Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases

Article abstract of DOI:10.1016/j.bmcl.2017.12.056

Rhomboid proteases form one of the most widespread intramembrane protease families. They have been implicated in variety of human diseases. The currently reported rhomboid inhibitors display some selectivity, but their construction involves multistep synthesis protocols. Here, we report benzoxazin-4-ones as novel inhibitors of rhomboid proteases with a covalent, but slow reversible inhibition mechanism. Benzoxazin-4-ones can be synthesized from anthranilic acid derivatives in a one-step synthesis, making them easily accessible. We demonstrate that an alkoxy substituent at the 2-position is crucial for potency and results in low micromolar inhibitors of rhomboid proteases. Hence, we expect that these compounds will allow rapid synthesis and optimization of inhibitors of rhomboids from different organisms.

Full text of DOI:10.1016/j.bmcl.2017.12.056

Accepted Manuscript
Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases
Jian Yang, Marta Barniol-Xicota, Minh T.N. Nguyen, Anezka Ticha, Kvido
Strisovsky, Steven H.L. Verhelst
PII:
S0960-894X(17)31230-1
https://doi.org/10.1016/j.bmcl.2017.12.056
BMCL 25512
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 November 2017
21 December 2017
23 December 2017
Please cite this article as: Yang, J., Barniol-Xicota, M., Nguyen, M.T.N., Ticha, A., Strisovsky, K., Verhelst, S.H.L.,
Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.056
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Benzoxazin-4-ones as novel, easily accessible
inhibitors for rhomboid proteases
Jian Yanga, Marta Barniol-Xicotaa, Minh T. N. Nguyenb, Anezka Tichac, Kvido Strisovskyc and Steven H. L.
Verhelsta,

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases
Jian Yanga, Marta Barniol-Xicotaa, Minh T. N. Nguyenb, Anezka Tichac, Kvido Strisovskyc and Steven H.
L. Verhelsta,b,*
a KU Leuven – University of Leuven, Laboratory of Chemical Biology, Department of Cellular & Molecular Medicine, Herestraat 49 Box 802, 3000 Leuven,
Belgium
bLeibniz Institute for Analytical Sciences ISAS, Otto-Hahn-Str. 6b, 44227 Dortmund, Germany
cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 166 10, Czech Republic
ARTICLE INFO
ABSTRACT
Article history:
Received
Rhomboid proteases form one of the most widespread intramembrane protease families. They
have been implicated in variety of human diseases. The currently reported rhomboid inhibitors
display some selectivity, but their construction involves multistep synthesis protocols. Here, we
report benzoxazin-4-ones as novel inhibitors of rhomboid proteases with a covalent, but slow
reversible inhibition mechanism. Benzoxazin-4-ones can be synthesized from anthranilic acid
derivatives in a one-step synthesis, making them easily accessible. We demonstrate that an
alkoxy substituent at the 2-position is crucial for potency and results in low micromolar
inhibitors of rhomboid proteases. Hence, we expect that these compounds will allow rapid
synthesis and optimization of inhibitors of rhomboids from different organisms.
Revised
Accepted
Available online
Keywords:
Rhomboid proteases
Intramembrane proteases
Benzoxazinones
2009 Elsevier Ltd. All rights reserved
.
Inhibitors
Activity-based protein profiling
Rhomboid proteases are amongst the most widespread
intramembrane protease (IMP) families, and have been found in
of adhesins that help apicomplexan parasites invade host cells,6, 7
and processing of Pink-1, a protein involved in mitophagy and
linked to Parkinson's disease.8-10 Potent and selective inhibitors
would be attractive research tools to examine these processes and
assess the drugability of rhomboids.
