Discovery of Novel 1,3,8-Triazaspiro[4.5]decane Derivatives That Target the c Subunit of F1/FO-Adenosine Triphosphate (ATP) Synthase for the Treatment of Reperfusion Damage in Myocardial Infarction

Article abstract of DOI:10.1021/acs.jmedchem.8b00278

Recent cardiology research studies have reported the role, function, and structure of the mitochondrial permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery, optimization, and structure-activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F₁/F_O-ATP synthase complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac function upon administration during reperfusion. The selected compounds did not show off-target effects at the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content despite interacting with the ATP synthase complex.

Full text of DOI:10.1021/acs.jmedchem.8b00278

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Article
Discovery of novel 1,3,8-triazaspiro[4.5]decane derivatives that
target the c subunit of F1/FO-adenosine triphosphate (ATP) synthase
for the treatment of reperfusion damage in myocardial infarction
Giampaolo Morciano, Delia Preti, Gaia Pedriali, Giorgio Aquila, Sonia Missiroli, Anna Fantinati, Natascia
Caroccia, Salvatore Pacifico, Massimo Bonora, Anna Talarico, Claudia Morganti, Paola Rizzo, Roberto
Ferrari, Mariusz Roman Wieckowski, Gianluca Campo, Carlotta Giorgi, Claudio Trapella, and Paolo Pinton
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00278 • Publication Date (Web): 30 Jul 2018
Downloaded from http://pubs.acs.org on July 31, 2018
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Discovery of novel 1,3,8-triazaspiro[4.5]decane derivatives that target the c subunit of F1/FO-
adenosine triphosphate (ATP) synthase for the treatment of reperfusion damage in myocardial
infarction
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Giampaolo Morciano 1,2,3,4$, Delia Preti 5$, Gaia Pedriali 2,3,4$, Giorgio Aquila 6, Sonia Missiroli 3,4, Anna
Fantinati 5, Natascia Caroccia 3,4, Salvatore Pacifico 5, Massimo Bonora 3,4, Anna Talarico 5, Claudia Morganti
3,4, Paola Rizzo 2,3, Roberto Ferrari 2,3,7, Mariusz R. Wieckowski 8, Gianluca Campo 2,7, Carlotta Giorgi 3,4
Claudio Trapella 4,5*, Paolo Pinton 2,3,4*
,
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1 Maria Pia Hospital, GVM Care & Research, 10132, Torino, Italy
2 Maria Cecilia Hospital, GVM Care & Research, 48033, Cotignola, Ravenna, Italy
3 Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and
Experimental Biology, University of Ferrara, 44121, Ferrara, Italy
4 Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121, Ferrara, Italy
5 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, 44121, Ferrara, Italy
6 Department of Medical Sciences, University of Ferrara, 44121, Ferrara, Italy
7 Cardiovascular Institute, Azienda OspedalieraꢀUniversitaria S. Anna, 44121, Cona, Ferrara, Italy
8 Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02ꢀ
093, Warsaw, Poland
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Abstract
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Recent cardiology research studies have reported the role, function and structure of the mitochondrial
permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of
myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological
approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery,
optimization and structureꢀactivity relationship (SAR) studies of the first smallꢀmolecule mPTP opening
inhibitors based on a 1,3,8ꢀtriazaspiro[4.5]decane scaffold that targets the c subunit of the F1/FOꢀATP synthase
complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects
in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac
function upon administration during reperfusion. The selected compounds did not show offꢀtarget effects at
the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content
despite interacting with the ATP synthase complex.
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Keywords: mPTP/inhibitors/mitochondria/reperfusion damage/apoptosis
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Introduction
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Programmed cell death (PCD) is a physiological, evolutionarilyꢀconserved event that is essential for both
intrauterine and postꢀembryonic development as well as tissue homeostasis through elimination of damaged
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cells . In humans, excessive PCD may be the basis of many diseases, including cardiovascular and
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neurodegenerative disorders. Several types of PCD exist, including mitochondrial permeability transition
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(MPT)ꢀdriven apoptosis , during which the inner mitochondrial membrane (IMM), which is typically highly
impermeable, exhibits increased permeability with a consequent osmotic influx of solutes in the mitochondrial
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matrix followed by loss of structural and functional characteristics of the affected mitochondria . This MPT
state is considered to be mediated by the mitochondrial permeability transition pore complex (PTPC), a
membrane multiprotein platform composed of poreꢀforming part(s) and modulators that contribute to its
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conformational state and thus its mechanism of action . Conditions favoring mitochondrial permeability
transition pore (mPTP) opening include increased intraꢀmitochondrial calcium concentrations ([Ca2+]),
oxidative stress and phosphates as well as high pH levels, all of which occur in ischemiaꢀreperfusionꢀbased
injuries 5, 6
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Although many components/modulators of the mPTP have been discovered in recent years 7, 8, the proteins
involved in the channelꢀforming fraction are still under intensive investigation. In two previous studies 9, 10, we
provided experimental evidence that the c subunit of F1/FOꢀATP synthase plays a pivotal role in mPTP activity
and mPTP formation, demonstrating a strong correlation between the mPTP functional state and c subunit
expression; indeed, depletion of the c subunit reduced channel opening in response to calciumꢀ and oxidative
stressꢀinduced stimuli, whereas its overexpression enhanced mPTP opening 9. Contrary to what was proposed
by another study 11, we previously showed that the MPT is linked to the dissociation of F1/FOꢀATP synthase
dimers. Moreover, a proper cꢀring conformation is required for MPT induction once F1/FOꢀATP synthase
dimers have dissociated 10. Other independent groups have confirmed and extended our observations, defining
the dissociation of F1 from FO during the MPT and recording a leak conductance within the purified c subunit
ring that may form the channel of the mPTP 12, 13, 14, 15. Indeed, major evidence for the soꢀcalled “c-ring
hypothesis” is derived from a comparison between a recent description of a voltageꢀsensitive pore represented
by reconstituted c subunit proteins and lacking cation selectivity 12 and the multiꢀconductance channel studied
in the 80sꢀ90s 16, where cꢀrings may be exposed during F1/FO uncoupling 12. However, these theories are not
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entirely supported by in silico simulations 17, and mathematical calculations may not have considered other
regulatory proteins that facilitate the channel formation process 14.
