Design and synthesis of tailored human caseinolytic protease P inhibitors

Article abstract of DOI:10.1039/c8cc05265d

Human caseinolytic protease P (hClpP) is important for degradation of misfolded proteins in the mitochondrial unfolded protein response. We here introduce tailored hClpP inhibitors that utilize a steric discrimination in their core naphthofuran scaffold to selectively address the human enzyme. This novel inhibitor generation exhibited superior activity compared to previously introduced beta-lactones, optimized for bacterial ClpP. Further insights into the bioactivity and binding to cellular targets were obtained via chemical proteomics as well as proliferation- and migration studies in cancer cells.

Full text of DOI:10.1039/c8cc05265d

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Design and synthesis of tailored human caseinolytic protease P
inhibitors†
Thomas F. Gronauera, Melanie M. Mandlb, Markus Lakemeyera, Mathias W. Hackla, Martina
Received 00th January 20xx,
Accepted 00th January 20xx
Meßnerb, Vadim S. Korotkova, Johanna Pachmayrb and Stephan A. Siebera*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Human caseinolytic protease P (hClpP) is important for degradation
of misfolded proteins in the mitochondrial unfolded protein
response. We here introduce tailored hClpP inhibitors that utilize a
steric discrimination in their core naphthofuran scaffold to
selectively address the human enzyme. This novel inhibitor
generation exhibited superior activity compared to previously
introduced beta-lactones, optimized for bacterial ClpP. Further
insights into the bioactivity and binding to cellular targets were
obtained via chemical proteomics as well as proliferation- and
migration studies in cancer cells.
Mitochondria are essential for cellular energy production and
represent a main source of reactive oxygen species (ROS).1
Stress caused by ROS results in accumulation of damaged
proteins which block essential functions and induce the
mitochondrial unfolded protein response (mtUPR).2 Human
caseinolytic protease P (hClpP) represents a major mediator of
cellular homeostasis in mtUPR by degradation of damaged
proteins into peptides.3,4 These peptides are transported across
the inner mitochondrial membrane to activate stress-
associated transcription factors.5,6 hClpP is a serine protease
expressed as inactive heptamers which assemble into an active
tetradecamer by binding to hClpX.7–9 Mice bearing a ClpP
knockout suffer from hearing loss, infertility and growth
defects. These effects are paralleled in humans by recessive
mutations of hClpP causing the Perrault syndrome.10 While
hClpP is not essential for viability of normal cells, a recent study
demonstrated its upregulation in patients with acute myeloid
leukemia (AML).11 Rapidly dividing cancer cells are exposed to
multiple mitochondrial stresses and require an efficient UPR to
Fig. 1 (a) Structures of previous ClpP inhibitors: beta-lactone D3 and phenyl ester
AV167. (b) Structures of phenyl ester derivatives prepared in this study. (c)
Structure of optimized probe TG42 used for labeling. (d) hClpP peptidase activity
assay (hClpP concentration 1ꢀµM, fluorogenic substrate: Suc-L-Y-AMC (200ꢀµM)).
Experiments were carried out in six technical replicates from two independent
experiments (mean ± standard deviation). (e) Intact protein mass spectrometry
results showing high modification (78%) of hClpP by phenyl ester AV167 (1ꢀµM
hClpP with 10-fold excess of AV167, 1 h incubation). (f) Results of apparent IC50
determination (§ 35% residual activity, see Fig. S3, ESI†).
