Novel highly selective inhibitors of ubiquitin specific protease 30 (USP30) accelerate mitophagy

Article abstract of DOI:10.1016/j.bmcl.2018.05.013

Mitophagy is one of the processes that cells use to maintain overall health. An E3 ligase, parkin, ubiquitinates mitochondrial proteins prior to their degradation by autophagasomes. USP30 is an enzyme that de-ubiquitinates mitochondrial proteins; therefore, inhibiting this enzyme could foster mitophagy. Herein, we disclose the structure–activity relationships (SAR) within a novel series of highly selective USP30 inhibitors. Two structurally similar compounds, MF-094 (a potent and selective USP30 inhibitor) and MF-095 (a significantly less potent USP30 inhibitor), serve as useful controls for biological evaluation. We show that MF-094 increases protein ubiquitination and accelerates mitophagy.

Full text of DOI:10.1016/j.bmcl.2018.05.013

Accepted Manuscript
Novel Highly Selective Inhibitors of Ubiquitin Specific Protease 30 (USP30)
Accelerate Mitophagy
Arthur F. Kluge, Bharat R. Lagu, Pranab Maiti, Mahaboobi Jaleel, Michael
Webb, Jyoti Malhotra, Ashley Mallat, P. Akhila Srinivas, James E. Thompson
PII:
S0960-894X(18)30402-5
https://doi.org/10.1016/j.bmcl.2018.05.013
BMCL 25830
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 March 2018
6 May 2018
7 May 2018
Please cite this article as: Kluge, A.F., Lagu, B.R., Maiti, P., Jaleel, M., Webb, M., Malhotra, J., Mallat, A., Srinivas,
P.A., Thompson, J.E., Novel Highly Selective Inhibitors of Ubiquitin Specific Protease 30 (USP30) Accelerate
Mitophagy, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.013
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Novel Highly Selective Inhibitors of Ubiquitin Specific Protease
30 (USP30) Accelerate Mitophagy
Arthur F. Kluge,a Bharat R. Lagu,*a Pranab Maiti,b Mahaboobi Jaleel,b Michael Webb,a Jyoti
Malhotra,a Ashley, Mallat,a P. Akhila Srinivasb and James E. Thompsona
aMitobridge, Inc. (an Astellas company), 1030 Massachusetts Avenue, Cambridge, Massachusetts 02138,
United States
bAurigene Discovery Technologies, Ltd., Hyderabad Telangana, India 500049.
KEYWORDS: mitophagy, deubiquitinase inhibitor, parkin, USP30
ABSTRACT: Mitophagy is one of the processes that cells use to maintain overall health. An
E3 ligase, parkin, ubiquitinates mitochondrial proteins prior to their degradation by
autophagasomes. USP30 is an enzyme that de-ubiquitinates mitochondrial proteins; therefore,
inhibiting this enzyme could foster mitophagy. Herein, we disclose the structure-activity
relationships (SAR) within a novel series of highly selective USP30 inhibitors. Two structurally
similar compounds, MF-094 (a potent and selective USP30 inhibitor) and MF-095 (a
significantly less potent USP30 inhibitor), serve as useful controls for biological evaluation. We
show that MF-094 increases protein ubiquitination and accelerates mitophagy.
Mitochondria have a complex life cycle that includes many processes, including: biogenesis,
fusion, fission, interaction with endoplasmic reticulum and organelle specific degradation by
mitophagy.1 Loss of the mitochondrial membrane electrochemical potential initiates the
mitophagy process. The depolarization is followed by stabilization of protein kinase PINK1 on
damaged mitochondria2; this is followed by recruitment of the E3 ubiquitin ligase parkin to
phosphorylated ubiquitin3, which results in increased ubiquitination of multiple mitochondrial
proteins and engulfment by the autophagosome.4 Parkin-mediated mitophagy is an important
process in mitigating cellular damage in many tissues, for example: 1) the liver induced toxicity
caused by alcohol and acetaminophen5 and 2) the kidney damage caused by cisplatin6 use.
Additionally, parkin loss of function is a causal event in autosomal recessive juvenile onset
parkinsonism7.
The ubiquitination of cellular proteins through the action of E3 ubiquitin ligases is opposed by
a family of de-ubiquitinating enzymes (DUBs).8 The DUB USP30, a cysteine protease,9 is
localized to mitochondria and has been shown to oppose the action of parkin-mediated
ubiquitination in clearing damaged mitochondria.10-11 USP30 is specific for isopeptide bonds
between the lysine-6 ε-amino group of ubiquitin and the c-terminus of the next member of the
polyubiquitin chain.12-13 In HeLa cells, USP30 has been shown to target the lysine-6 linked
ubiquitin chains on specific outer membrane proteins while leaving others intact.12
Previously reported inhibitors of USP30 include dodecapeptides and aptamers14-15 and the
triterpene 15-oxaspiramilactone (Figure 1).9 Recently some N-cyano pyrrolidines (Figure 1) and
other heterocycles as USP30 inhibitors have been reported in the patent literature.16 Some of the
N-cyano pyrrolidines in the earlier patent applications were reported to be dual inhibitors of
USP30 and UCLH1 (ubiquitin C-terminal hydrolases 1) but selectivity data on the other
compounds have not been reported. These structures bear some resemblance to the known

