/TEA
R1
N
O
NH
NH2
N
O
Cl
5 benzoyl
chlorides
5 amides
100), and size, as assessed by molecular weight (MW < 500).
Results:
%
SUMO
Inc.
R1
R1
Cmpd
1
logP
O
O
N
S
2.1
43 ±4.6
O
S
O
NH
2
3
4
CH3
3.0
1.6
2.5
29 ±3.6
28 ±3.0
25 ±2.6
N
HN
O
H
Scheme 1. The sulfonamide hit class was followed up via synthesis of 60
derivatives from 3 subclasses. The results of this SAR leaded the lead
compound 1 which increases SUMOylation by 43%. Data are represented
as mean ± SD.
Figure 3. The 3 classes of SUMOylation activators chosen for follow-up
from HTS. Compounds were chosen based on nascent SAR and drug-
likeness. Shown are the average percentages of increase in the SUMOylation
signal over baseline across all representatives of each scaffold.

sulfonamide, resulted in a loss of activity.
Amongst the benzoyl substituents, hydrophobic and
heteroaromatic groups were tolerated. The preferred compound 8
Chemical Series 2: Benzothiazoles. The optimization plan,
synthesis, and resultant SAR for our benzothiazole series of
SUMO activators are depicted in Scheme 2. There were 2 sub-
series synthesized, the hydrazides and amides, using
commercially available building blocks and standard reaction
conditions including reaction of a benzoyl chloride and either
hydrazine or amine, 1:1, in DMF with 1.2 equivalents of TEA at
room temperature for 4 hours. This afforded 50 hydrazides and
Optimization Plan:
R1
N
R2 DMF/TEA
N
R1
N
S
NH
N
NH2
O
Cl
S
O
5 amino
thiazoles
5 benzoyl
chlorides
2
25 amides R
R1
N
2
5 amides, with the top activators shown in the table. As can be
R2
DMF/TEA
N
O
R1
seen in the table, a limited number of substitutions were tolerated
on the benzothiazole portion, mainly methyl groups or methoxy,
with halogens or electronic withdrawing groups being inactive.
The most active derivatives of this class. Compound 5 was
progressed to neuroprotection in cell-based assays. It is notable
that all of the compounds of the amide sub-series were inactive,
thus there is a requirement for the hydrazide moiety.
N
N
O
S
Cl
S
NH
S
NH2
O
S
O
R2
5 amino
thiazoles
5 sulfonyl
chlorides
25 sulfonamides
Results:
%
logP SUMO
Inc.
Cmpd
R1
O
O
8
9
2.6
3.4
3.2
2.6
53 ±3.6
37 ±2.6
25 ±3.0
20 ±2.7
N
S
S
Br
Cl
N
S
NH
10
O
R1
O
11
O
Scheme 3. The aminothiazole hit class was followed up via synthesis of 50
amides and sulfonamides. All sulfonamides were inactive. The results of this
SAR leaded the lead compound 8 which increases SUMOylation by 53%.
Data are represented as mean ± SD.
was progressed to cell-based neuroprotection assays.
Representative compounds 1, 5, and 8 were tested in dose
response assays in a neuronal cell line using CSM14.1 cells.
Additionally, all reported compounds 1-11 were tested in Buffalo
green monkey kidney (BGMK) cells. In both assays, the
compounds were tested for their ability to rescue cells from
conditions of stress that are associated with stroke and
neurodegenerative pathophysiology. This served as a validated in
vitro assessment of these compounds to serve as neuroprotective
drug candidates for further development. Endoplasmic reticulum
Scheme 2. The benzothiazole hit class was followed up via synthesis of 75
derivatives in 2 subclasses. The results of this SAR leaded the lead
compound 5 which increases SUMOylation by 38%. Data are represented as
mean ± SD.
(
in stroke, ischemia, and neurodegeneration.
ER) stress has been established as a major cause of neuronal loss
Thus, we used a
23-25
known ER stress-causing agent thapsigargin (TG) to model the
compromised state in vitro. In the absence of our SUMOylation
activators, TG caused the complete death of both cell lines.
Chemical Series 3: Aminothiazoles. The optimization plan,
synthesis, and results for the aminothiazole series of SUMO
activators is depicted in Scheme 3. There were 2 sub-series
synthesized that consisted of amides and sulfonamides. Again,
we used commercially available building blocks and standard
reaction conditions including reaction of a benzoyl chloride or
sulfonyl chloride and thiazole amine, 1:1, in DMF with 1.2
equivalents of TEA at room temperature for 15 hours. We
generated 50 diverse aminothiazole derivatives, with the top
activators shown in the table. All top actives had the
unsubstituted ortho-pyridyl derivative, and no substitution was
tolerated on the ring. Replacement with unsubstituted phenyl
produced a weakly active derivative. Optimization of the other
site showed that the sulfonamide derivatives were all inactive.
To assess the ability of the compounds to rescue neurons
from ER stress-induced cell death, the rat striatal neuroprogenitor
2
6
cell line CSM14.1 was used. Briefly, cells were pre-treated for
2 hours with compound or buffer followed by injury with TG, the
known inducer of ER stress. Measurement of cellular ATP
content was used as a surrogate marker of cell viability. Controls
consisted of TG with no added compound as the negative control
(100% cell death) and DMSO without TG as the positive control
(100% cell viability). The known anti-ER stress compound
Salubrinal was used as a positive control as well. The results of
the cell viability assays with our SUMOylation enhancers are
shown in Figure 4. As can be seen, there is a clear dose-response
rescue with the added compounds, and some compounds rescued