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While extensive effort has been invested in the synthesis and testing of new mPTP opening inhibitors 18, 19, to
the best of our knowledge, none of these inhibitors were designed to target the c subunit expressed in
eukaryotic cells or for cardioprotective purposes in the treatment of myocardial infarction (MI); however,
Danshensu (DSS), which is isolated from a traditional Chinese herb, is reported to modulate c subunit protein
expression and have cellꢀprotective effects 20.
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Among ATP synthase inhibitors, oligomycin A was identified in 1958 21. Its presence is sensed by a subunit
important for the functional and structural coupling between FO and F1: the oligomycin sensitivityꢀconferring
protein (OSCP). Recently, an important role of OSCP as a site of interaction with cyclophilin (CyP) D, a
matrix protein that can modulate mPTP opening, was discovered. Intriguingly, in the early 2000s, two reports
described the potential of oligomycin A to inhibit mPTP opening induced by selenite 22 and its additive effect
on cyclosporine A (CsA)ꢀdependent mPTP activity 23. Therefore, in addition to the finding that oligomycin A
binds to the cꢀring of yeast mitochondrial ATP synthase as shown in an highꢀresolution crystal structure model
24, this compound increased the protective effects against MPT conferred by F1/FOꢀATP synthase, again
demonstrating its role in membrane permeability.
With the aim of identifying a new pharmacological approach for the treatment of ischemiaꢀreperfusionꢀrelated
damage, in this manuscript, we describe the discovery, optimization and structureꢀactivity relationship (SAR)
studies of the first smallꢀmolecule mPTP inhibitors to target the c subunit of the F1/FOꢀATP synthase complex.
These compounds, which are based on a 1,3,8ꢀtriazaspiro[4.5]decane scaffold, may lead to the development of
new cardio/neuroprotective agents for the clinical management of ischemic events.
Results
Identification of 1-phenyl-8-tosyl-1,3,8-triazaspiro[4.5]decan-4-one (PP11) as a novel small-molecule
inhibitor of the mPTP. Oligomycin A is classified as an mPTP opening inhibitor that targets the c subunit of
F1/FOꢀATP synthase 25; therefore, it was selected as the reference compound for this project. Indeed,
oligomycin A is known to establish several Van der Waals interactions with c subunits, forming a hydrogen
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bond with Glu through a water molecule bridge through which the carboxyl group of a leucine residue is
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also recruited (Figure 1A) 24. In particular, the southern part of oligomycin A (Figure 1B) is closely engaged
by the binding cavity of the cꢀring 24. Considering this information and the need to simplify this natural
compound to facilitate easy, accessible synthesis, we explored the activity of a series of small molecules
mimicking the southern 1,7ꢀdioxaspiro[5.5]undecane moiety of oligomycin A. To test the hypothesis that the
spiro bicyclic fragment may be mandatory for mPTP inhibition, we initially screened a small internal library
of spiro derivatives that somewhat resemble the functional structure of the natural compound. These
preliminary investigations resulted in the identification of 1ꢀphenylꢀ1,3,8ꢀtriazaꢀspiro[4,5]decanꢀ4ꢀone
derivative 1 (PP11, Figure 1C). To assess the efficacy of PP11 in inhibiting Ca2+ꢀmediated mPTP opening, we
first measured its biological activity in HeLa cells by calceinꢀcobalt assay 26. mPTP opening was stimulated by
the addition of the ionophore ionomycin, and the resulting kinetics were compared. As shown in Figure 1D,
the addition of 1 µM ionomycin to cells pretreated with 1 µM PP11 resulted in desensitization of mPTP
opening by approximately 50% as assessed by the slope of the curve after stimulation, which was significantly
less than that observed in vehicleꢀtreated cells. Moreover, PP11 was able to inhibit the mPTP opening in a
manner very similar to that of oligomycin A 24, 27, 28 as a direct derivative but at a tenfold lower concentration.
These data demonstrated that the smallꢀmolecule inhibitor derived from the known compound oligomycin A
was able to efficiently inhibit mPTP opening. Therefore, PP11 was selected as the starting point for further
studies.
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PP11 accumulates selectively in mitochondria. To monitor and better characterize PP11’s biological
activity in cells, we investigated its subcellular localization, focusing on the amount localized within
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mitochondria, the hypothetical site of action. As illustrated in Figure S1AꢀC, fractionation of living cells
treated with vehicle (A) or PP11 (B) resulted in successful isolation of pure mitochondrial, cytosolic and
endoplasmic reticulum (ER) fractions. Samples from each subcellular compartment were analyzed by HPLCꢀ
HRMS to detect the compound. As shown in Figure 2C, PP11 selectively accumulated in the mitochondria
without detectable traces in the other subcellular fractions (Figure 2D, E); however, a residual amount of the
spiro derivative was detectable in the supernatant fraction (Figure 2F). The same investigation performed after
administration of oligomycin A (Figure S1C) revealed a less selective distribution of the compound, with
significant amounts detected in all the isolated subcellular compartments (Figure S2).
