Inhibitors of heat shock protein 70 (Hsp70) with enhanced metabolic stability reduce tau levels

Article abstract of DOI:10.1016/j.bmcl.2021.128025

The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau). Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduces tau levels. However, the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to be useful tools for studying tau homeostasis.

Full text of DOI:10.1016/j.bmcl.2021.128025

Bioorg. Med. Chem. Lett. 41 (2021) 128025
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of heat shock protein 70 (Hsp70) with enhanced metabolic
stability reduce tau levels
a
,
*
b
,
c
c
b,
c
b,c
Hao Shao , Xiaokai Li , Shigenari Hayashi , Jeanette L. Bertron , Daniel M.C. Schwarz
,
c
b,c,*
Benjamin C. Tang , Jason E. Gestwicki
a
Hunan Key Laboratory of Molecular Precision Medicine, Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, United States
Institute for Neurodegenerative Disease, University of California San Francisco, San Francisco, CA 94158, United States
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative
diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau).
Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduces tau levels. However,
the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a
chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically
introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal
pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it
retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to
be useful tools for studying tau homeostasis.
Molecular chaperone
Tauopathy
Neurodegeneration
Protein folding
Metabolism
Hsc70
Hsp72
Introduction
microtubules or promote its degradation.18 Despite the similarities be-
1
9
tween the orthologs, Hsp72 is more tightly linked to tau degradation.
Tau is an intrinsically disordered, microtubule-associated protein
Pharmacological studies have also supported this idea. Specifically, a
brain penetrant, irreversible Hsp72 inhibitor, methylene blue (MB),
reduces levels of insoluble tau and improves learning and memory in
1
,2
that is abundantly expressed in neurons.
When tau’s microtubule
3
,4
binding is disrupted, by either mutation or post-translational modi-
fications, such as phosphorylation5 and acetylation7,8, it is aberrantly
localized to the cytosol. This dissociated tau is then prone to aggregate,
where it can form neurofibrillary tangles (NFTs) that are associated with
neuronal cell death9 and neurodegenerative disorders, including Alz-
heimer’s disease (AD) and frontotemporal dementia (FTD). These ob-
servations have driven interest in therapeutic approaches that modulate
,6
20
tauopathy mouse models. Likewise, other classes of Hsp70 inhibitors,
including dihydropyridines and rhodacyanines, which likely bind to
2
1
both Hsp72 and Hsc70, reduce tau levels in cellular and tissue mod-
22–24
els.
These molecules appear to work by stabilizing a conformation
of Hsp70s that promotes tau degradation.25 Unlike MB, which has pro-
miscuous activity, the rhodacyanines are relatively selective for Hsp70
tau homeostasis,1
0,11
including stabilizing microtubule , reducing tau
12
family members in cells, so there is interest in further developing them
26
8
,13
post-transcriptional modifications , and enhancing tau degradation
as chemical probes.
through inhibition of the molecular chaperones Hsp90 and Hsp701
Scheme 1.
4–16
.
One rhodacyanine, MKT-077, was first identified as an inhibitor of
Hsp70s in cancer models, but it was later shown to decrease both
phosphorylated and total tau levels in HeLaC3 cells.23 We found that
MKT-077 binds to an allosteric site that is conserved among Hsp70
isoforms21 and it is considered to be a pan-Hsp70 inhibitor. Here, the
term “inhibitor” is somewhat incomplete, because, while binding to this
allosteric site inhibits ATP turnover, it also stabilizes the conformation
Heat Shock Protein 70 (Hsp70) is a molecular chaperone that binds
to tau soon after its release from microtubules.17 There are two major,
cytoplasmic orthologs of Hsp70 that are thought to perform this func-
tion: Hsc70 (HSPA8) and Hsp72 (HSPA1A). These isoforms are highly
conserved and, after they bind tau, they either stabilize its re-binding to
*