2
virtually all sequenced organisms.1, They utilize a serine-
histidine dyad to cleave their substrates, which are also
membrane proteins, in a transmembrane helix (TM) or a
juxtamembrane region. Rhomboid proteases were originally
discovered in Drosophila melanogaster.3 In this organism,
Rhomboid-1 is a vital player in the epidermal growth factor
receptor pathway. Various biological roles in other organisms
have been discovered since then, such as the mediation of
quorum sensing by processing of a component of the twin
arginine translocase in the bacterium Providencia stuartii4 and
ER associated degradation of membrane proteins.5 More
medically relevant roles of rhomboid proteases include cleavage
After protocols had been established for the expression and
12
purification of rhomboid proteases in detergent micelles,11,
several different types of inhibitors were discovered,13, 14 all of
which contain an electrophile that reacts with the serine
nucleophile in the active site. These inhibitors include 4-chloro-
16
18
20
isocoumarins,15,
fluorophosphonates,17,
β-lactams,19,
β-
lactones,21 and peptidyl chloromethyl ketones,22 aldehydes23 and
ketoamides (Figure 1A).24

Figure 1. (a) Overview of known rhomboid inhibitors. The general serine
protease inhibitor 3,4-dichloroisocoumarin (top left), the 4-chloro-
isocoumarin scaffold with a 7-amino group (top middle), β-lactams and β-
lactones (top right), the fluorophosphonate CAPF (bottom left), peptide
chloromethyl ketones (bottom middle) and peptide aldehydes (bottom right).
(b) Comparison of the isocoumarin and benzoxazin-4-one scaffolds. (c)
Mechanism of the inactivation of serine proteases by benzoxazinones and
their reactivation upon hydrolyss of the inhibitor acyl-enzyme complex.
Whereas
3,4-dichloroisocoumarin
(DCI)
and
the
fluorophosphonate-based activity-based probe FP-Rh are pan-
inhibitors of rhomboid proteases, β-lactones and other chloro-
isocoumarins have shown some degree of selectivity.25 During
the course of this work, ketoamides were reported as a scaffold
with selectivity against the E. coli protease GlpG over other,
soluble serine proteases.24
variety of other substituents to explore the influence on the
potency against rhomboid proteases.
Scheme 1. Synthesis of benzoxazin-4-ones from anthranilic acids. Reagents
and conditions: (a) Acetic anhydride, 130 °C, 86-87%. (b) Acid chloride,
triethylamine, dichloromethane, 0 °C to room temperature, 13-98%. (c)
Chloroformate, pyridine, room temperature, 6-57%.
Synthetically, most of these rhomboid inhibitors are not very
easily accessible and require multistep
organic synthesis. We therefore decided to
seek for an inhibitor scaffold that would be
easier to synthesize. Benzoxazin-4-ones
(Figure 1B) are heterocyclic, mechanism-
based inhibitors of soluble serine
proteases.26 Attack on the carbonyl by the
active site serine leads to acylation of the
serine side chain, similar as with
isocoumarins (Figure 1C). Usually, the
ester bond between protease and inhibitor
hydrolyses over time, and the potency of
benzoxazin-4-ones in part depends on the
rate of deacylation (Figure 1C).26 Conveniently, benzoxazin-4-
ones can be made in one synthetic step from substituted
anthranilic acids and acid anhydrides, acid chlorides or
chloroformates. The two fused aromatic ring structures in this
class of inhibitors offer the possibility of broad chemical
variation and optimization for a particular target protease. Since
the first report of benzoxazin-4-ones as serine protease
Scheme 1 outlines the synthesis of the benzoxazinones we
designed above. An overview of all synthesized compounds is
given in Figure 2. Compounds 1a-3a were made from the
corresponding anthranilic acid derivative and acetic anhydride
under reflux conditions (Scheme 1). The other benzoxazinones
were constructed by reacting the anthranilic acids with acid
o
inhibitors,27 various publications have followed, for example on
chlorides or chloroformates at 0 C to room temperature, using
29
inhibition of human leukocyte elastase,28,
cathepsin G,30
triethylamine or pyridine as a base. All designed benzoxazinones
were successfully synthesized, except for compound 1e. The
reason for this is unclear, as the close analogs 2e and 3e were
both obtained in 86% yield.