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Moreover, to directly monitor target engagement inside cells, we performed a cellular thermal shift assay
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(CETSA)
using a protocol based on ligandꢀinduced thermal stabilization of the target protein, the c
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subunit of FOꢀATP synthase. Thermal shifts at high compound concentrations are known to correlate with
median inhibitory concentration (IC50) values and affinities, as measured by other methods 32, 33; therefore, by
using higher concentrations of PP11, we were able to detect its binding with the c subunit of FOꢀATP synthase
(Figure S1D) between 60ꢀ80°C. Despite the low sensitivity of the assay and an inability to perform any
molecular docking assays, Figure S1D shows that the c subunit protein was thermally stabilized by PP11
pretreatment (redꢀdashed line) and that this effect was absent in other mitochondrial proteins, such as ATP5A
(blackꢀdashed line). To further confirm the putative selective PP11ꢀc subunit binding, a UPLCꢀXEVOꢀTQD
(Waters, UK) mass spectrometry investigation of the c subunit protein overexpressed in and
immunoprecipitated from isolated mitochondria of PP11ꢀpretreated cells was performed. The results from this
experiment are reported in Figure S3 and indicate that a significant amount of PP11 was clearly detectable and
bound to the c subunit.
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Together, these data confirm the ability of PP11 to selectively enter mitochondria and bind the c subunit to
inhibit mPTP opening.
Design, synthesis and biological characterization of PP11 analogs as inhibitors of the mPTP. Encouraged
by the promising biological profile of PP11 and considering the higher chemical stability of the azaꢀspiro
moiety compared with the spiroꢀketal function of oligomycin A, we initiated an SAR investigation of a new
bicyclic core to improve compound potency. We applied a versatile synthetic approach that resulted in a series
of PP11 derivatives that were differentially substituted at the N1 and N8 positions (Figure 3). We first
investigated the effect of replacing the tosyl function at the N8 position of PP11 with a benzyl group
(compound 6a) or with different sulfonamide (8ꢀ10), carboxamide (13, 14), urea (15 and 16) or thiourea (17)
moieties. The results of the biological assay indicated that the compounds with the benzyl group or
(hetero)arylsulfonamide N8 substitutions exhibited the best performance; these compounds were therefore
combined with N1 phenyl rings at the para/meta positions with both electron donating or withdrawing groups
(compounds 6bꢀg, 11, 12). The common synthetic pathway for the preparation of all the investigated final
compounds is depicted in Figure 3. αꢀAminonitriles 3a-g were synthesized by a Strecker reaction from the
commercially available Nꢀbenzylpiperidone 2, which was reacted with (substituted) anilines in the presence of
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trimethyl silyl cyanide (TMSCN). The nitrile group was then hydrolyzed to the corresponding amide function
(4a-g) by treatment with concentrated sulfuric acid. Spirocyclization was then performed with
dimethylformamide dimethyl acetal (DMFꢀDMA), which resulted in the generation of the unsaturated
intermediates 5a-g. The latter derivatives were also screened as mPTP inhibitors to evaluate the effect of the
C2ꢀN3 double bond constraining the 5ꢀmembered ring of the bicyclic template. Subsequently, the
imidazolinone ring was efficiently reduced with NaBH4 to yield the final compounds 6aꢀg. Debenzylation at
the N8 position of 6a and 6b was performed via hydrogenation under Pd/C catalysis and yielded 7a and 7b,
respectively, which were employed as key intermediates for the selective functionalization of the N8 position.
Specifically, the sulfonamide derivatives 1 and 8-12 were obtained by treatment of 7a-b with appropriate
sulfonyl chlorides, while N8 acetylation (13) or benzoylation (14) was achieved by standard treatment with
acetic anhydride or benzoylchloride, respectively. In addition, the urea derivatives 15ꢀ16 and the thiourea 17
were prepared from 7a and the properly substituted isocyanates or isothiocyanates.
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To investigate the effectiveness of the new, synthesized PP11 analogs against mPTP opening, we used the
mitochondrial swelling assay as previously described 34; this protocol is faster than microscopy analysis for the
screening of multiple compounds. PP11 significantly inhibited mPTP opening (Figures 1D and 4B). To
demonstrate specificity in measuring Ca2+ꢀinduced mPTP opening, we added two positive controls, CsA and
ruthenium red (RR), a known mPTP inhibitor and a mitochondrial calcium uptake blocker, respectively.
Freshly isolated mouse liver mitochondria were first evaluated to ensure high purity (Figure 4A) and were then
stimulated with 500 µM Ca2+, and absorbance at 540 nm, at which a decrease was indicative of swelling, was
monitored for 10 minutes (Figure 4B, C). Under these conditions, mitochondria treated with vehicle exhibited
marked membrane swelling (red bar), which was significantly prevented by pretreatment with 1 µM CsA and
5 µM RR (green and blue bars, respectively). As shown in Figure 4B, most of the small molecules showed
good inhibitory potential compared to the control mitochondrial sample (red bar). In particular, compounds 5c,
6g and 10 were significantly more potent than PP11 in inhibiting mPTP opening. Additional details are shown
in panel 4D, including the mean percentages of mPTP inhibition. Figure 4E shows that 1 ꢁM was the lowest
dose with the best effectiveness in inhibiting mitochondrial swelling for each selected compound.
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Biological profile of the most promising compounds. Overall, mPTP assays revealed a set of three very
potent inhibitors (5c, 6g and 10) with the greatest effects on reducing mitochondrial swelling (Figure 4B)
compared to PP11.
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Given the crucial role of the c subunit in ATP synthase assembly and cellular energy production as a
component of the membrane rotor, the basal mitochondrial ATP content and agonistꢀinduced ATP production
were monitored by the luciferinꢀluciferase assay 35. Treatment of living cells with 1 µM PP11 slightly but
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significantly depleted mitochondrial ATP levels in resting conditions (Figure 5A); similarly, the relative
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amount of ATP remained low even upon histamineꢀinduced calcium uptake
(Figure 5B). The PP11
derivatives 5c, 6g and 10 synthesized with various specific chemical groups, such as electron withdrawing
atoms (Cl) or moieties (CF3) in the aromatic substituents in positions 1 and 8 of the spirodecane scaffold,
allowed us to successfully overcome this drawback and stepꢀlimiting factor. Indeed, use of the 6g and 10
inhibitors did not affect ATP levels, but treatment with compound 5c induced a small decrease in the basal
mitochondrial ATP content, which was completely recovered upon Ca2+ꢀinduced ATP generation (Figure 5A,
B).
