New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting

Article abstract of DOI:10.1016/j.bmcl.2018.05.031

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.

Full text of DOI:10.1016/j.bmcl.2018.05.031

Accepted Manuscript
New TRAP1 and Hsp90 chaperone inhibitors with cationic components: pre-
liminary studies on mitochondrial targeting
R. Rondanin, G. Lettini, P. Oliva, R. Baruchello, C. Costantini, C. Trapella, D.
Simoni, T. Bernardi, L. Sisinni, M. Pietrafesa, G. Ponterini, M.P. Costi, T.
Vignudelli, R. Luciani, D.S. Matassa, F. Esposito, M. Landriscina
PII:
S0960-894X(18)30432-3
https://doi.org/10.1016/j.bmcl.2018.05.031
BMCL 25848
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 January 2018
4 May 2018
14 May 2018
Please cite this article as: Rondanin, R., Lettini, G., Oliva, P., Baruchello, R., Costantini, C., Trapella, C., Simoni,
D., Bernardi, T., Sisinni, L., Pietrafesa, M., Ponterini, G., Costi, M.P., Vignudelli, T., Luciani, R., Matassa, D.S.,
Esposito, F., Landriscina, M., New TRAP1 and Hsp90 chaperone inhibitors with cationic components: preliminary
studies on mitochondrial targeting, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.05.031
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New TRAP1 and Hsp90 chaperone inhibitors with cationic components: preliminary
studies on mitochondrial targeting
a*
b
a
a
a
a
a
Rondanin R., Lettini G., Oliva P., Baruchello R., Costantini C., Trapella C., Simoni D., Bernardi
a
b
b
c
c
c
c
d
T., Sisinni L., Pietrafesa M., Ponterini G., Costi M.P., Vignudelli T., Luciani R., Matassa D.S.,
d*
b,e*
Esposito F., Landriscina M.
a
Dep. of Chemical and Pharmaceutical Sciences, University of Ferrara, Italy
b
Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata,
Rionero in Vulture, Italy
c
Dep. of Life Sciences, University of Modena and Reggio Emilia, Italy
d
Dep. of Molecular Medicine and Medical Biotechnology, University of Napoli Federico II, Italy
e
Dep. of Medical and Surgical Sciences, University of Foggia, Italy
Corresponding authors:
Prof. Franca Esposito, Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli
Studi di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy. Tel.: +39 081 7463145; fax: +39 081
7
464359; E-mail: franca.esposito@unina.it.
Prof. Matteo Landriscina, Dipartimento di Scienze Mediche e Chirurgiche, Università degli Studi di
Foggia, Viale Pinto, 1, 71100 Foggia, Italy. Tel.: +39 0881 736241; fax: +39 0972 726482; E-mail:
matteo.landriscina@unifg.it
Keywords. TRAP1 inhibitors; Mitochondrial targeting; Anticancer activity; Lipophylic cations;
Apoptosis.
Abstract.
TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural
similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial
TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding
the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent
cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as
vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to
accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a
guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell
growth and in inducing apoptotic cell death in colon carcinoma cells.
TRAP1 (Tumor Necrosis Factor-Associated Protein 1, a mitochondrial paralog of Hsp90 chaperone
family, also known as Hsp75) is a component of a mitochondrial pathway, selectively up-regulated in
tumor cells and responsible for maintenance of mitochondrial integrity, thus favoring cell survival.
Studies demonstrated that mitochondrial TRAP1, together with Hsp90, interacts with cyclophilin D (Cyp
D), a regulator of permeability transition pore, and antagonizes the Cyp D-dependent apoptotic cascade,
1
likely via a protein (re)folding mechanism . In a context of cancer cells with TRAP1 overexpression, its