Cathepsin C inhibitors and are likely covalent inhibitors with cysteine forming an adduct with
the N-cyano group.17
High-throughput screen led to the identification of racemic phenylalanine derivative 1 as a
USP30 inhibitor. Compound 1 has an IC50 <1 µM and does not inhibit USP’s 1, 8 and 9 at 10 M
concentration (Figure 1).18-20 We report herein the results of a structure-activity relationship
(SAR) study of the family of analogs derived from 1 leading to identification of several potent
and highly selective inhibitors of USP30. These inhibitors are expected to be non-covalent
inhibitors of USP30.
Figure 1. Inhibitors of USP30
A rapid SAR exploration of 1 was enabled by the modular synthetic approach shown in Figure
2 and Tables 1 - 4. Analogs of 1 were prepared using a general synthetic route (or with minor
modifications for compounds 2 - 4) shown in Scheme 1. The compounds were tested for USP30
inhibition using a biochemical assay that measures the effect of an inhibitor on USP30 cleavage
of the substrate ubiquitin-rhodamine-110-glycine to give a fluorescent product: rhodamine-110.21
Compounds with IC50< 1 µM were screened for inhibition of USP’s 1, 8 and 9 at 10 M. The
data on selected compounds is summarized in Table 1.
Scheme 1. General Method for Synthesis of Analogs of 1
A series of positional modifications of 1 established several key structural requirements for
USP30 inhibitory activity. The racemate and the enantiomers of 1 have similar IC50s suggesting
that the chirality of the phenylalanine moiety does not contribute significantly to the binding
affinity to the protein. The three exchangeable hydrogens (two amides and one sulfonamide) are
essential, since replacement of any one by a methyl group (compounds 2 - 4) results in drastic
drop in USP30 inhibition. The SAR in the sulfonamide region seemed to be fairly restrictive in
terms tolerated substitutions. Compound 5 with an additional methylene group between the