To identify possible side effects of the compounds on living cells that may preclude their use in future studies,
we evaluated other important mitochondrial parameters. Mitochondrial membrane potential (MMP) and
calcium homeostasis are critical factors for the maintenance of physiological functions in cells, including
proper respiratory chain function and a wide range of intracellular signaling pathways, respectively. As shown
in Figure 5C and D, pretreatment of cells with the 5c, 6g and 10 inhibitors did not affect basal MMP or
mitochondrial calcium uptake. Moreover, mitochondrial morphology, the number of mitochondria in cells and
their network volume were unchanged (Figure 5E).
In addition, the results previously described were further confirmed by the 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) assay, which showed that treatment of living cells with micromolar
concentrations of 5c, 6g and 10 for 24 h and 48 h did not induce significant MTT reduction compared with
treatment with vehicle (Figure 5F). Only prolonged (72 h) treatment slightly but significantly decreased cell
viability in vitro (Figure 5F).
The permeability transition pore has been postulated to play an important role in cytochrome c (CytC) release
37; therefore, we explored the ability of the selected compounds to prevent CytC release from mitochondrial
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cristae. To correlate mPTP desensitization with apoptotic cell death prevention, we detected the amount of
CytC in the cristae of mitochondrial fractions from the mitochondrial swelling assay upon calcium stimulation.
Pretreatment with 5c, 6g and 10 prevented CytC release as assessed by immunoblot analysis (Figure 5G).
Taken together, these findings demonstrated that inhibition of mPTP opening may inhibit CytC release to the
cytosol and consequently prevent apoptotic cell death (Figure 6E).
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Next, to understand the molecular mechanism by which the selected compounds inhibit mPTP complex
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activity, we used a previously tested proximity ligation assay (PLA)ꢀbased procedure to verify the F1/FOꢀ
ATP synthase dimerization grade in living cells. Vehicleꢀtreated cells exhibited a considerable amount of redꢀ
dotted staining that colocalized with the mitochondrial protein translocase of outer mitochondrial membrane
20 (TOMM20). We showed the F1/FOꢀATP synthase dimerization status via a PLA intensity profile upon
ionomycin administration alone or in the presence of smallꢀmolecule inhibitors. As recently published by our
lab, the MPT was accompanied by a significant decrease in the PLA signal following 1ꢀµM ionomycin
treatment 10; however, the MPT did not occur with the same intensity in the presence of 1 µM 5c, 6g and 10
(Figure 5H). These findings suggest that mPTP desensitization by the synthesized compounds is correlated
with stabilization of F1/FOꢀATP synthase dimers.
Cardioprotective effect of compound 10 in a model of reperfusion injury. Considering the ability of these
inhibitors to desensitize mPTP opening (Figure 4B) by stabilizing ATP synthase dimers (Figure 5H) and
prevent CytC release (Figure 5G) without affecting longꢀterm cell viability (Figure 5F) and the mitochondrial
ATP content (Figure 5A, B), we investigated the effects of compound 10 in an animal model of cardiac
reperfusion injury. We isolated beating rat hearts and placed them in a Langendorff system, which was
continuously perfused with KrebsꢀHenseleit buffer (KHB) bubbled with oxygen at 37°C. The ex vivo protocol
included stabilization of the heart for 20 min, and then, retrograde perfusion was progressively stopped to
induce 30 min of global ischemia followed by 1 hour of reperfusion (Figure 6A). After stabilization, the left
ventricular developed pressure (LVDP) was 89.8±3 mmHg in the I/R vehicle group. Following reperfusion,
the LVDP decreased to 66±5 mmHg, with a mean reduction of 36±9%, indicating successful induction of
ischemia (as previously reported 38). No difference in LVDP was identified among the experimental groups
during stabilization. As indicated in panel 6A, compound 10 was administered in the reperfusion phase during
the first 10 minutes of reflow. The dose of 10 µM of compound 10 was selected based on previous
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experiments to identify the highest dose that could be perfused in the heart without toxicity (data not shown).
In isolated hearts, perfusion with a constant volume of derivative 10 resulted in decreases in coronary
perfusion pressure (CPP) (ꢀ17.5±3.4%) and endꢀdiastolic pressure (EDP) (ꢀ72±9.86%) (Figure 6B, D) with an
increase in LVDP (+36.4±3.9%) (Figure 6C), indicating reduced diastolic stiffness, vasoconstriction and
deterioration of myocardial performance, respectively. At the end of the procedure, cell death was analyzed in
the hearts by terminal deoxynucleotidyl transferase dUTP nickꢀend labeling (TUNEL) assays. In the
I/R+vehicle group, 64% of the cardiomyocytes were TUNELꢀpositive; however, the number of TUNELꢀ
positive cardiomyocytes was significantly reduced in the presence of spiro derivative 10 (Figure 6E).
These findings confirmed the ability of cꢀringꢀtargeting agents to inhibit mPTP opening and to protect against
cell death in a cardiovascular model.
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Discussion and Conclusions
Myocardial infarction is an ischemic heart disease in which mPTP opening is widely accepted as a crucial step
in the development of myocardial damage, which is better known as ischemiaꢀreperfusion injury (IRI) 6. Since
up to 50% of the final infarct size is due to IRI, targeting the mPTP complex may be a valuable
pharmacological adjunct to reduce infarct size. Currently, although satisfying results have been achieved in in
vitro and in vivo animal models, manipulation of the PTPC and, more generally, mitochondrial targeting do
not appear to influence the mortality of affected patients, but they reduce hospital readmission for heart failure
39, 40
39. Instead, drugs with a broadꢀspectrum mechanism of action are currently utilized in clinical practice
.
The apparent lack of translational value among mitochondrialꢀtargeting drugs can be ascribed first to limited
knowledge regarding the exact molecular structure of mPTP; therefore, the appropriate mitochondrial target
possibly has not yet been considered. Second, the multifactorial nature of MI should be considered, and the
adoption of combined strategies targeting multiple intracellular signaling pathways is always recommended 41.