silencing was demonstrated to cause sudden growth inhibition and apoptosis, and this correlated with
altered mitochondrial function and modified protein expression, thus suggesting that this pathway may
2
represent a novel molecular target for anticancer therapy . In this perspective, an attractive idea has been
recently proposed regarding the delivery of TRAP1 inhibitors inside mitochondria, as a tool to conjugate
3
anticancer activity together with selectivity toward cancer cells with high mitochondrial TRAP1 levels .
TRAP1 is also involved in protein homeostasis through an extramitochondrial quality control pathway
involving the proteasome regulatory particle TBP7, and this function is relevant for TRAP1 antiapoptotic
4
-6
role . Thus, several mechanisms are involved in multifaceted roles of TRAP1 in adaptive processes of
7
-10
cancer cells
.
Several attempts have been made in recent years to increase the efficacy of anticancer therapy through a
specific subcellular compartmentalization delivery of drugs. In this field, mitochondria have been
considered an attractive target for their relevant metabolic roles altered in cancer models. An appropriate
1
1-16
and specific drug-delivery system is required to design mitochondria-targeted drugs.
Indeed, many
structures able to direct pharmacological compounds to mitochondria share the presence of a basic
component or a permanent cationic lipophilic group, in order to cross over membranes by exploiting a
very favorable electric gradient. For example, groups such as polyamines, protonated at physiological pH,
have been successfully employed to carry and internalize biologically active compounds through
1
7
mitochondrial membranes into the organelle . These structures, indeed, not only allow mitochondrial
membrane crossing, but also favor specific accumulation in the organelle. In the field of Hsp90 inhibitors,
the so-called Gamitrinibs, a family of geldanamycin derivatives (17-AAG) linked to cyclic guanidines or
triphenylphosphonium groups, have been proposed as a novel class of mitochondria-directed
1
8,19,3
TRAP1/Hsp90 inhibitors
. Our study took advantage from the availability of a TRAP1 crystal
2
0
structure recently provided by Sung and co-workers . In fact, a further issue, besides the specific
mitochondrial delivery, deals with the selectivity of TRAP1/Hsp90 ATPase inhibition. To this aim we
searched for novel putative TRAP1 antagonists and, in this study, a number of derivatives, as compounds
21
1
and 2 in Figure 1, already known to be non- or partly ionizable Hsp90 inhibitors, were selected since
they were predicted to interfere also with TRAP1 activity, based on sequence homology between Hsp90
and TRAP1 structures. Furthermore, the Hsp90 inhibitors 1 and 2 were modified to enhance their
accumulation into mitochondria.

N
N
CONHEt
O
CONHEt
O
O
O
HN
HN
HO
OH
HO
OH
N
1
OCH3
2
O
N
CONHEt
O
O
HN
SPACER
Charge(s) +
HO
OH
Figure 1. Known Hsp90 inhibitors and general structure of potential mitochondrial targeting derivatives.
The feasibility of the project was first investigated by preparing a small explorative sample of a three-
portion structure so equipped:
1
) a common 3,4 isoxazole diamide structure, as seen in Figure 1, derived from known compounds active
as Hsp90 inhibitors. The nitrogen at position 4 of isoxazole is particularly suitable to link a multitude of
different appendages;
2
3
) a cationic head, either as a permanent ion or as an ionizable group at physiologic pH;
) a spacer between the portions above described, that can be chosen of various lengths.
In particular, a series of triphenylphosphonium as well as of pyridinium salts, and a guanidinium or a
polyamine appendage have been considered as cationic heads to be linked to a common intermediate (4,
2
1.
Scheme 1), and were obtained as previously reported. Conjugate derivatives were comparatively
evaluated by testing inhibition of recombinant TRAP1 ATPase activity, as well as accumulation in
mitochondria, cell viability and induction of apoptosis in colorectal carcinoma cells.
The synthetic procedure is depicted in Schemes 1 and 2.

HOOC
ClOC
HOOC
ClOC
COOCH3
COOCH3
COOCH3
COOCH3
3
9
i
i
N
CONHEt
NH2
O
BnO
OBn
4
ii
ii
N
N
CONHEt
O
CONHEt
O
O
O
HN
HN
COOCH3
BnO
OBn
BnO
OBn
6
5
1
0
iii
iii
COOCH3
N
N
CONHEt
O
CONHEt
O
O
O
HN
HN
OBn
COOH
BnO
OBn
BnO
6
1
1
6
iv
iv
COOH
N
O
N
CONHEt
O
CONHEt
O
O
NH2
NH
HN
OBn
HN
O
HN
OBn
BnO
BnO
6
1
2
7
v
v
NH
O
NH2
HN
N
N
CONHEt
O
CONHEt
O
O
O
NH2
NH
HN
HN
OH
13
HN
O
HO
OH
HO
6
8
NH
O
NH2
HN
Scheme 1. Synthesis of guanidine bearing derivatives 8 and 13. Reagents and conditions: i): oxalyl
chloride, DMF, CH Cl , 4 h, rt; ii): CH Cl , TEA, 16 h, rt; iii): MeOH, 1N NaOH, H O, 24 h, 70 °C; iv):
2
2
2
2
2
DMF, CDI, 1 h, rt, CH N · HCl, 16 h, rt; v): 1 M BCl , CH Cl , - 78 °C, 1 h.
5
3
3
2
2