chymase,31 and the complement system C1r serine protease.32 We
hypothesized that benzoxazinones may also inhibit rhomboid
proteases, because of the structural analogy to 4-chloro-
isocoumarins (Figure 1B). From medicinal chemistry point-of-
view, it is of interest to note that benzoxazinones with similar
substitution pattern as 4-chloro-isocoumarins have a slightly
lower ClogP (Supplemental Figure 1). Rhomboid inhibitors of
the 4-chloro-isocoumarin class carry small and large hydrophobic
groups at the 3-position, which corresponds to the 2-position in
the benzoxazin-4-one scaffold. We therefore selected a range of
chloroformates and activated carboxylic acids to yield these type
of substituents and increase the likelihood of obtaining potent
rhomboid inhibitors.
Figure 2. Overview of benzoxazin-4-ones used in this study.
Having the benzoxazinones at hand, we performed an
inhibition assay against the E. coli rhomboid GlpG and the B.
subtilis rhomboid YqgP by competitive activity-based protein
profiling (ABPP). In competitive ABPP, protease samples are
first treated with the compound to be tested and then treated with
an activity-based probe (ABP) to label the remaining amount of
We also chose a number of commercially available anthranilic
acid derivatives to introduce variety at the aryl ring. For soluble
serine proteases, the substituents at the aryl ring influence the rate
of deacylation. It was reported that steric bulk at the 5-position
slows down the deacylation step for human leukocyte elastase.28
A 5-methyl group slows down this step for cathepsin G.30 For
HSV-1 protease, a 5-chloro substituent generally led to lower
IC50 values.33 We therefore chose the anthranilic acid precursors
to include 5-chloro and 5-methyl groups, but we also selected a

active protease (Figure 3A). Depending on the tag of the ABP,
detection can take place in various manners. Here, we used FP-
Rh, an ABP that reacts with the majority of all serine
hydrolases34 by means of its fluorophosphonate reactive group. It
is also a suitable pan-rhomboid probe.25 Hence, GlpG or YqgP
were first incubated with the different benzoxazinone analogs for
30 min, subsequently incubated with FP-Rh for an additional 60
min, followed by SDS-PAGE. Whereas labeling of DMSO
vehicle treated GlpG gave clear labeling by FP-Rh, the known
isocoumarin inhibitor S006, which reacts with the active site
serine and an additional histidine,16 led to almost complete
abrogation of the signal (Figure 3B). To our delight, we found
that several benzoxazinones also inhibited GlpG and YqgP
(Figure 3B and 3C).
Interestingly, some benzoxazinones are able to discriminate
between the different rhomboids. For example, 2f inhibits GlpG
with an IC50 value in the low micromolar range, while being only
moderately active against YqgP, representing a selectivity of 15
fold. Compound 5 shows an apparent IC50 of 0.47 0.16 µM for
YqgP and approximately 60 µM for GlpG, corresponding to a
selectivity of 130 fold. Since benzoxazinones have been reported
as inhibitors of soluble serine proteases, we checked whether the
rhomboid hits also inhibit two model serine proteases: bovine
trypsin and chymotrypsin. We found that these benzoxazinones
displayed higher potencies against these proteases than against
rhomboids. Nevertheless, compound 3d shows inhibition in the
same order of magnitude for both rhomboids and soluble
proteases, and for compound 2d, the potency for YqgP and
trypsin is comparable. In an accompanying paper (see co-
submitted paper by Goel, Weggen and colleagues),
benzoxazinones with a 2-styryl substituent show some selectivity
against rhomboids, but have lower potency than the here
described compounds with a 2-alkoxy substituent. This illustrates
that the substituents on the benzoxazinones have an influence on
both potency and selectivity, and further derivatization may lead
to better properties. To gain further insight into this, the
selectivity of benzoxazinones against a broad panel of serine
proteases and hydrolases should be explored in future
experiments.