9, 10
Based on our previous studies on the role of the cꢀring in mPTP opening
and to validate a new
pharmacological approach for the treatment of ischemiaꢀreperfusionꢀrelated damage, in this manuscript, we
describe the discovery, optimization and SAR studies of the first smallꢀmolecule mPTP opening inhibitors that
target the c subunit of the F1/FOꢀATP synthase complex.
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To the best of our knowledge, this is the first attempt to inhibit the mPTP by targeting the eukaryotic c subunit
of ATP synthase using novel small molecules for therapeutic purposes. Indeed, treatment during reperfusion
with one of the most promising screened compounds (10) showed beneficial effects in an ex vivo model of MI,
with a reduction of apoptotic cell death upon IRI (Figure 6E). The decreases in CPP and EDP (Figure 6B, D)
with an increase in LVDP (Figure 6C) in the isolated heart reflected significant reductions in diastolic
stiffness, vasoconstriction and deterioration of myocardial performance. Moreover, the ex vivo beneficial
effects of compound 10 together with strong inhibition of mPTP opening by the selected compounds in vitro
can be achieved without altering mitochondrial parameters (Figure 5AꢀE) or shortꢀterm toxicity in living cells.
Indeed, these compounds weakly but significantly disrupted cell viability only with 72 h of treatment (Figure
5F).
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The compounds were derived using the scaffold of the known c subunit inhibitor oligomycin A; we first
identified PP11, a compound with a strong desensitization effect on mPTP opening (Figure 1D) and an
inhibitory effect comparable to that of oligomycin A, but it is functionally active at a tenfold lower
concentration (Figure 1D). The increased performance at lower concentrations can be ascribed to its better
localization to the mitochondrial compartment (Figure 2), the putative site of action, as assessed by HPLCꢀ
HRMS. Indeed, PP11 accumulates exclusively in the pure mitochondrial fraction (Figure 2 and Figure S1)
without detectable traces in other subcellular compartments. In contrast, although oligomycin A is present in
the mitochondrial compartment where it exerts known biological effects, a significant amount diffuses into the
cytoplasm and ER organelles (Figure S2), likely lowering its potency and conferring a better drugꢀlike profile
9, 10, 14
to PP11. In addition, given its effectiveness in targeting the c subunit for mPTP inhibition
and the
exclusive mitochondrial localization of its ligand, PP11 (and derivatives) use led to fewer side effects
compared to other known mPTP inhibitors, such as CsA. Indeed, CsA offꢀtarget effects 42, 43 are derived from a
42, 43
diffuse intracellular localization pattern characterized by cytosolic
prompting research on alternative methods to target the mPTP.
and nuclear interactions 44, thus
Criticism could arise from the use of putative ATP synthase disruptors as ligands of the membrane rotor
components, as confirmed in this work by the suboptimal effect of PP11, a preliminary derivative of
oligomycin A, on the mitochondrial ATP content 45. Indeed, although PP11 is considered a good mPTP
opening inhibitor (Figure 1D), it has a slight deleterious effect on the production of basal ATP (Figure 5A);
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however, this effect is reduced upon Ca2+ꢀdependent stimulation in the mitochondria (Figure 5B). When
designing PP11 derivatives, we tried to evaluate different chemical modifications at several positions of the
parent compound to identify possible structural determinants that can abolish any undesired effects. Among
the most promising compounds screened in Figure 4B, 6g and 10 did not affect the basal mitochondrial ATP
content; only compound 5c promoted a small decrease in the basal mitochondrial ATP content, which was
completely recovered upon Ca2+ꢀinduced ATP generation. A possible explanation for such experimental
evidence may be faster reversibility of the interaction of 6g and 10 with the postulated cellular target compared
to PP11.
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The main findings of this work are consistent with those of our previously published studies and those of
independent groups 12ꢀ14, 46, confirming the importance of the roles of i) the cꢀring as a core component in
mPTP activity and ii) the dimer versus monomer transition of ATP synthase linked to the mPTP and the
resulting apoptotic cell death. Indeed, a PLAꢀdedicated assay suggested dimer stabilization upon Ca2+ꢀ
triggered mPTP opening when cells were pretreated with the 5c, 6g and 10 compounds (Figure 5H) as a
molecular mechanism by which these inhibitors may exert antiꢀapoptotic effects.
We are aware that our data appear to disagree with the hypothesis from Walker JE’s group on the persistence
of the MPT in a cell clone lacking the c subunit of human ATP synthase 47; however, as a very exhaustive
commentary on this discrepancy explains 48, in the absence of the primary mechanisms in mPTP opening (e.g.,
c subunit expression), the MPT can presumably occur through other pathways involving misfolded proteins of
the mitochondrial membrane 48, 49
.
In conclusion, we propose smallꢀmolecule c subunit inhibitors as a new pharmacological approach for the
treatment of IRI. Future perspectives will be oriented towards the study of ATP synthase conformational
changes (if detectable) in terms of modulation of its dynamic assembly into supercomplexes upon inhibitor
treatment (for instance, by using native gels) and understanding the exact binding sites between inhibitors and
mitochondrial protein(s). Photoaffinity labeling of the most promising compounds identified here may lead to
useful tools that will facilitate future binding site deconvolution studies advancing structureꢀbased drug design
approaches in this field.
