Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00208

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Full text of DOI:10.1021/acsmedchemlett.6b00208

Letter
pubs.acs.org/acsmedchemlett
Discovery of Fluoromethylketone-Based Peptidomimetics as
Covalent ATG4B (Autophagin-1) Inhibitors
Zongxing Qiu,‡ Bernd Kuhn,§ Johannes Aebi,§ Xianfeng Lin,‡ Haiyuan Ding,‡ Zheng Zhou,‡ Zhiheng Xu,‡
Danqing Xu,‡ Li Han,‡ Cheng Liu,‡ Hongxia Qiu,‡ Yuxia Zhang,‡ Wolfgang Haap,§ Claus Riemer,§
Martin Stahl,§ Ning Qin,‡ Hong C. Shen,‡ and Guozhi Tang*,‡
‡Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Shanghai, 720 Cailun Road,
Shanghai 201203, China
§Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center Basel, F. Hoffmann-La Roche
Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
S
* Supporting Information
ABSTRACT: ATG4B or autophagin-1 is a cysteine protease
that cleaves ATG8 family proteins. ATG4B plays essential
roles in the autophagosome formation and the autophagy
pathway. Herein we disclose the design and structural
modifications of a series of fluoromethylketone (FMK)-based
peptidomimetics as highly potent ATG4B inhibitors. Their
structure−activity relationship (SAR) and protease selectivity
are also discussed.
KEYWORDS: ATG4B, autophagy, covalent inhibitor, fluoromethylketone, peptidomimetics
utophagy is an evolutionarily conserved process essential
for cell homeostasis and housekeeping by catabolizing
inhibitor with an IC50 of 51 μM in an assay detecting the
cleavage of LC3-GST.10
Previously, we reported the identification of Z-FA-FMK (1,
Figure 1) as a covalent active-site directed ATG4B inhibitor
A
aggregated proteins and damaged cellular components.1 The
hallmark of autophagy is the formation of autophagosomes and
subsequent fusion with lysosomes to achieve degradation of
their contents. Dysregulation of autophagy has been recently
described in the pathogenesis of a variety of diseases such as
cancer, neurodegenerative and metabolic disorders, and viral
infections.2,3 Modulation of autophagy has become a very active
area of preclinical and clinical research, and particularly, there is
high interest to identify potent and specific autophagy
inhibitors.4
Figure 1. Structures of peptidomimetic ATG4B inhibitors.
ATG4 or autophagins are a class of cytosolic cysteine
proteases that cleave ATG8 family proteins, such as light chain
3 (LC3). ATG4 plays essential roles in the formation and
maturation of autophagosomes.5,6 Among all four human
ATG4 orthologues, ATG4B is functionally dominant, and it is
the sole enzyme reported to efficiently cleave LC3 precursors
and to deconjugate lipid from membrane bound LC3-
phosphatidylethanolamine (LC3-PE).7 ATG4B has been
considered as a potential therapeutic target in the development
of chemosensitizer for the treatment of certain cancer types.
Recently, a number of small molecule ATG4B inhibitors such
as Z-L-Phe-chloromethylketone and NSC185058 were reported
to have modulatory effects on the autophagy process.8−10
However, because of its high chemical reactivity, the
chloromethylketone moiety is usually associated with significant
cytotoxicity. NSC185058, however, is only a weak ATG4B
from a TR-FRET based focused library screening.11 The hit
expansion of 1 led to the discovery of Z-FG-FMK (2), which
was 10-times more potent than 1 with an IC50 of 1.2 μM in the
biochemical assay.11 Herein we disclose the structure-guided
optimization of 2 toward highly potent fluoromethylketone
(FMK)-based ATG4B inhibitors.