sulfonamide and the phenyl ring results in complete loss of USP30 inhibition. The
ethylsulfonamide analog 6 does not inhibit USP30, whereas compounds containing t-
butylsulfonamide (1) and isopropylsulfonamide (7) inhibit USP30 with IC50s around 0.5 M.
When the sulfonamide group is replaced with an amide group, the resulting compound ((S)-8
does not inhibit USP30. The complete loss of inhibition seen with amide (S)-8 indicates a critical
role the SO2 moiety through either direct binding to USP30 or through its tetrahedral geometry at
the binding site. Based on these results, the two primary amides and the t-BuNHSO2 group were
kept constant during exploration of SAR in the other regions.
The structural correlation between potency with the size of the substituent at the -chiral center
is shown in Table 1. Benzyl (1) as well as its 4-hydroxy (9) and 4-methoxy (10) analogs, and
cyclohexylmethyl (17) all show good inhibition; however, the 4-methyl substitution (11) is not
tolerated. Analogs in which the L-phenylalanine moiety is replaced with glycine, alanine, valine
and phenylglycine (R1 = H, Me, i-Pr and Ph respectively) (12 - 15 in Table 1) moieties do not
inhibit USP30 up to 10M concentration. Compound 16, where R1 = phenethyl, weakly inhibits
USP30 (2.2 M). Further SAR was then explored by keeping the (S)-phenylalanine and the
naphthyl- t-butylsulfonamide moieties unchanged.
Table 1. SAR
Cmpd.
R2
X
R6
USP30 IC50
(M)*
0.37
0.27±0.028
0.25
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
SO2
SO2
SO2
SO2
SO2
SO2
CH2SO2
SO2
SO2
CO
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
Et
(R,S)-1
(S)-1
(R)-1
(R,S)-2
(R,S)-3
(R,S)-4
(R,S)-5
(R,S)-6
(R,S)-7
(S)-8
>10
>10
>10
>10
>10
0.51
>10
Bn
Bn
i-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
4-hydroxybenzyl
4-methoxybenzyl
4-methylbenzyl
0.47±0.21
0.09
(S)-9
(S)-10
(S)-11
(S)-12
(S)-13
(S)-14
(S)-15
(S)-16
(S)-17
>10
>10
>10
>10
>10
2.27
0.67
H
Me
iPr
Ph
Phenethyl
cyclohexylmethyl

*The assay was performed in duplicates and the values were typically within 10% of each other. For select
compounds, two or more experiments were performed independently. In those instances, the data is reported as
Mean ± SD
Table 2. SAR for N-acyl substituent
Cmpd.
R1
USP30 IC50
(M)
0.27±0.028
0.83
Cmpd.
R1
USP30
IC50 (M)*
>10
phenyl
benzyl
2-phenylethyl
benzyloxy
cyclopropyl
cyclopentyl
1
26
27
28
29
30
31
32
33
34
4-chlorophenyl
4-fluorophenyl
4-methylphenyl
4-methoxyphenyl
4-cyanophenyl
4-hydroxyphenyl
2-pyridyl
>10
>10
>10
0.37
18
19
20
21
22
23
24
25
0.049
2.5
9.0
0.27
>10
cyclohexyl
4,4-difluorocyclohexyl
cycloheptyl
0.12±0.0.026
0.10
0.16
0.18
4.3
>10
2-thiazolyl
adamantyl
*The assay was performed in duplicates and the values were typically within 10% of each other. For select
compounds, two or more experiments were performed independently. In those instances, the data is reported as
Mean ± SD
The data summarized in Table 2 demonstrate a significant dependence of inhibition on the
structure of R1. Substitutions at the 4-position of the phenyl ring affect USP30 inhibition, e.g.
the 4-fluorophenyl analog (19) has an IC50 = 0.05 M and compound 23 (R1 = 4-hydroxyphenyl)
showed no inhibition (at 10 µM). Other compounds such as 18, 20 and 22 with R1 = 4-
chlorophenyl, 4-methylphenyl and 4-cyanophenyl, respectively, inhibit USP30 with IC50s
between 2.5 M and 0.27 M. Electron withdrawing substituents (e.g. F, CN) on the phenyl ring
are preferred over the electron donating groups (e.g., OMe, OH). Electron deficient
heteroaromatic rings are preferred in this region as shown by 2-pyridyl (24) and 2-thiazolyl (25)
with IC50s of 0.16 M and 0.18 M, respectively. For further exploration of the lipophilic
pocket, the phenyl group was replaced with benzyl (26), phenylethyl (27) and benzyloxy groups
(28). None of these compounds inhibit USP30 (at 10 µM). Non-aromatic cycloalkyl groups were
tolerated in this region as evidenced by the USP30 inhibition observed with cyclopentyl (30),
cyclohexyl (31) and 4,4-difluorocyclohexyl (32) groups. Smaller rings such as cyclopropyl (29)
or larger rings such as adamantyl (34) resulted in complete loss of USP30 inhibition while the
cycloheptyl analog (33) showed an order of magnitude decrease in USP30 inhibition.
As a strategy to reduce the molecular weight and the number of aromatic rings in the
compounds, 5-amino-naphthalene sulfonamide at the C-terminus of the amino acid core was
replaced with 4-aminobenzene sulfonamide moiety (Table 3). The N-acyl group and the L-
phenylanine moiety were simultaneously modified using the insights gained from the SAR in the
naphthylsulfonamide series. Therefore, optimum substituents from Table 1 and Table 2 (R1
=
phenyl, cyclohexyl and 2-pyridyl) were matched with the optimum R2 substituents (benzyl, 4-
fluorobenzyl, and cyclohexylmethyl). As anticipated, several compounds inhibit USP30 with
IC50s < 100 nM as shown in Table 3. All the compounds with IC50s < 1 M were screened for