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Experimental Section
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Chemistry. Reaction progress and product mixtures were monitored by thinꢀlayer chromatography (TLC) on
silica gel (precoated F254 MachereyꢀNagel plates) and visualized with a UV lamp (254ꢀnm light source). The
organic solutions from extractions were dried over anhydrous sodium sulfate. Chromatography was performed
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on Merck 230ꢀ400 mesh silica gel or using Isolera One (Biotage Sweden). H, 13C, DEPT, bidimensional
(gCOSY) and heterocorrelated (gHMQC, gHMBC) NMR spectra were recorded on a VARIAN Mercury Plus
400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) using the peak of
deuterated solvents as an internal standard, and coupling constants (J) are reported in Hertz. Splitting patterns
are designed as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and b, broad. Melting points for
purified products were determined in a glass capillary on a Stuart Scientific electrothermal apparatus SMP3
and are uncorrected. Mass spectra were recorded by an ESI single quadrupole mass spectrometer Waters ZQ
2000 (Waters Instruments UK). For analytical controls, Beckmann System Gold 168 HPLC was used with an
LC column Kinetex 5ꢀµm EVO C18 100 Å (250 X 4.6 mm) and a variable wavelength UV detector fixed to
220 nm. The analysis was conducted using two solutions, A and B, containing 100:0.1 H2O:TFA and
40:60:0.1 H2O:CH3CN:TFA, respectively, with a gradient elution of 0ꢀ50% solution B over 30 minutes. The
purity of all compounds was determined by HPLC and was greater than 95%.
LCꢀHRMS analysis of crude mitochondria and cytosol of PP11ꢀtreated cells and final compounds was
performed with an ESIꢀQꢀTOF Nano HPLCꢀCHIP Cube® Agilent 6520 instrument (Agilent Technologies
USA) using a linear gradient (0.4 µL/min) from 0% solvent A (97% water/3% acetonitrile/0.1% formic acid)
to 80% solvent B (97% acetonitrile/3% water/0.1% formic acid) over 10 minutes and from 80% to 5% solvent
B over 5 minutes using a Zorbax C18 Column (43 mm X 75 µm, 5 µm) equipped with an enrichment column
(4 mm, 40 nL). The UPLCꢀMS analysis was performed in an Acquity UPLC equipped with a triple quadrupole
mass spectrometer XEVOꢀTQD (Waters UK). Chromatographic separation was carried out using a BHE C18
column (50 mm X 2.1 mm i.d. 1.8ꢁm) from Waters (Waters, Milford, USA) heated at 40°C. The mobile phase
consisted of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (Solvent B). An
8ꢀminute gradient elution at 0.3 mL/min was performed as follows: from 100% solvent A to 80% solvent A
over 4 minutes, from 80% A to 20% A over 2 minutes, and from 20% A to 0% A over 2 minutes. Mass
spectrometric detection was carried out using an electrospray interface (ESI) operated in positive ionization
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mode with multiple reaction monitoring (MRM) for the PP11 analyte. Nitrogen was used as a desolvation gas
at a 650ꢀL/h flow rate with the desolvation temperature set at 200°C and the source temperature set at 150°C.
The collision gas (argon) flow was set at 0.1 mL/min. The capillary voltage was set at 4 kV; the collision
energy and con voltage were optimized to maximize the signal corresponding to the major transition observed
in the MS/MS spectra following fragmentation of the [M+H]+ ion corresponding to the PP11 molecule.
Calcein-cobalt assay. HeLa cells were pretreated with DMSO (vehicle), 10 ꢁM oligomycin A, 1 ꢁM CsA or 1
ꢁM PP11 and then loaded with calcein acetoxymethyl ester and Co2+ as previously described 26. Staining
solution was added to the cells for 15 minutes at 37°C in a 5% CO2 atmosphere. Image acquisitions were
performed with a motorized Olympus IX81ꢀZDC inverted microscope with a 40×/1.30ꢀN.A. UPlanFLN oilꢀ
immersion objective and Cell MT20E xenon lamp. Ionomycin (1 µM) was administered 30 seconds after the
beginning of the experiment to induce mPTP opening.
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Mitochondrial isolation and swelling assay. Mitochondria were isolated by conventional procedures
involving differential centrifugation. Freshly excised SV129 mouse livers were washed and then homogenized
in medium containing 50 mmol/L TrisꢀHCl, 25.67 g/L sucrose, and 40.98 g/L DꢀMannitol (pH 7.4)
supplemented with 0.5 mmol/L EGTA and 5 g/L bovine serum albumin (BSA). The homogenate was then
transferred to microcentrifuge tubes and centrifuged at 0.8 rcf for 5 minutes for at least 2 cycles; the
supernatants were collected, and the pellets were discarded. Subsequently, the sample was centrifuged at 10.0
rcf for 10 minutes to separate the mitochondrial fraction; the pellet was resuspended and ground in a looseꢀ
fitting glass Potter Elvehjem homogenizer for a fixed number of times. Samples were then centrifuged at 10.0
rcf for 10 minutes, and the pellet (mitochondria) was resuspended in 1 mL of Respiration Buffer (pH 7.4, Trisꢀ
HCl 50 mmol/L; 25.67 g/L sucrose; 40.98 g/L DꢀMannitol) supplemented with 5 mmol/L succinate.
Mitochondria were quantified and diluted to a final concentration of 1 mg/mL for each mitochondrial swelling
assay to monitor the changes in absorbance at 540 nm as previously described 34. Incubations with small
molecules were carried out at 25°C, and mPTP opening was induced by the addition of 500 µM Ca2+.
Ex vivo model. I/R was studied ex vivo using the Langendorff model with minor modifications 10. In brief,
upon euthanasia, the hearts of Wistar rats weighing 270ꢀ280 g at inclusion into the study were rapidly excised,
immediately arrested in iceꢀcold KHB (pH 7.4; 4°C), cannulated and retrograde perfused at a fixedꢀflow rate
(11 mL/min) through the aorta with warm KHB (37°C) bubbled with 95% O2 and 5% CO2. Upon removal of
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the left atrial appendage, a latex fluidꢀfilled balloon was inserted into the left ventricular chamber through the
atrium to obtain an isovolumetrically beating preparation and connected to a pressure transducer (APT300,
HugoꢀSachs, Grünstraße, Germany) by a fluidꢀfilled polyethylene catheter to monitor performance. An
additional transducer above the aortic cannula monitored the CPP. At the start of each experiment, the fluid in
the balloon was increased incrementally to achieve a constant EDP of 4±1 mmHg. The LVDP was then
measured. The LVDP, EDP and CPP were continuously recorded using a programmable acquisition system
(HSE Isoheart Software for Isolated Heart, HugoꢀSachs, Grünstraße, Germany).