Figure 3. Inhibition assay by competitive ABPP. (a) Schematic
representation of competitive ABPP for rhomboid proteases. A purified
rhomboid protease is treated with
a benzoxazinone. Subsequently, the
residual active rhomboid is labeled by means of the ABP FP-Rh. (b) Active
GlpG is efficiently labeled by FP-Rh and visualized by in-gel fluorescent
scanning. Treatment with GlpG inhibitor S006 or different benzoxazinones
leads to various degrees of inhibition. (c) The residual activities of GlpG and
YqgP treated with the indicated benzoxazinones, as quantified by gel band
density and visualized in a heat map representation.
For compounds that displayed lower than 5% residual activity
in the initial screen, we determined the apparent IC50 value
against the two rhomboids and two commercially available
soluble serine proteases (Table 1). The potency against the two
rhomboids is in the low micromolar to submicromolar range.
Compound 2f, the most potent benzoxazinone for GlpG, displays
an apparent IC50 of 1.0 0.64 µM. Benzoxazinone 5 turned out
to be the most potent compound for YqgP with an apparent IC50
of 0.47 0.16 µM. These potencies are comparable with those of
the most potent 4-chloroisocoumarins16 and β-lactams.19, 20
For some of the most potent benzoxazinones, we checked
whether these were able to inhibit GlpG in vivo using E. coli cells
with a permeabilized outer membrane.35, 36 Although compounds
3c, 3d, 3f did not show any or only weak (1f) inhibition, we
found that compound 2f showed an IC50 value of 8.5 0.17 µM
(see Supplemental Figure 2), illustrating the capacity of
benzoxazinones to inhibit rhomboid proteases in a membrane
environment of living cells.
Table 1. Apparent IC50 values (µM) of selected benzoxazin-4-
ones.
For soluble proteases, benzoxazin-4-ones are covalent
inhibitors with a reversible mechanism: they acylate the active
site serine to give an acyl intermediate, which can be hydrolyzed
to re-form the active protease species (Figure 1C). To get insight
into the mechanism of inhibition of rhomboids by
benzoxazinones, we checked whether the covalent O-acyl
enzyme-inhibitor complex was able to hydrolyze and regain
activity. Therefore, we incubated GlpG with compounds DCI, 1f,
2f, 3c, 3d or 3f at a concentration that gives full inhibition. Then,
the excess of inhibitors was removed from the solution by a gel
filtration spin column, followed by 0 min, 30 min, 1 h, or 4 h
incubation in order to allow hydrolysis of the acyl intermediate.
Subsequently, the residually active GlpG was detected by ABPP
using the FP-Rh probe as in our previous experiments. As shown
in Figure 4, GlpG treated with DCI regained 50% of its activity
after 4h of incubation. Only with compound 1f, GlpG regained
activity more rapidly: in 30 min, almost the full activity was
restored. For the other benzoxazinones, the hydrolysis of the O-
acyl intermediate was much slower and after 4 h, most inhibition
was maintained. Interestingly, the only difference between the
fast hydrolyzing 1f and the more stable 2f and 3f is the presence
of a 5-methyl or 5-chloro-substituent, respectively. It has been
suggested that after reaction with the active site serine and ring
opening of the benzoxazinone scaffold, the carbonyl group
Compound
GlpG
YqgP
Trypsin
< 0.1
Chymotrypsin
< 0.1
1f
2d
2f
3c
3d
3f
4
2.5 0.34
N.D.a
3.7 1.2
2.8 0.59
15 6.0
7.1 1.5
6.2 2.6
1.6 0.38
4.6 1.6
1.1 0.11
< 0.1
< 0.1
1.0 0.64
2.2 0.47
1.7 0.34
1.2 0.45
N.D.