The guanidine vehicle was introduced using two linkers of different length: the terephthalic acid
monomethyl ester and the 7-(4-methoxycarbonylphenyl)-heptanoic acid were converted into acyl
chlorides and reacted with intermediate 4, obtaining compounds 5 and 10, respectively. Ester hydrolysis
and coupling with guanidine (obtained from the hydrochloride treated with potassium tert-butoxide) gave
intermediates 7 and 12, finally deprotected with BCl to yield compounds 8 and 13.
3
Similarly, compound 4 was reacted with hexanedioic acid chloride monoethyl ester to obtain compound
2
2
1
5, then hydrolized and coupled with the protected amine 17 (obtained as described in literature ). The
deprotection of compound 18 gave the final polyamine derivative 19.
Finally, the permanent cationic derivatives were synthesized reacting compound 4 with 6-bromohexanoic
acid chloride, then deprotecting 21 with BCl and displacing bromine of 22 with either pyridine (as
3
refluxing solvent) or triphenylphosphine in refluxing 1,4-dioxane to give compounds 23 and 24,
respectively.

N
CONHEt
NH2
O
BnO
OBn
4
O
O
4
O
i
vi
Br
5
EtO
OH
Cl
1
4
20
N
N
CONHEt
O
CONHEt
O
O
O
O
HN
HN
Br
5
4
BnO
OBn
BnO
OBn
OEt
21
1
5
vii
ii
N
O
CONHEt
O
N
CONHEt
O
O
O
HN
HN
OBn
Br
5
HO
OH
4
BnO
OH
2
2
1
6
viii
ix
Boc
N
H N
Boc
2
N
N
H
N
O
N
Boc
CONHEt
O
1
7
CONHEt
O
O
iii
Br
Ph
Ph
P
Ph
HN
HN
N
5
N
CONHEt
HO
OH
HO
OH
O
5
O
O
23
24
Br
HN
OBn
4
BnO
HN
Boc
N
1
8
iv, v
N
Boc
H
N
N
Boc
CONHEt
O
O
O
HN
4
HO
OH
HN
1
9
N
H
H
N
NH2
Scheme 2. Synthesis of derivatives with polyamine (19), pyridinium (23) and phosphonium (24)
appendages. Reagents and conditions: i): oxalyl chloride, DMF, CH Cl , 4 h, rt, TEA, CH Cl , 16 h, rt;
2
2
2
2
ii): THF, LiOH · H O, 2 h, 70 °C; iii): CDI, THF, 24 h, rt; iv): TFA, CH Cl , 1 h, rt; v): 1 M BCl ,
2
2
2
3
CH Cl , - 78 °C, 1 h; vi): CH Cl , TEA, 12 h, rt; vii): CH Cl , 1 M BCl , - 78 °C; viii): Pyridine, 18 h, rf;
2
2
2
2
2
2
3
ix): Ph P, 1,4-dioxane, 150 °C, 40 min.
3

All newly-synthetized compounds were tested for inhibition of recombinant TRAP1 ATPase activity and
cell viability, accumulation in mitochondria, and induction of apoptosis in colon carcinoma HCT116
cells.
Based on the structural homology between TRAP1 and Hsp90, the newly synthesized compounds were
tested in comparison with the isoxazole-amide 1, recently emerged from our studies on Hsp90
21
inhibitors. Two well-known potent Hsp90 inhibitors, i.e., AUY922 and Hsp990, obtained from
Novartis, were also tested since they were also proposed as potentially acting on the TRAP1 ATPase
2
3,24
domain.
In preliminary experiments, the ability of the reported compounds to accumulate inside mitochondria was
investigated. To this purpose, a mass spectrometric analysis on separated mitochondrial and cytosolic
fractions purified from colorectal carcinoma HCT116 cells exposed to 1 M of each agent for 12 h was
used as a qualitative technology to assess the intracellular distribution of our compounds. We found
molecular peaks corresponding to compounds 8, 13, 19 and 24, but not to the reference compounds 1 and
Hsp990, in the mitochondrial fractions of HCT116 cells. On the other hand, in the experimental
conditions adopted, we did not find any of the tested compounds in the cytosolic compartment.
Additional experiments are however necessary to quantitatively address the issue of the intracellular
distribution of these compounds.
Data about ATPase activity inhibition of recombinant TRAP1 are reported in Table 1. The guanidine 8,
with an IC of 500nM, showed an inhibitory activity comparable to that of the reference inhibitor 1 (IC
5
0
50
=
400 nM), as well as of Hsp990 and AUY922. The guanidine analogue with a longer linker 13 resulted
six times less active, whereas a more substantial drop of activity was observed upon testing the polyamine
9 (5M), and the pyridinium 23 (10M). The phosphonium analogue 24 was almost inactive (25M),
similarly to the reference inhibitor 2.
1