a.Center for integrated Protein Science Munich, Department of Chemistry,
Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany
* E-mail: stephan.sieber@tum.de
b.Paracelsus Medizinische Privatuniversität Salzburg, Strubergasse 21, 5020
Salzburg, Austria
maintain cell homeostasis. Accordingly, silencing of hClpP in
† Electronic Supplementary Information (ESI) available: Methods, Fig. S1-S7, Tables
T1, T2, Schemes S1-S4, Table S1 (Excel file). Proteomic data are available via AML cell lines increased mitochondrial ROS levels and thereby
ProteomeXchange with identifier PXD010277. See DOI: 10.1039/x0xx00000x
attenuated cell viability. hClpP thus represents a promising anti-
This journal is © The Royal Society of Chemistry 20xx
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an 8-methoxynaphthofuran moiety
(TG11) Voierw AdrtiicflfeeOrnelinnet
substituted benzofurans (TG24
-
TG27)DOreI:s1u0l.t1e0d39/Cin8CCin0a5c2t6i5vDe
compounds (Fig. 1b, Scheme S1, ESI†). Thus, modifications at a
different site were subsequently explored by introducing a
bromine substituent (TG43) and an alkyne tag at 2-position
(TG42) (Fig. 1b, c, Scheme 1). Synthesis of these derivatives
started with Pechmann reaction of 2-naphthol and ethyl
4-chloroacetoacetate to the corresponding β-naphthocoumarin
Scheme 1 Synthesis of compound TG42: (a) ethyl 4-chloroacetoacetate (1.14ꢀeq.),
H2SO4 (98%), 0ꢀ°C, 3ꢀh, 92%; (b) 6ꢀM NaOHaq, 80ꢀ°C, 20ꢀh, 96%; (c) H2SO4 (5.30ꢀeq.),
MeOH, 65ꢀ°C, 5ꢀh, 91%; (d) NBS (1.09ꢀeq.), CHCl3/CH3CN (1:1), -ꢀ8ꢀ°C, 1ꢀh, 90%; (e)
1. Pd(PPh3)4 (1.8ꢀmol%), CuI (0.27ꢀeq.), TMS-acetylene (2.00ꢀeq.), NEt3 (1.10ꢀeq.),
THF, rt, 20ꢀh, 86%, 2. 1ꢀM LiOHaq, THF/H2O (3:1), rt, 24ꢀh, 93%; (f) EDC ∙ HCl (1.50ꢀeq.),
DMAP (0.50ꢀeq.), 1,3,4 oxadiazolyl phenol (TG05, 1.20ꢀeq.), CH2Cl2, rt, 18ꢀh, 49%.
(
TG04
)
which rearranged under basic conditions to
TG06). Ester protection and
naphthofuranylacetic acid
(
subsequent electrophilic bromination with NBS led to the
precursor (TG38) for Sonogashira coupling with TMS-protected
acetylene. Deprotection of the carboxylic ester and
TMS-protecting group could be achieved in one step with
aqueous LiOH solution. Esterification with oxadiazolyl phenol
cancer target.12 Previous experiments with beta-lactone
inhibitors, originally discovered and optimized for bacterial
ClpPs,13 induced death of leukemia cells and even reduced their
growth in xenografted mice.11 We recently discovered a next
generation of bacterial ClpP inhibitors based on a phenyl ester
scaffold.14 Of note, one molecule, AV167, displayed superior
inhibitory activity against hClpP exceeding that of previous
beta-lactones (Fig. 1a).
We here systematically dissect the AV167 core structure and
identify novel inhibitors with superior potency for hClpP
inhibition compared to the gold standard lactones. The
inhibitors showed anti-proliferation, anti-migratory and
apoptotic effects against cancer cells. Corresponding activity-
based protein profiling (ABPP) suggested that multiple targets
are likely responsible for these effects. Given the clinical need
for novel hClpP inhibitors our scaffolds provide a unique starting
point to further fine tune selectivity.
(TG05), derived from methyl paraben via two step synthesis,
resulted in compound TG42 (Scheme 1). Satisfyingly, probe
TG42 and its bromo precursor TG43 exhibited elevated potency
compared to AV167 (1.54ꢀµM) with apparent IC50s of 0.39 and
0.57ꢀµM, respectively (Fig. 1b, f, S3, ESI†). Intact MS revealed
about 50ꢀ% modification to be sufficient to achieve full
inhibition (Fig. S1, ESI†). As AV167 bears an activated ester
moiety we systematically tested the inhibition profiles of
various ester derivatives (Fig. 1b, d). As expected alcohols
exhibiting electron-donating properties such as phenol and p-
methoxyphenol (TG28
while derivatives with electron withdrawing groups including p-
nitro- and p-methylsulfonylphenol (TG30 TG51 TG52 TG54
, TG29) turned out to be poor inhibitors
,
,
,
)
showed inhibition profiles comparable to the parent
p-oxadiazolylphenol of AV167. All compounds with activated
ester moieties and naphthofuran substituents in 2-position
In a previous high-throughput screen against bacterial ClpP,
AV167
,
bearing
a
characteristic naphthofuran moiety,
(TG42, TG43, TG53) displayed potent IC50s ranging from 0.33 -
protruded as the best hClpP inhibitor out of six phenyl ester
hits.14 We thus selected this scaffold for further optimization.