As an early step in the autophagosome formation, ATG4B
cleaves proLC3 to expose the C-terminal Gly120 for
subsequent PE-conjugation and autophagosome membrane
insertion.5 The cocrystal structures of catalytically inert C74S or
Received: May 18, 2016
Accepted: June 25, 2016
© XXXX American Chemical Society
A
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H280A mutant Atg4B in complex with the LC3(1−124)
peptide revealed that the substrate induces a conformational
change of both the regulatory loop (258−262) and Trp142
resulting in an open protein conformation in which the C-
terminal ETFG residues at the processing site of LC3 make
several intermolecular contacts with the catalytic pocket of
ATG4B.12 Molecular modeling13 of compounds 1 and 2 into
the open conformation of ATG4B, using the TFG residues of
LC3 as template, suggests a potential steric clash of the
additional methyl group in 1 with Trp142, which likely explains
the inferior anti-ATG4B activity of this molecule (Figure 2).
Table 1. Inhibition of ATG4B by 1−3 and 4a−n
Figure 2. Overlay of the X-ray crystal structure (PDB code: 2Z0D)12
of the C-terminal LC3 peptide (Glu117-Gly120, salmon) in human
ATG4B (orange) with a model of Z-FG-FMK 2 (cyan).
Intermolecular hydrogen bonds for 2 are shown as red lines and the
covalent interaction with Cys74 is shown in blue. Models were built
using the modeling software MOLOC.13 See also Figure S1 in the
Supporting Information for a surface representation of the binding site.
In the predicted binding mode, the FMK warhead makes a
covalent bond with the S atom of Cys74 and the planar P1
glycine moiety stacks onto the π-system of the indole ring of
Trp142. The benzyl substituent nicely overlaps with the P2
phenylalanine of LC3 and is engaged in dispersion interactions
with lipophilic side chains in the S2 pocket as well as stacking
interactions with the π-surface of a β-turn. The amide linker
connecting P1 and P2 fragments uses its carbonyl oxygen atom
to form a hydrogen bond to a buried water molecule. In
contrast, in the amide linker between P2 and P3 the NH
functionality makes a hydrogen bond with the carbonyl oxygen
atom of Tyr143, and the carbonyl group is solvent-exposed.
Additionally, the Cbz phenyl ring stacks onto the backbone
amide of Tyr143−Gly144.
To investigate the SAR of the C-terminal LC3 peptides, we
synthesized and tested a number of FMK-based peptidomi-
metics including Ac-ETFG-FMK (3a, Figure 1) for their
ATG4B inhibitory activities. We find that 3a is about 15 times
less active than 2 in the TR-FRET assay and inactive in the
cellular based actin-LC3-dNGluc cleavage assay (or luciferase
release assay, LRA),11 probably due to limited cell permeability
(Table 1). Moreover, it should be noted that the FMK warhead
at the P1 position is important for ATG4B inhibition as
peptides like Ac-ETFG (3b) and Ac-TFG are inactive in both
TR-FRET and LRA assays.
a
IC50s [μM] are mean values of TR-FRET experiments in duplicate
b
runs, with variation 10%. IC50s [μM] are mean values determined
by luciferase-release assay (LRA) with Actin-LC3B-dNGLuc construct
used in HEK-293T cells. Experiments were run in duplicate with
variation 15%. Compounds were not cytotoxic to HEK-293T cells
c
(CC50 > 100 μM) in 24 h treatment. ND, not determined.
Information) and tested in both TR-FRET and LRA assays.
The resulting SAR reveals that formation of a covalent bond to
Cys74 is critical for anti-ATG4B activity as direct structural
changes of the FMK moiety completely abolish anti-ATG4B
activities (examples 4a−b in Table 1), in agreement with the
results of 3a and 3b. In addition, we find that many covalent
warheads such as pyrazole amide (example 4c), aldehyde (4d),
nitrile (4e−f), and Michael acceptor-like 4g are not tolerated
either. We hypothesize that this is due to an insufficient fit to
the binding site around Cys74 and/or too low reactivity for the
formation of a covalent bond.
The chemistry space at the P2 position was explored with a
variety of amino acids. For example, (S)-α-methyl substitution
and amino acids such as (S)-phenylglycine, (S)-valine, and (R)-
Phe were incorporated but resulted in a complete loss of anti-
ATG4B activity (examples 4h−k). We find that halogen
substituents can be tolerated at the meta-position of the phenyl
group of 2. For example, meta-Cl analogue 4m shows an IC50 of
1.1 μM in the TR-FRET assay. In contrast, substituents at
In the first optimization phase we focused on modifications
of the warhead and the Phe residue at the P2 position of 2.