< 0.1
< 0.1
< 0.1
1.2 0.12
< 0.1
0.41 0.24
< 0.1
< 0.1
< 0.1
5
63 18
2.4 1.2
0.47 0.16 < 0.1
7.3 2.3 < 0.1
< 0.1
9
< 0.1
aNot determined.
From the series of compounds tested, we were able to derive a
basic structure-activity relationship. The oxygen substituent on
the 2-position seems to be crucial for the potency, as all
compounds that showed low micromolar activity, carried an
alkoxy substitutent at the 2-position. Notably, compounds 2f and
3f, which both carry an OCH2Ph substituent at the 2-position,
display low micromolar activity, whereas the isosteric
compounds 2h and 3h, which have a CH2CH2Ph group at this
position, did not even make the cut-off in the general screen at
100 µM compound concentration (Figure 3C), clearly indicating
the requirement for the oxygen atom.

6. Baker RP, Wijetilaka R, Urban S. Two Plasmodium rhomboid
proteases preferentially cleave different adhesins implicated in all
invasive stages of malaria. PLoS Pathog. 2006;2(10): e113.
7. O'Donnell RA, Hackett F, Howell SA, et al. Intramembrane
proteolysis mediates shedding of a key adhesin during erythrocyte
invasion by the malaria parasite. J Cell Biol. 2006;174(7): 1023-
1033.
moves out of the plane of the ring and can be shielded by the
substituent in the 5-position against attack by a water molecule.28
Another possibility is the binding of the 5-substituent in the S1
pocket,15, 22 yielding a more stable complex and possibly blocking
the access of water from the water retention site. Only a structure
of GlpG with a bound benzoxazinone may give an answer to this
question. All in all, the results shown here indicate that most
benzoxazinones are slowly reversible rhomboid inhibitors with
an off-rate lower than DCI.
8. Jin SM, Lazarou M, Wang C, Kane LA, Narendra DP, Youle RJ.
Mitochondrial membrane potential regulates PINK1 import and
proteolytic destabilization by PARL. J Cell Biol. 2010;191(5):
933-942.
9. Meissner C, Lorenz H, Weihofen A, Selkoe DJ, Lemberg MK.
The mitochondrial intramembrane protease PARL cleaves human
Pink1 to regulate Pink1 trafficking. J Neurochem. 2011;117(5):
856-867.
Figure 4. Residual activities of GlpG, respectively, 0, 30, 60 and 240 min
after removing the excess of inhibitors from the enzyme-inhibitor complex.
10. Shi G, Lee JR, Grimes DA, et al. Functional alteration of PARL
contributes to mitochondrial dysregulation in Parkinson's disease.
Hum Mol Genet. 2011;20(10): 1966-1974.
In conclusion, we have shown in this paper that benzoxazin-4-
ones are micromolar inhibitors of rhomboid proteases.
Importantly, all compounds can be obtained by a one-step
synthesis from commercially available and low-cost starting
materials. In this way, we have synthesized 29 benzoxazin-4-one
structures and tested them against two model rhomboids. The
most potent activities of the compounds synthesized here are in
the micromolar range and one compound displayed in vivo
activity. Inspection of the active structures revealed that the
substituents have substantial impact on the potency. Specifically,
a 2-alkoxy substituent seems critical for activity. We have also
shown that the off-rate of the covalent ester intermediate is on the
timescale of hours. Because of the easy synthesis, we expect that
benzoxazin-4-ones offer the possibility for future optimization of
potency and selectivity against rhomboid proteases.
11. Lemberg MK, Menendez J, Misik A, Garcia M, Koth CM,
Freeman M. Mechanism of intramembrane proteolysis
investigated with purified rhomboid proteases. EMBO J.
2005;24(3): 464-472.
12. Urban S, Wolfe MS. Reconstitution of intramembrane proteolysis
in vitro reveals that pure rhomboid is sufficient for catalysis and
specificity. Proc Natl Acad Sci USA. 2005;102(6): 1883-1888.