USP1, USP8 and USP9 inhibition. None of the compounds inhibit these proteases, which
demonstrate the selectivity of this series of compounds.
Table 3. SAR of Benzenesulfonamide Series for inhibition of USP30
USP30
IC50 (M)*
Compd
R1
R2
phenyl
phenyl
cyclohexyl
cyclohexyl
benzyl
cyclohexylmethyl
benzyl
cyclohexylmethyl
benzyl
cyclohexylmethyl
cyclohexylmethyl
cyclohexylmethyl
cyclohexylmethyl
2-thienylmethyl
2-thienylmethyl
4-pyridylmethyl
benzyl
>10
0.23
35
36
37
38
39
40
41
42
43
44
45
46
47
48
0.06 ±0.012 (n=3)
0.08
0.02
0.02
0.46
0.06
0.03
0.02
0.03
0.02
0.25
0.12
4-fluorophenyl
4-fluorophenyl
2-fluorophenyl
3-fluorophenyl
3,4-difluorophenyl
4-fluorophenyl
cyclohexyl
4-fluorophenyl
2-pyridyl
2-pyridyl
cyclohexylmethyl
*The assay was performed in duplicates and the values were typically within 10% of each other. For select
compounds, two or more experiments were performed independently. In those instances, the data is reported as
Mean ± SD
Scheme 2. Synthesis of compounds 29 and 31

Reagents and conditions: a) Acetic anhydride, aq. NaOH (2N), toluene, 100 °C, 2 h; b) Chlorosulfuric
acid, rt, 2.5 h; c) 2-methylpropan-2-amine, Et3N, dry THF, rt, 16 h; d) aq. NaOH (5N), MeOH, 80 °C,
16 h.
e) Methyl-L-phenylalaninate hydrochloride, EDC-HCl, HOBT, DIPEA, DMF, rt, 16 h; f) LiOH-2H2O,
THF, MeOH, H2O, rt, 6 h; g) 5-amino-N-(tert-butyl)naphthalene-1-sulfonamide (E), EDC-HCl, HOBt,
DMF, rt, 48 h.
Since structurally related analogs 31 (MF-094) and 29 (MF-095) have significantly different
inhibitory activity for USP30, the compounds were used to probe the biology of USP30. The
synthesis of 31 and 29 is shown in Scheme 2. Both compounds have < 30% inhibitory activity
for a panel of 22 ubiquitin specific protease assays up to 10 µM at Ubiquigent Ltd (Dundee,
UK).22 The compounds were used to evaluate binding to the active site on the surface of isolated
mitochondria.23
Mitochondria were isolated from C2C12 myoblasts, which are of murine origin, and the intact
organelles were exposed to a DUB specific activity based probe, biotin-UbVME (biotin-
ubiquitinylated methyl vinyl ether).24 USP30 enzymatic activity results in covalent modification
of the active site cysteine with biotinylated ubiquitin, which was detected after SDS-PAGE and
transfer to a membrane as shown in Figure 2. The upper band in Figure 2 has been assigned
previously as methylcrotonyl CoA carboxylase and propionyl CoA carboxylase;25 these enzymes
were endogenously biotinylated and so appear irrespective of biotin-UbVME. Compound 31
protected USP30 from modification at concentrations from 0.2 to 5 µM whereas 29 did not
protect mitochondrial USP30 from modification.
Figure 2. Compound 31 protects the active site of endogenous USP30 from modification by
biotin-UbVME, an activity-based probe. USP30 is indicated by the arrow.
The effects of 31 and 29 were examined in a recently described mitophagy assay that tracks the
disappearance of bromodeoxyuridine-labelled mitochondrial DNA in terminally differentiated
C2C12 cells (Figure 3).26 Inhibition of USP30 is expected to increase the net rate of ubiquitin
accumulation on mitochondria by slowing the de-ubiquitination of outer membrane proteins. In
turn, faster accumulation of ubiquitin will lead to an increased rate of mitophagy. Compound 31
accelerated the disappearance of BrdU from the mitochondrial DNA after 6 hours. In contrast, 29
did not elicit a significant effect.