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Proximity ligation assays. Cells were fixed in 4% PFA for 10 min at 37°C, washed in PBS, placed in a jar
containing 1 mmol/L EDTA buffer (pH 8.0) for 20 min at 100°C (to improve epitopeꢀantibody binding) and
then placed at room temperature for an additional 10 min. Then, the cells were permeabilized with 0.05%
Triton Xꢀ100 for 10 min at 37°C, and unspecific binding sites were blocked by incubating the cells in 0.05%
Triton Xꢀ100 supplemented with 2% BSA for 45 min at 37°C. Upon overnight incubation with ATP5Hꢀ
specific antibodies that were previously conjugated to + or ꢀ PLA oligonucleotide probes per the instructions
of the Duolink In Situ® Probemaker kits, detection was performed as follows. A ligationꢀligase solution was
added to each sample for 30 min at 37°C followed by 2 washes for 2 min each with Duolink In Situ® Wash
Buffer A; an amplificationꢀpolymerase solution was added and incubated for 100 min at 37°C followed by 2
washes for 10 min each with 1x Duolink In Situ® Wash Buffer B and 1 wash for 1 min with 0.01x Duolink In
Situ® Wash Buffer B. Cells were fixed again in 4% PFA for 10 min at 37°C and then blocked in 0.05% Triton
Xꢀ100 supplemented with 2% BSA for 10 min at 37°C. Finally, the samples were incubated overnight with
primary antiꢀTOM20 antibody, and the next day, the primary antibodies were revealed by incubation with the
appropriate goat antiꢀrabbit Alexa Fluor 488® secondary antibodies. The slides were then stained with
Duolink In Situ® Detection Reagent Red and mounted using DAPIꢀcontaining Duolink In Situ® Mounting
Medium. Protein proximity was evaluated on an Axiovert 200M fluorescence microscope equipped with a
40X water immersion objective (N.A. 1.2, from Carl Zeiss Microscopy, LLC) as a function of mitochondrialꢀ
localized red signal intensity.
Statistical analysis. The statistical method included oneꢀway ANOVA with multiple comparisons performed
by GraphPad Prism. P values are reported in the figure legends.
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ASSOCIATED CONTENTS
Supporting Information
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Supplementary Methods; General procedures for the preparation of compounds 3a-g, 4a-g, 5a-g, 6a-g, 7a-b, 1
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and 8-17; H, 13C, DEPT NMR spectra of compounds 1, 8-17, and 6a-g; HRMS spectra of compounds 5a-g,
6a-g, 1, and 8-17; Figures S1, S2 and S3 (.PDF)
Molecular formula strings for the final compounds 5a-g, 1, 8-17 (.CSV).
AUTHOR INFORMATION
Corresponding Authors
*Paolo Pinton
Email: paolo.pinton@unife.it
*Claudio Trapella
Email: claudio.trapella@unife.it
Author Contributions
$ G.M., D.P. and G.P. equally contributed to this manuscript.
ACKNOWLEDGMENTS
PP is grateful to Camilla degli Scrovegni for continuous support. PP is supported by the Italian Ministry of
Education, University and Research, the Italian Ministry of Health, Telethon (GGP15219/B), the Italian
Association for Cancer Research (AIRC: IGꢀ18624), and by local funds from the University of Ferrara. CG is
supported by local funds from the University of Ferrara, the Italian Association for Cancer Research (AIRC:
IGꢀ19803), the Italian Ministry of Health, and by a Fondazione Cariplo grant. GM would like to acknowledge
Pierangelo M. for his support. DP is supported by the fund FIR (Fondo per l’Incentivazione alla Ricerca) of
the University of Ferrara (Year: 2016; code: FIR1679708). MRW is supported by the Polish National Science
Centre (UMOꢀ2014/15/B/NZ1/00490).
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ABBREVIATIONS USED
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CETSA: cellular thermal shift assay, CsA: cyclosporine A, CPP: coronary perfusion pressure, CytC:
cytochrome c, EDP: endꢀdiastolic pressure, IMM: inner mitochondrial membrane, KRB: KrebsꢀHenseleit
buffer, LVDP: left ventricular developed pressure, MI: myocardial infarction, MMP: mitochondrial membrane
potential, MPT: mitochondrial permeability transition, PCD: programmed cell death, PLA: proximity ligation
assay, PTPC: permeability transition pore complex, RR: ruthenium red, SAR: structureꢀactivity relationship,
TUNEL: terminal deoxynucleotidyl transferase dUTP nickꢀend labeling.
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Conflict of interest: The authors declare no competing financial interest.
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paradigm of pore structure and function? FEBS Lett 2002, 512, 1ꢀ7.
Figure legends
Figure 1. Discovery of PP11 as the first small-molecule inhibitor of the mPTP with a spirocenter
template that mimics the spiroketal moiety of oligomycin A.
A: Graphical representation of the key amino acid residues defining the binding pocket of the known mPTP
inhibitor oligomycin A to the cꢀring.
B: Structure of the southern portion of oligomycin A showing a 1,7ꢀdioxaspiro[5.5]undecan moiety that is
closely involved in the interaction with the cꢀring.
C: Structure of PP11, a novel smallꢀmolecule inhibitor of the mPTP with a 1,3,8ꢀtriazaꢀspiro[4.5]decanꢀ4ꢀone
template.
D: Calceinꢀcobalt assay in living cells pretreated with vehicle, oligomycin A, PP11 and CsA. mPTP opening
was stimulated by ionomycin administration, and representative kinetics data are reported.
***: p value<0.001; ****: p value<0.0001. CsA: cyclosporine A.
Figure 2. PP11 selectively localizes in mitochondria.