Corresponding authors at: No 110, Xiangya Road, Kaifu District, Changsha, Hunan 410008, China, (H. Shao). 675 Nelson Rising Lane, San Francisco, CA 94158,
United States (J.E. Gestwicki).
E-mail addresses: haoshaodream@hotmail.com, hao.shao@ucsf.edu (H. Shao), Jason.gestwicki@ucsf.edu (J.E. Gestwicki).
https://doi.org/10.1016/j.bmcl.2021.128025
Received 22 November 2020; Received in revised form 30 March 2021; Accepted 4 April 2021
Available online 9 April 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Shao et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128025
NH2
X
OTs
S
N
S
S
(a)
(b)
N
S
(c)
N
S
S
R
R
S
R
R
N
X= F, Cl
O
O
O
X2
1. X = N, X = CH
1 2
N
O Br N
N
2
. X = CH,X = N
1
2
X1
N
Br
O
(e)
S
N
S
(d)
N
S
R
or
N
R
S
N
S
OTs
O
O
◦
◦
◦
Scheme 1. Reaction conditions:(a) 1) potassium ethylxanthate, DMF, 140 C, h; 2) Mel, Net3, EtOH, 80 C, 1 h; (b) p-TsOMe, anisole, 125 C, 4 h; (c) 3-ethyl
◦
◦
, MeCN, 70 ◦C, 3 h; 2) DCM/MeOH, aq⋅NH3, r.t., 1 h.
rhodanine, NEt3, MeCN, 25 C, h; (d) p-TsOMe, DMF, 135 C, 3h; (e) 1) Net
3
Fig. 1. Previously reported allosteric Hsp70 inhibitors and their metabolism. (A) Chemical structures of MKT-077, YM-01 and YM-08. (B). The benzothiazole is
known to be the metabolically liable site.
of Hsc70 and Hsp72 that promotes degradation of bound proteins, such
as tau. Thus, molecules in the chemical series could be useful probes in
neurodegenerative disease models because they accelerate tau degra-
dation. Towards that goal, recent medicinal chemistry efforts have
started to improve the potency of MKT-077. For example, a close MKT-
suggested that the benzothiazole is located in a hydrophobic pocket that
would accept modification by halogens. In this study, we reported the
synthesis of seventeen YM-08 analogs and the selection of JG-23 as a
molecule with improved metabolic stability and tau-reducing activity.
0
77 derivative, YM-01, was found to have improved membrane
Results and discussion
permeability and it promotes tau degradation at concentrations ~1 µM.
Importantly, treatment with YM-01 leads to preferential degradation of
pathogenic tau, while normal, microtubule-associated tau is resistant, in
a mouse brain slice model.24. While these findings are promising, the
quaternary amine in YM-01 leads to its accumulation in mitochondria
and limits its blood–brain barrier (BBB) permeability. To solve this issue,
a neutral analog, YM-08 (Fig. 1), was synthesized by replacing the
pyridinium with a pyridine. YM-08 retained binding to Hsp70s and it
still had the ability to decrease tau levels in HeLaC3 cells, while it also
had improved brain exposure.16 However, YM-08 is rapidly metabolized
and its half-life is less than three minutes in mouse liver microsome
assays, significantly limiting its applications in vivo.
To understand which sites might enhance metabolic stability, we
sought to systematically install halogens at the 3-, 4–5- and 6-ring po-
sitions in the benzothiazole. In addition, we envisioned swapping the
position of the pyridine nitrogen to reveal any distal, electronic effects.
Accordingly, seventeen YM-08 analogs were designed and synthesized
2
627
using a previously reported method as shown in Scheme 1.
Briefly,
the synthesis started from the cyclization of substituted anilines with
potassium ethyl xanthate, followed by methylation with methyl iodide
under mild basic condition. The resulting substituted 2-(methylthio)
benzothiazole was reacted with methyl p-toluenesulfonate to afford its
methylthioiminium salt, which was subsequently condensed with 3-eth-
ylrhodanine. The resulting compounds were activated by methyl p-tol-
uenesulfonate, followed by the condensation of either 1-((1,3-
dioxoisoindolin-2-yl)methyl)-2-methylpyridin-1-ium bromide or 1-
In a metabolite identification study, we had previously identified the
benzothiazole in YM-08 as the major site for Phase I oxidation (Fig. 1).16
However, it was not clear which site was oxidized. ed Here, we explored
whether introducing halogens to specific sites on the benzothiazole ring
may block this oxidation and increase the lifetime of YM-08 analogs.
This approach seemed feasible because docking of YM-08 to Hsc70
(
(1,3-dioxoisoindolin-2-yl)methyl)-4-methylpyridin-1-ium
bromide.
The protecting group on the pyridine nitrogen was then removed in the
presence of catalytic amount of aqueous ammonium hydroxide to yield
2