2 0 0
1 5 0
1 0 0
5 0
ns
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0
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)
)
)
)
)
O
M
M
M
M
2
M
M
u
u
u
u
u
u
S
3
1
3
3
.
1
3
(
.
M
(
(
(
0
0
D
(
1
1
(
9
9
3
3
2
2
1
9
3
Figure 3. C2C12 myotubes were incubated with compounds for 24 hours and the mitochondrial
DNA was labeled with BrdU for 2 hours. Six hours after labeling, 31 enhanced mitophagy as
measured by loss of BrdU from mtDNA (p < 0.05 at all concentrations). Compound 29 did not
enhance mitophagy under the same conditions (ANOVA with Kruskal Wallis post test).
We have identified a chemical series of highly selective USP30 inhibitors. Two structurally
similar compounds 31 (MF-094) and 29 (MF-095), which inhibit USP30 with IC50s that are at
least two orders of magnitude different, were used to demonstrate the role of USP30 inhibition in
accelerating mitophagy in C2C12 myotubes. We believe that compounds 31 and 29 represent
unique tools to probe the underlying biology of USP30 and further work will be reported in due
course.
Acknowledgement: Authors thank Dr.s Michael Patane, Effie Tozzo and Yoh Terada for
valuable discussions.
AUTHOR INFORMATION
Corresponding Author
* E-mail: Blagu@mitobridge.com
Author Contributions
The manuscript was written through contributions of all authors, all authors have given approval
to the final version of the manuscript.
ABBREVIATIONS
USP = Ubiquitin specific peptidase; DUB = deubiquitinase; biotin-UbVME = biotin-ubiquitin-
vinylmethyl ester; BrdU = bromodeoxyuridine.

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by pre- incubating 20nM of USP30 with varying concentration of NCE (the compound ) in the
assay buffer (PBS (pH 7.4), 1 mM DTT, 0.01% Tween 20, 0.01% BSA , DMSO final conc :1%)
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22. USPs tested were USP1, USP2, USP4, USP5, USP6, USP7, USP8, USP9, USP11, USP14,
USP15, USP16, USP19, USP20, USP21, USP25, USP27, USP28, USP35, USP36, USP45,
CYLD.
23. Some of the biological results were presented previously. JE Thmpson, J. E.; Terada, Y.;
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24. Mitochondria were isolated from C2C12 myoblasts using the mitochondrial isolation kit for
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compound 29, compound 31 or DMSO and 1.5 µM biotin-UbVME (UbiQ, Amersterdam,
Netherlands) in 6 mM phosphate buffer pH 7.4, 150 mM NaCl, 50 µM tris(2-
carboxyethyl)phosphine, 1% DMSO at ambient temperature for 5 minutes. Reactions were
quenched by addition 0.33 volumes of 4x SDS-PAGE sample buffer (Bio-rad, Hercules,
California), 14 mM beta-mercaptoethanol and heating at 95 °C for five minutes. After SDS-
PAGE and transfer to polyvinyldifluoride the membrane was blocked with 1% fish gelatin

blocking buffer (Boston Bioproducts, Ashland, Massachusetts). The membrane was incubated
with 100 ng/ml streptavidin dylight (800 nm emission maximum) in 1% fish gelatin buffer with
detergent (Boston Bioproducts, Ashland, Massachusetts) for 60 minutes, rinsed and the image
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1. Selective USP30 inhibitors
2. Increase protein ubiquitination
3. Accelerates mitophagy

Graphical Abstract