Thus, analogues 4a−n were synthesized (see the Supporting
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a
Scheme 1. Synthesis of FMK-Based ATG4B Inhibitors 8a−k and 9a−h
a
Reagents and conditions: (a) HATU, DIPEA, DMF, rt, 97%; (b) HCl, EtOAc, 30 °C, 30 min, quant.; (c) carboxylic acids, HATU, DMF, rt; (d)
DMP, DCM, rt.
ortho- and para-positions are clearly detrimental to anti-ATG4B
activity (examples including 4l).
that aryl substituents, especially those at the meta-substitution,
are well tolerated revealing a rather flat SAR (examples 8g−i).
Subsequently, bicyclic aromatic groups were used at the P3
position, and the naphthalene-1-carboxamide analogue 9a
showed strong inhibition of ATG4B, with IC50 values of 80
and 73 nM in the TR-FRET and cellular-based LRA assays,
respectively (Table 3). In comparison, β-substituted analogue
Next, structural explorations were carried out to optimize the
terminal N-Cbz group. By using the method shown in Scheme
1, (S)-N-Boc phenylalanine was coupled with 1-amino-3-fluoro-
propan-2-ol hydrochloride (5) in the presence of HATU and
DIPEA to give amide product. The N-Boc protective group was
removed by treating the intermediate with a solution of HCl in
EtOAc, and the resulting amine hydrochloride salt 6 was used
for further functional transformations. For instance, amine 6
was coupled with selected carboxylic acids to give amides 7.
The FMK group was generated by treating 7 with Dess−Martin
periodinane (DMP), and inhibitors 8a−k were evaluated for
their anti-ATG4B activities.
a
Table 3. Inhibition of ATG4B by 9a−j
We find that the N-Boc analogue 8a shows comparable
activity to 2 with an IC50 of 2.0 μM in TR-FRET assay (Table
2). Importantly, the carbamate moiety can be replaced by more
a
Table 2. Inhibition of ATG4B by 8a−k
a
Definitions are the same as those described in Table 1.
9b is more than 10 times less active than 9a probably due to
less optimal π-stacking of the β-naphthyl group with the
backbone amide. The physicochemical properties of 9a show
moderate solubility (LYSA: 41 μg/mL) and high human and
mouse liver microsome clearances of 13.9 and 70 mL min−1
kg−1, respectively. Finally, halogen-substitutions and a nitrogen
atom walk were carried out on the α-naphthyl group. We find
that 5-F substituted analogue 9d has the best anti-ATG4B
activity in our series with an IC50 of 15 nM in the cellular LRA
assay. We also note that aza-decorations are well tolerated at
the 3- and 4-positions but not at the 8-position of the naphthyl
group (examples 9e−g). Both quinoline-4-carboxamide ana-
logue 9e and cinnoline-4-carboxamide analogue 9g show strong
anti-ATG4B activities and improved solubility compared to 9a
with LYSA values of 188 and 230 μg/mL, respectively. In
a
Definitions are the same as those described in Table 1.
rigid and smaller amide moieties, exemplified by benzamide
analogue 8e and cyclopentanecarboxamide 8f, which demon-
strate good anti-ATG4B activities with IC50 values of 1.21 and
1.86 μM, respectively. Pyrazine-2-carboxamide analogue 8j
shows promising potency and physicochemical properties, such
as a high water solubility of 129 μg/mL in a lyophilized
solubility assay (LYSA). Further structural explorations suggest
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a
Table 4. Selectivity of FMK-Based ATG4B Inhibitors
protease family
protease
1
8e
9a
Asp. protease
Ser. protease
cathepsin D
>100
>100
>100
>100
>100
>100
43.5
>100
>100
>100
>100
>100
>100
>100
>100
0.7
>100
>100
>100
>100
>100
>100
75.1
cathepsin G
tryptase, βII
neutrophil elastase
20S proteasome
caspase 2
Compl. protease
Cys. proteases
caspase
caspase 3
caspase 7, 8, 9
cathepsin B
>100
0.3
>100
0.2
cathepsin
others
calpain
0.6
0.6
0.096
>100
>100
metalloprotease
ACE, ECE-1
MMP1, MMP3, MMP9
>100
>100
>100
>100
a
IC50 apparent [μM] are mean values of protease inhibition from duplicate runs, with variation 15%. Assays were performed by Genscript
according to their standard protocols, see the Supporting Information (www.genscript.com).