Subcellular localization of PP11 was investigated via HPLCꢀHRMS analysis of samples from different
cellular compartments before and after treatment with the compound.
A: The HPLCꢀHRMS profile of PP11 analyzed in physiological solution revealed broad band absorption (7ꢀ
8.5 min) with maximum absorption at a retention time of 8.03 min (top panel). The blue region of the HPLC
spectrum corresponds to a unique mass peak at [M+H]+ m/z of 386.15339 (bottom panel, [M+H]+ calculated
for PP11 386.1533).
B: The HPLC profile of the mitochondrial fraction isolated from untreated cells revealed a relatively clean
zone in the detection region of PP11 (from 7 to 8.5 min, top panel). The gray band on the HPLC spectrum
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corresponds to the mass spectra area between the retention time of 7 to 8.5 minutes enlarged in the bottom
panel.
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C: The HPLCꢀHRMS profile of the mitochondrial fraction isolated from cells pretreated with PP11 clearly
indicated the presence of the PP11 compound with an HPLC band at a retention time of 8.2 min (top panel).
The red area of the signal corresponds to the enlarged TIC signal for the mass peaks (bottom panel), among
which the [M+H]+ m/z of 386.15339 indicated that the presence of PP11 was not detectable in the
corresponding untreated fraction (panel B).
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D: No detectable amount of PP11 was found by HPLCꢀHRMS in the cytosolic fraction of PP11ꢀtreated cells.
E: The HPLC profile of the ER fraction isolated from PP11ꢀtreated cells revealed a relatively clean zone in the
detection region of PP11 (from 7 to 8.5 min, top panel). The gray band of the HPLC spectrum corresponds to
enlargement of the TIC signals of mass peaks detected by HRMS analysis (bottom panel).
F: The HPLCꢀHRMS profile of the supernatant fraction isolated from cells pretreated with PP11 indicated the
presence of the compound.
Figure 3. Synthesis of PP11 and related spiro bicyclic derivatives.
Figure 4. Screening of PP11 analogs.
A: Immunoblot detection of GAPDH (cytosolic marker) and TIM23 (mitochondrial marker) proteins in the
homogenate (H) of the whole liver and in the mitochondrial (Mito) fraction.
B: Mitochondrial swelling assay for the screening of compounds in freshly isolated mouse liver mitochondria.
Data were obtained by recording changes in absorbance (540 nm), and then, the data were converted into
percentages. Black bar: untreated and unstimulated mitochondria; red bar: untreated mitochondria stimulated
with 500 µM Ca2+; green bar: pretreatment with 1 µM CsA and stimulation with 500 µM Ca2+; blue bar:
pretreatment with 5 µM RR and stimulation with 500 µM Ca2+; cyan bar: pretreatment with 1 µM PP11 and
stimulation with 500 µM Ca2+; and white bars: pretreatment with 1 µM PP11 analogs and stimulation with
500 µM Ca2+. Red arrows indicate the three selected compounds.
C: Kinetics and statistical data from the mitochondrial swelling assay for the vehicle, CsA, RR, 5c, 6g and 10
experimental conditions.
D: Mean percentages of mPTP inhibition with the tested compounds.
E: Doseꢀresponses for the 5c, 6g and 10 compounds.
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Oneꢀway ANOVA was used for statistical analysis; *: p value<0.05; **: p value<0.01; CsA: cyclosporine A;
RR: ruthenium red.
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Figure 5. Biological profile of the most promising compounds.
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A: Mitochondrial ATP content in living cells at resting conditions using the luciferaseꢀluciferin method.
B: Mitochondrial ATP generation upon agonistꢀinduced Ca2+ uptake in living cells using the luciferaseꢀ
luciferin method. His indicates 100 µM histamine, the agonist used to induce Ca2+ꢀdependent ATP production.
C: TMRM fluorescence as an index of mitochondrial membrane potential changes in living cells.
D: Mitochondrial calcium uptake in living cells using a mitochondriallyꢀtargeted aequorin probe; histograms
of statistical and representative kinetics data; 100 µM histamine (His) addition is indicated.
E: Evaluation of mitochondrial morphology parameters using an mtCHERRY probe.
F: MTT assay in living cells to evaluate the toxicity of compounds at 24 h, 48 h and 72 h and at different
concentrations. Black bar: cells treated with vehicle; and green bar: 1 ꢁM compound; yellow bar: 5 ꢁM; red
bar: 10 ꢁM. The data were standardized to the vehicle condition.
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G: Immunoblot detection of cytochrome c in both mitochondrial cristae and respiration buffer from the
mitochondrial swelling assay. HSP60 was used as a mitochondrial marker, and GAPDH was used as a
cytosolic marker.
H: PLAꢀbased assay for assessment of the ATP synthase dimerization status. Blue: nuclei by DAPI detection;
green: mitochondria by TOM20 detection; red spots: ATP5H detection as described in the methods.
Representative images are shown below.
a.u.: arbitrary units; CytC: cytochrome c; n.s: not significant; *: p value<0.05; **: p value<0.01; and ****: p
value<0.0001; IB: immunoblotting.
Figure 6. Beneficial effects of compound 10 in a cardiac reperfusion injury model.
A: Diagram depicting the standard Langendorff ex vivo protocol.
B: Coronary perfusion pressure recording of a rat heart in the stabilization, ischemia and reperfusion phases.
Compound 10 is used at a concentration of 10 µM.
C: Left ventricular peak developed pressure recording of a rat heart in the stabilization, ischemia and
reperfusion phases.
D: Endꢀdiastolic pressure recording of a rat heart in the stabilization, ischemia and reperfusion phases.
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E: TUNEL assay for apoptosis evaluation under experimental conditions and representative images. Red:
nuclei detected by TOꢀPRO; green: apoptotic nuclei detected by TUNEL enzyme.
**: p value<0.01; and ****: p value<0.0001. The green bar in panels AꢀD indicates vehicle or compound
administration time.
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