First, covalent inhibition of hClpP was validated for AV167 and
compared to one of the best previous beta-lactone ClpP
inhibitors (D3) (Fig. 1a). As quantitative acylation kinetics,
represented in kobs/[I] values, could not be resolved for AV167
due to its rapid binding velocity (data not shown), we
performed two alternative assays assessing the reactivity and
potency. ClpP inhibitors have been previously characterized by
their inhibition of ClpP peptidase activity at three different
concentrations (100, 10 and 1ꢀµM).14–16 AV167 significantly
reduced hClpP turnover already at 10ꢀµM and intact mass-
spectrometric (MS) analysis of the modified enzyme revealed
about 80ꢀ% binding (Fig. 1d, e, S1, ESI†). Contrary, D3 only
achieved 65ꢀ% inhibition at the highest concentration tested
(Fig. 1d). This trend is also reflected by corresponding apparent
IC50 values determined at identical incubation times. D3
displayed a 4.5-fold higher value compared to AV167 and again
retained 35ꢀ% residual enzyme activity at saturating
concentrations indicating restricted binding (Fig. 1f, S3, ESI†).
Based on these promising properties, AV167 was selected for
structure-activity relationship (SAR) studies to i) dissect the
structure for essential enzyme recognition motifs and ii) identify
possible sites for functionalization with a benign tag for target
identification via ABPP.17–19 Synthesis of five derivatives bearing
0.57ꢀµM, indicating sub-stoichiometric inhibition of hClpP (used
at 1ꢀµM in this assay) (Fig. 1f, Scheme S1-S5, ESI†). Contrary to
AV167, these compounds showed only minor inhibition of
S. aureus ClpP, demonstrating a crucial discrimination of species
selectivity caused by substituents attached to the naphthofuran
ring at 2-position (Fig. 1f, S2, ESI†).
To assess the ability of these compounds to block proteolytic
activity hClpP was activated via an acyldepsipeptide derived
chaperone mimic.20,21 Binding of this activator fragment to
hydrophobic pockets induces pore opening and digest of FITC
casein. While beta-lactone D3 showed no activity, AV167
, TG42
and TG52 TG54 efficiently blocked proteolysis (Fig. 2a). This
-
result was further corroborated by a second proteolysis assay in
presence of the ClpX chaperone.22,23 Escherichia coli ClpX
(EcClpX) forms a functionally active complex with hClpP and is
preferred in these assays compared to the largely
uncharacterized human ClpX.7 For enzyme activity assessment,
degradation of green fluorescent protein (GFP) tagged with an
EcClpX recognition sequence was monitored.23 All new
derivatives exhibited strong inhibition down to 1ꢀµM
concentration (Fig. 2b), a remarkable result given the previous
difficulties of chemical inhibitors to overcome conformational
2 | J. Name., 2012, 00, 1-3
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Fig. 2 (a) FITC-casein degradation assay with hClpP, acyldepsipeptide fragment and inhibitors at three different concentrations (1.0ꢀµM hClpP, 10ꢀµM acyldepsipeptide fragment,
2ꢀµM FITC-casein. Each data-set represents results from six replicates obtained from two independent experiments. (b) hClpP GFP protease assay (0.2ꢀμM hClpP in the presence
of 0.4ꢀμM EcClpX and 0.4ꢀμM GFP-SsrA) with compounds as indicated. Each data-set represents results from three replicates (mean ± standard deviation) (c) ABPP-labeling of
recombinantly expressed hClpP in E. coli Rosetta2 expression strains. SDS-PAGE of soluble fraction shows distinct bands at ≈ 32ꢀkDa. For Coomassie stained SDS-PAGE gels see
Fig. S7 ESI†. (d) Schematic representation of the ABPP workflow: Cells are incubated with electrophilic compounds which react with reactive amino acids of target proteins.