13. Wolf EV, Verhelst SH. Inhibitors of rhomboid proteases.
Biochimie. 2016;122: 38-47.
14. Strisovsky K. Rhomboid protease inhibitors: Emerging tools and
future therapeutics. Semin Cell Dev Biol. 2016;60: 52-62.
15. Vinothkumar KR, Strisovsky K, Andreeva A, Christova Y,
Verhelst S, Freeman M. The structural basis for catalysis and
substrate specificity of a rhomboid protease. EMBO J.
2010;29(22): 3797-3809.
16. Vosyka O, Vinothkumar KR, Wolf EV, Brouwer AJ, Liskamp
RM, Verhelst SHL. Activity-based probes for rhomboid proteases
discovered in a mass spectrometry-based assay. Proc Natl Acad
Sci U S A. 2013;110(7): 2472-2477.
Acknowledgments
17. Xue Y, Ha Y. Catalytic mechanism of rhomboid protease GlpG
probed by 3,4-dichloroisocoumarin and diisopropyl
fluorophosphonate. J Biol Chem. 2012;287(5): 3099-3107.
18. Xue Y, Chowdhury S, Liu X, Akiyama Y, Ellman J, Ha Y.
Conformational change in rhomboid protease GlpG induced by
inhibitor binding to its S' subsites. Biochemistry. 2012;51(18):
3723-3731.
We thank Dr. E. Ruijter and Prof. R.V.A. Orru (Vrije
Universiteit Amsterdam) for providing some initial
benzoxazinones for activity screening. We acknowledge funding
by the Chinese Scholarship Council (to JY), H2020 (Marie Curie
Fellowship to MBX), the Deutsche Forschungsgemeinschaft, the
the Ministerium für Innovation, Wissenschaft und Forschung des
Landes Nordrhein-Westfalen, the Senatsverwaltung für
Wirtschaft, Technologie und Forschung des Landes Berlin and
the Bundesministerium für Bildung und Forschung. KS
acknowledges support from EMBO (Installation Grant no. 2329),
Ministry of Education, Youth and Sports of the Czech Republic
(projects no. LK11206 and LO1302), Marie Curie Career
Integration Grant (project no. 304154), and the National
Subvention for Development of Research Organisations (RVO:
61388963) to the Institute of Organic Chemistry and
Biochemistry.
19. Pierrat OA, Strisovsky K, Christova Y, et al. Monocyclic beta-
lactams are selective, mechanism-based inhibitors of rhomboid
intramembrane proteases. ACS Chem Biol. 2011;6(4): 325-335.
20. Vinothkumar KR, Pierrat OA, Large JM, Freeman M. Structure of
rhomboid protease in complex with beta-lactam inhibitors defines
the S2' cavity. Structure. 2013;21(6): 1051-1058.
21. Wolf EV, Zeissler A, Vosyka O, Zeiler E, Sieber S, Verhelst SH.
A new class of rhomboid protease inhibitors discovered by
activity-based fluorescence polarization. PLoS One. 2013;8(8):
e72307.
22. Zoll S, Stanchev S, Began J, et al. Substrate binding and
specificity of rhomboid intramembrane protease revealed by
substrate-peptide complex structures. Embo J. 2014;33(20): 2408-
2421.
References and notes
23. Cho S, Dickey SW, Urban S. Crystal Structures and Inhibition
Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug
Design Implications for Rhomboid Proteolysis. Mol Cell.
2016;61(3): 329-340.
1. Koonin EV, Makarova KS, Rogozin IB, Davidovic L, Letellier
MC, Pellegrini L. The rhomboids: a nearly ubiquitous family of
intramembrane serine proteases that probably evolved by multiple
ancient horizontal gene transfers. Genome Biology. 2003;4(3):
R19.
2. Lemberg MK, Freeman M. Functional and evolutionary
implications of enhanced genomic analysis of rhomboid
intramembrane proteases. Genome Res. 2007;17(11): 1634-1646.