the cells to nearly 100% at higher concentrations. All EC50
values were in the single-digit micromolar range (1.2-9.8µM).
shown to protect against IL-1β-induced cell death via reduction
28
of caspase 3 cleavage. Furthermore, increased sumoylation
decreased cytochrome c release via a reduction in outer
mitochondrial membrane binding and subsequent fission.
To assess an orthogonal cell line and further validate the
ability of SUMOylation activators to rescue varied cell types, the
compounds were also profiled for cytoprotection in BGMK
29
Finally, pharmacologically increasing sumoylation levels in
SHSY5Y cells protected against oxygen/glucose deprivation-
induced cell death. These reports indicate that sumoylation
2
7
cells. Briefly, cultured BGMK cells were pretreated with
SUMOylation enhancers 1-11 for 2 hours. Treated cultures were
then stressed with 150 nM of thapsigargin to mimic the effects of
activating the unfolded protein response and initiate the ER stress
cascade that eventually leads to apoptosis. The cytoprotective
effect of each compound was assessed using CCK-8 colorimetric
readings to measure cell viability. Representative SUMOylation
activator compounds show significant protection against
thapsigargin-induced cell death (Figure 4D). As a positive
control, Salubrinal was tested and rescued the injured cells to
30
levels are intimately linked with apoptosis.
Our novel approach of treating CNS trauma and
via
neurodegeneration
by enhancing
neuroprotection
pharmacological activation of SUMOylation provides a new
avenue of intervention for urgently needed medications. There
have been few reports of drug-like small molecules that increase
SUMOylation. We have validated a novel target for neuronal loss
both biologically in that SUMOylation has been shown to be
neuroprotective, and chemically in that we are able to modulate
this process with small molecules. The ability of these molecules
to rescue degenerative cells provides hope for the development of
therapeutics.
4
7.22% viability. It is also noted that the level of cell rescue does
not directly correlate to the sumoylation activation seen in the
biochemical screening assay. In the cellular assay we are also
measuring the ability of compounds to reach their biological
target in addition to measuring potential for sumoylation
increase. Thus, any discrepancy is most likely due to the physical
properties of the respective compound classes affecting their
ability to dissolve in the aqueous cellular assay buffer and
subsequently cross cellular membranes. In order to accomplish
this, the compounds must have a balanced physicochemical
profile. Analysis of the average cell rescue ability for the
compounds reported herein shows that the quinoline-
sulfonamides and benzothiazoles are relatively comparable at
about 50% of cell viability, whereas the aminothiazole class has
an average cell rescue ability of about 66% viability. Therefore, it
can be argued that the aminothiazoles have a better balance of
physical properties that enable improved solubility and ability to
penetrate cell membranes and can be described as being more
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Figure 4. Neuroprotective and cytoprotective assessment of SUMO
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2. In 1536-well white plates, 2µl of assay buffer was dispensed into
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2
columns 3-48. Using a HighRes biosolutions pintool, 70nl of

2
7
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4
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Katherine Krajnak, Russell Dahl*