Following cell lysis, click reaction to affinity tag bitoin-N3 and enrichment on avidin beads, proteins are tryptically digested and analyzed by LC-MS/MS. (e) Volcano plot
representation of gel-free quantitative ABPP experiment with TG42 in Jurkat cells (20ꢀµM TG42, 1ꢀh labeling) depicting enrichment and significance of enrichment from four
technical replicates. Color codes show connection to certain cell compartment (GOCC) and biological processes (GOBP) by gene ontology analysis. Two sample student’s t-test
was performed by comparison of labeled group against DMSO as single control group with Benjamini-Hochberg FDR correction set to 0.05. Cut-off lines were set at a minimum
log2 change of 2 and a minimum p-value of 0.05.
rearrangements within ClpP upon chaperone or small molecule despite its superior in vitro potency and labeling performance
activator binding.22 Reactivity and selectivity of all alkynylated compared to previous inhibitors, further optimization of TG42
compounds was tested against recombinant hClpP expressed in is required to enhance cellular selectivity for mitochondrial
E. coli cells. Intact cells were incubated with the probes, lysed, hClpP. Nevertheless, given the crucial role of hClpP and several
clicked to rhodamine azide24,25 and analyzed via fluorescent of the identified targets such as Reticulon28 and Protein LYRIC29
SDS-PAGE. hClpP was clearly labeled by TG42, TG52 and TG53 in apoptosis (Fig. 2e, S5, ESI†) we investigated the biological
with TG42 bearing the best signal to noise ratio (Fig. 2c). As consequences of TG42 in Huh7 cancer cells and demonstrated
hClpP is located in mitochondria, a specific inhibitor needs to concentration-dependent cell death with induction of apoptosis
permeate two membrane barriers for target engagement. To (Fig. 3a, b). Cancer cell migration could also be significantly
test if TG42 is able to address mitochondrial hClpP in living reduced at 30ꢀµM TG42 treatment (Fig. 3c).
cancer cells, we performed a gel-free whole proteome LC- Selective eradication of cancer cells is an important task in drug
MS/MS analysis (Fig. 2d). For this intact Jurkat and Huh7 cells, development to reduce side effects of chemotherapy. hClpP is
shown to express hClpP by western blot (Fig. S4, ESI†), were a promising target in this endeavor lacking inhibitors for
incubated with the probes, lysed, clicked to biotin azide,24,25 therapeutic applications. AV167 represented a starting point to
enriched via binding to avidin beads and finally released by achieve this goal bearing a characteristic curved naphthofuran
tryptic digest.14 In parallel the same procedure was carried out moiety suitable to durably inhibit ClpP peptidolytic as well as
with DMSO-treated cells as control. Peptides were subjected to proteolytic activities. Importantly, substitution at 2-position of
high-resolution LC-MS/MS and multiple biological replicates the naphthofuran moiety not only enhanced in vitro potency
were analyzed via label-free quantification (Fig. 2d).26 TG42 but also represented a crucial signature motif differentiating
significantly enriched hClpP in Jurkat cells, however, a large between binding into human or bacterial ClpP isoforms. TG42
number of other prominent targets were additionally obtained, exhibited superior inhibition and selectivity towards hClpP
suggesting that the electrophilic probe is of either limited compared to a reference beta-lactone. Further fine-tuning of
selectivity in human proteomes or has restricted access to the inhibitor scaffold is needed to enhance its cellular
mitochondrial compartments (Fig. 2e, S5, ESI†). In contrast, selectivity.
labeling of Huh7 cells (Fig. S6, ESI†) and Jurkat cells27 with This work was supported by the Deutsche Forschungs-
lactone D3 resulted in no enrichment of hClpP at all. Thus, gemeinschaft SI1096/8-1 (ClpP) and CIPSM.
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Büchel, T. Adler, M. Hrabé de Angelis, D. H. Busch,VAie.wZAertlilc,leAO. nSl.ine
DOI: 10.1039/C8CC05265D
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Fig. 3 (a) hClpP inhibitors reduce cell viability in Huh7 cells after treatment for 48ꢀh
and 72ꢀh (CellTiterBlue® assay). Diagrams depict results of nine technical replicates
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± standard deviation). (b) ClpP
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treatment. Apoptosis was measured by flow cytometry using Nicoletti Assay (cell
permeabilization, propidium iodide staining, gating of subG1 population, * p<0.05,
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inhibitors decrease Huh7 cell migration after incubation for 16ꢀh with TG42 and
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Conflicts of interest
There are no conflicts to declare.
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Table of contents
To expedite functional studies of human ClpP we introduce tailored small molecule
inhibitors. These compounds are active against the proteolytic ClpXP complex. Target
identification elucidates anti-proliferative effects against cancer cells.