3. Urban S, Lee JR, Freeman M. Drosophila Rhomboid-1 defines a
family of putative intramembrane serine proteases. Cell.
2001;107(2): 173-182.
24. Tichá A, Stanchev S, Vinothkumar KR, et al. General and
Modular Strategy for Designing Potent, Selective, and
Pharmacologically Compliant Inhibitors of Rhomboid Proteases.
Cell Chem Biol. in press.
25. Wolf EV, Zeissler A, Verhelst SH. Inhibitor fingerprinting of
rhomboid proteases by activity-based protein profiling reveals
inhibitor selectivity and rhomboid autoprocessing. ACS Chem
Biol. 2015;10(10): 2325-2333.
26. Powers JC, Asgian JL, Ekici OD, James KE. Irreversible
inhibitors of serine, cysteine, and threonine proteases. Chemical
Reviews. 2002;102(12): 4639-4750.
27. Teshima T, Griffin JC, Powers JC. A new class of heterocyclic
serine protease inhibitors. Inhibition of human leukocyte elastase,
porcine pancreatic elastase, cathepsin G, and bovine chymotrypsin
A alpha with substituted benzoxazinones, quinazolines, and
anthranilates. J Biol Chem. 1982;257(9): 5085-5091.
4. Stevenson LG, Strisovsky K, Clemmer KM, Bhatt S, Freeman M,
Rather PN. Rhomboid protease AarA mediates quorum-sensing in
Providencia stuartii by activating TatA of the twin-arginine
translocase. Proc Natl Acad Sci U S A. 2007;104(3): 1003-1008.
5. Fleig L, Bergbold N, Sahasrabudhe P, Geiger B, Kaltak L,
Lemberg MK. Ubiquitin-dependent intramembrane rhomboid
protease promotes ERAD of membrane proteins. Mol Cell.
2012;47(4): 558-569.

28. Krantz A, Spencer RW, Tam TF, et al. Design and synthesis of
4H-3,1-benzoxazin-4-ones as potent alternate substrate inhibitors
of human leukocyte elastase. J Med Chem. 1990;33(2): 464-479.
29. Gutschow M, Kuerschner L, Neumann U, et al. 2-
(diethylamino)thieno1,3oxazin-4-ones as stable inhibitors of
human leukocyte elastase. J Med Chem. 1999;42(26): 5437-5447.
30. Gutschow M, Neumann U. Inhibition of cathepsin G by 4H-3,1-
benzoxazin-4-ones. Bioorg Med Chem. 1997;5(10): 1935-1942.
31. Neumann U, Schechter NM, Gutschow M. Inhibition of human
chymase by 2-amino-3,1-benzoxazin-4-ones. Bioorg Med Chem.
2001;9(4): 947-954.
32. Hays SJ, Caprathe BW, Gilmore JL, et al. 2-amino-4H-3,1-
benzoxazin-4-ones as inhibitors of C1r serine protease. J Med
Chem. 1998;41(7): 1060-1067.
33. Jarvest RL, Parratt MJ, Debouck CM, et al. Inhibition of HSV-1
protease by benzoxazinones. Bioorganic & Medicinal Chemistry
Letters. 1996;6(20): 2463.
34. Bachovchin DA, Ji T, Li W, et al. Superfamily-wide portrait of
serine hydrolase inhibition achieved by library-versus-library
screening. Proc Natl Acad Sci USA. 2010;107(49): 20941-20946.
35. Ruiz N, Falcone B, Kahne D, Silhavy TJ. Chemical conditionality:
a genetic strategy to probe organelle assembly. Cell. 2005;121(2):
307-317.
36. Ticha A, Stanchev S, Skerle J, et al. Sensitive Versatile
Fluorogenic Transmembrane Peptide Substrates for Rhomboid
Intramembrane Proteases. J Biol Chem. 2017;292(7): 2703-2713.
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