addition, they show increased intrinsic metabolic stability
compared to 9a. For example, the human and mouse liver
microsome clearances of 9g are about 9.9 and 39 mL min−1
kg−1, respectively.
a focused-library screening hit. Naphthalene-1-carboxamide
analogue 9a and its derivatives inhibit ATG4B with IC50 values
<100 nM in both a TR-FRET assay and a cell-based luciferase-
release assay. The FMK class of ATG4B inhibitors are inactive
in a panel of different proteases involving cysteine proteases like
caspase 2, 3, 7, 8, and 9. However, they inhibit cathepsin B and
calpain with submicromolar IC50 values. The SAR of the
current FMK series and biostructure differences between
ATG4B and other cysteine-proteases support further structural
modifications to increase target selectivity. An LC-MS/MS
study demonstrated that analogue 9a forms an irreversible and
selective covalent bond with Cys74 and thus inactivates
ATG4B proteolytic activity. A binding mode of our series in
ATG4B was constructed by molecular modeling revealing key
protein−ligand interactions. Efforts including modulation of
warhead reactivity and bioisostere replacements of the P1
amide group as well as autophagy modulation effects by
analogues of 9a will be the subject of future publications.
ATG4B has several solvent-exposed cysteine residues.
Nevertheless, ATG4B formed a 1:1 association with FMK
analogues after 4 h of coincubation.11 A subsequent LC−MS/
MS study indicated that 9a formed an irreversible covalent
bond with the more reactive thiol group of Cys74 located at the
catalytic site of ATG4B. This finding and additional SAR
supports our hypothetical binding mode (Figure 2). For
example, in our model the P1 amide NH group is not involved
in direct ATG4B interactions, while the P2 NH moiety and the
backbone carbonyl oxygen of Tyr143 form a hydrogen bond. In
line with this binding mode, blocking of both ligand NH groups
through N-methylation (9i and 9j) is well tolerated at the P1
position (9i) but not on the phenylalanine P2 part (9j, Table
3). Based on a binding model of 9a in ATG4B (Figure S2) we
postulate that the improved activities of naphthyl analogues of
this topology are due to more efficient stacking of the extended
bicyclic π-system with the backbone amide group of Tyr143−
Gly144.14 This should be sensitive to the relative orientation of
the naphthyl ring to the plane of the adjacent amide linker. This
is indeed observed as the preferred orthogonal orientation in
the case of α-substitution (9a) leads to significantly better
ATG4B activity than for β-substitution (9b) in which the
preferred orientation is close to planar.
EXPERIMENTAL METHODS
■
Molecular Docking of FMK Analogues. Model building was
performed with the cocrystal structure of human ATG4B with LC3
(1−120) as template (PDB code: 2Z0D)12 using the following
procedure: (1) in silico mutation of the TFG residues of LC3 into Z-
FA(G)-FMK; (2) rebuilding of A280H using the structure 2Z0E12 to
reproduce the human sequence; (3) removal of the fluorine atom of Z-
FA(G)-FMK and formation of a covalent bond between the sulfur
atom of Cys74 and the reactive methylene unit of FMK. Here, the
second, alternate conformation of the side chain of Cys74 in the
crystal structure 2Z0D was used as this is closer to the FMK group. (4)
Minimization of the ligand within the mostly rigid protein allowing for
relaxation of only few selected residues (e.g., Tyr54, Cys71, Trp142,
Asn261) in close contact to the small molecule.
FMK analogues 1, 8e, and 9a were selected for target
selectivity profiling against a panel of proteases (Table 4).