H. Shao et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128025
Table 1
10–13) improved microsome stability (~13 to 33% remaining). As in
the previous series, replacement of fluorine with chlorine further
improved metabolic stability. Specifically, 52% and 39% of compounds
Chemical structures and liver microsome stabilities of YM-08
1
5 (JG-23) and 16 were remaining, respectively. Compounds 14 and 17
had poor solubility and were thus excluded from further study (n/t;
Table 1). Based on these results, we chose the top 4 compounds and
measured their T1/2 in mouse liver microsome assays (Fig. 2). The
calculated T1/2 values from these experiments correlated with the single
time point measurements: the half-lives of compounds 5, 6 and 16 are
around 20 min, and JG-23 (15) has the longest T1/2 value (36 min).
Finally, we tested the ability of JG-23 to decrease total tau (t-tau)
levels in the HeLaC3 cell model. Cells were treated with JG-23 for 24 h
at the indicated concentrations, and the t-tau levels were analyzed by
Western blot. We found that JG-23 significantly decreased t-tau levels at
concentrations above 10 µM (Fig. 3a). Also, JG-23 did not activate
cellular stress, as determined by the constant levels of Hsp72 and Hsp90
in the treated cells. Next, we asked whether JG-23 might also reduce t-
tau levels in SH-SY5Y cells. This experiment was important because the
HeLaC3 model over-expresses 0N4R tau, while it is expressed at physi-
ological levels in the SH-SY5Y cells. Satisfyingly, we found that JG-23
had similar effects on t-tau, Hsp72 and Hsp90 in this model (Fig. 3b),
reducing t-tau by ~80%.
analogs.
Compound
R
1
R
2
Percentage atT30min
YM-08
H
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
2-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
4-pyridinyl
–
12
19
5
1
2
3
4
5
6
7
8
9
3-F
4-F
5-F
6-F
3-Cl
4-Cl
5-Cl
6-Cl
H
12
38
37
13
17
6
1
1
1
1
1
1
1
1
0
3-F
4-F
5-F
6-F
3-Cl
4-Cl
5-Cl
6-Cl
33
23
13
30
n/t
52
39
n/t
1
2
3
4
5 (JG-23)
6
7
Conclusions
Allosteric inhibitors of Hsp70, which favor a pro-degradation
conformation of the chaperone, are potentially promising chemical
probes for understanding tau homeostasis. However, the poor metabolic
stability of earlier molecules, such as YM-08, had limited such studies.
Here, guided by the knowledge that P450-mediated metabolism occurs in
the benzothiazole ring, we focused on introducing halogens at the 3-, 4-,
the final products 1–17 in overall yields between 20 and 30% and purity
greater than 97%. The compounds were purified by flash chromatog-
1
raphy and characterized by LC-MS and H-NMR.
To probe which benzothiazole position might be most important for
microsome stability, we first measured the amount of unmodified
compound remaining after incubation with mouse liver microsomes for
5
- and 6-positions. Two series of compounds were designed and syn-
◦
28
3
0 min at 37 C, using dextromethorphan as a positive control . First,
thesized, revealing that a 4-chloro modified analog, JG-23, is 12-fold
more stable than YM-08. One of the interesting aspects of these find-
ings was that shifting the heteroatom in the distal pyridine generally
improves stability, despite the fact that metabolism does not occur in
this ring. It seems possible that the pyridine impacts either P450 binding
or oxidation activity, perhaps mediated by electronic effects through the
conjugated pi system. Importantly, these modifications did not seem to
affect target binding, as compound JG-23 retained the ability to pro-
mote t-tau degradation in two cellular models. Future work will focus on
improving potency and brain exposure, with the goal of creating a next-
generation Hsp70 probe for studying tau turnover in the brain.
we confirmed that YM-08 is rapidly metabolized in this experiment,
such that its levels were below the limit of detection at 30 min (Table 1).
Then, we found that introduction of fluorine at any of the 3-, 4-, 5- or 6-
positions on YM-08 (compounds 1–4) modestly increased this stability
(
~5 to 20% remaining after 30 min). The analogs containing chlorine at
the corresponding positions (compounds 5–8) were generally more
stable. For example, the 5 and 6 were approximately 2- to 3-fold more
stable than 1 and 2 (37 to 38%). In addition, across this series, the
compounds with substituents at the 3- and 4-positions were generally
better than those with halogens at the 5- and 6-positions.
Next, we examined the effects of switching the pyridine nitrogen
from the ortho (YM-08) to para position (compound 9–17). In early ex-
periments, we found that compound 9 was modestly more stable than
YM-08 (Table 1), so it seemed worth exploring this series in more detail.
Introduction of a fluorine at any of the four positions (compounds
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
Fig. 2. Microsomal stability of selected compounds. Dextrometorphan is used as a positive control.
3

H. Shao et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128025
Fig. 3. Compound JG-23 led to degradation of t-tau without eliciting a heat shock response in HeLaC3 (a) and SH-S55Y (b) cells. HeLaC3 or SH-SY5Y cells were
treated with JG-23 for 24 h at the indicated concentrations, lysed and western blots performed. Results are representative of at least two independent experiments.
the work reported in this paper.
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Alzheimer’s disease and related tauopathies. Nat Rev Drug Discovery. 2009;8:
7
83–793.
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