These ATG4B inhibitors have no activity against the aspartic
protease, serine proteases, metalloproteases, and the 20S
proteasome from our panel. In addition, they show very low
inhibition of caspases, which belong to the cysteine protease
TR-FRET Assay. Five microliters of serial-diluted test compounds
(1% DMSO) in assay buffer was added into 5 μL of purified ATG4B
(final 0.1 nM) in 384-well plate, and the solution was incubated at rt
for 30 min. Then, 5 μL of His-GATE-16-GST (final 20 nM) was
added into the wells, and the solution was incubated at rt for another
30 min. Five microliters of detection solution (final 2 nM of Eu-anti-
His and 20 nM of Ulight-anti-GST) was added, and the resulting
mixture was incubated at rt for 40 min. The fluorescence signal was
measured with Envision4 (Ex, 340 nm; Em, 665/615 nm) and data
was analyzed by Genedata Screener (Genedata AG).
Luciferase Release Assay (LRA). A similar procedure has been
reported before.15 Basically, HEK-293T cells (human embryonic
kidney cells, ATCC, CRL-11268) were seeded in 6-well plate with a
density of 70% confluence. One microgram of enzyme expression
plasmid and 2 μg of substrate expression plasmid were cotransfected
family. However, the FMK analogues hit a few cysteine
(app)
proteases such as cathepsin B and calpain with IC50
values
<1 μM. While the sequence identity is very low between
ATG4B and these cysteine proteases, the result can likely be
explained by the significant reactivity of the FMK group and the
relatively open binding pockets of these proteases. Compound
9a is slightly more active on ATG4B compared to cathepsin B
and calpain. In this study, we did not attempt to further
increase the selectivity window through target-based molecular
design.
In summary, we discovered a series of highly potent FMK-
based covalent ATG4B inhibitors by structural modifications of
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using 9 μL of X-tremeGENE HP DNA Transfection Reagent (Roche
diagnostics cat#06366236001). Three combinations (pCNDA3.1
vector and dNGluc, HTRA4 and Actin-dNGluc, and ATG4B and
Actin-LC3-dNGluc) were used in the transfections. After 24 h, the
transfected cells were trypsinized, suspended, and transferred to 96-
well plates (Corning, can#3599), serial-diluted compounds in assay
buffer were added into the platesm and the mixture was incubated for
24 h. Fifty microliters of cell supernatant was transferred to a 96-well
white plate (Corning, Cat#3917), and luciferase signal was detected by
using BioLux Gaussia luciferase assay kit (NEW ENGLAND BioLabs,
Cat#E3300L) and measured with Envision4.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00208.
(12) Satoo, K.; Noda, N. N.; Kumeta, H.; Fujioka, Y.; Mizushima, N.;
Ohsumi, Y.; Inagaki, F. The structure of Atg4B-LC3 complex reveals
the mechanism of LC3 processing and delipidation during autophagy.
EMBO J. 2009, 28, 1341−50.
Synthetic procedures and analytical data for compounds
2−5, 8a−k, and 9a−j; predicted binding models of Z-
FG-FMK (2) and 9a; general assay protocols for
evaluation of protease selectivity (PDF)
(13) Gerber, P.; Muller, K. MAB, a generally applicable molecular
̈
force field for structure modelling in medicinal chemistry. J. Comput.-
Aided Mol. Des. 1995, 9, 251−268.
(14) Harder, M.; Kuhn, B.; Diederich, F. Efficient stacking on protein
amide fragments. ChemMedChem 2013, 8, 397−404.
(15) Ketteler, R.; Seed, B. Quantitation of autophagy by luciferase
release assay. Autophagy 2008, 4, 801−806.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86 21 28946723. E-mail: gordon.tang@roche.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Qinshan Gao, Yujuan Gao, and Jin Ge for
purification of final compounds.
ABBREVIATIONS
■
ATG4B, autophagy-related gene 4B; Cbz, carbobenzyloxy or
benzyloxy carbamate; DMP, Dess−Martin periodinane; LC3,
light chain 3; LRA, luciferase release assay; LYSA, lyophilized
solubility assay; PE, phosphatidylethanolamine; TR-FRET,
time-resolved fluorescence energy transfer
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