Table 1. Compound IC s upon recombinant TRAP1
5
0
ATPase inhibition assay and MTT viability assay in
HCT116 cells.
TRAP1 ATPase
MTT assay
Compound
IC (M)
IC (M)*
5
0
50
NVP-AUY922
0.5
Nd
Hsp990
0.2
0.4
20
0.5
3
Nd
1
2
8
0.87±0.08
Nd
1.32±0.11
6.17±0.56
26.7±2.1
7.86±1.04
5.12±0.68
1
1
2
2
3
9
3
4
5
10
25
*
Nd: not done
Then we focused our attention on the possible dual activity of new compounds toward both TRAP1 and
Hsp90 proteins and measured the affinities (K ) of our best new TRAP1 inhibitor, compound 8, towards
d
TRAP1 and Hsp90. To this aim, we used compound 8 and geldanamycin-FITC (G-FITC) in a
fluorometric competitive binding assay (see refs. 25 and 26 and the details in Supplementary data). First,
we determined the dissociation equilibrium constants for the G-FITC/protein complexes using
fluorescence anisotropy as the observable (results are shown in Figure S1 of the Supplementary data). We
obtained K s 37 (±14) nM with Hsp90 and 48 (±20) nM with TRAP1. The first value is consistent with
d
2
5,27
the 23 and 30 nM K previously reported for the geldanamycin/Hsp90 complex.
Thus, G-FITC has
d
essentially the same affinity for the two proteins.
The decrease in the emission anisotropy of the FITC probe measured following increasing amounts of
compound 8 to the G-FITC and either Hsp90 or TRAP1 protein solutions is shown in Figure 2. The
anisotropy values calculated according to eq. 2 in the Supplementary data are shown as solid lines. The
best fitting K s were 1.5 (± 0.5) and 10 (± 4) nM for the complexes of compound 8 with Hsp90 and
d
TRAP1, respectively. The corresponding changes in standard free energy (T = 26 °C) are -50.5 (± 1.0)
-1
and -45.8 (± 1.2) kJ mol . To compare our result for the 8/Hsp90 complex with previous IC data
5
0
obtained using a very similar fluorescence polarization approach with several other structurally closely
2
1
related 3,4-isoxazolediamides, but lacking the guanidine appendage, we recalculated the anisotropy r,
using the above K value (1.5 nM), as a function of the concentration of the incoming ligand at the
d
concentrations of G-FITC (5 nM) and Hsp90 (30 nM) employed in that work. The IC50 value thus
obtained (see Figure S2 in the Supplementary data) is 21 nM and lies well within the range of IC values
5
0

reported for 29 substituted 3,4-isoxazolediamides. Overall, the K and G° values found for the two
d
complexes indicate similar affinities of compound 8 for Hsp90 and TRAP1, with only a slight preference
for the former protein.
Figure 2. Emission anisotropy of the FITC probe in competitive binding experiments of compound 8 and
geldanamycin-FITC (G-FITC) to proteins Hsp90 and TRAP1. Top: [Hsp90]=70 nM, [G-FITC]=12 nM.
Bottom: [TRAP1]=100 nM, [G-FITC]=35 nM.  =470 nm, emiss=525 nm. The solid curves represent
exc
anisotropies calculated according to eq. 2 in Supplementary data.
In subsequent experiments, all compounds were tested for their potential to inhibit cell proliferation, by
MTT assay, upon 24 h exposure of colon carcinoma HCT116 cells to increasing concentrations of the
drugs whose IC values are reported in Table 1. The MTT assay data showed that the guanidine 8 is
5
0
active in the low micromolar range (IC 1.32 µM) with a cell growth inhibitory activity comparable to
5
0
that of the reference 1 (IC 0.87µM), as previously demonstrated for other well-known mitochondria-
5
0
1
8
delivered compounds . By contrast, the phosphonium 24 (IC 5.12 µM), the guanidine analogue 13
5
0
(
IC 6.17 µM), the pyridinium 23 (IC 7.86 µM) and the polyamine 19 (IC 26.7 µM) demonstrated
50 50 50
lower or minimal activity.

Induction of apoptosis was evaluated in HCT116 cells upon 24 h treatment with increasing concentrations
of reference and modified compounds (Figure 3). Compared to the reference compounds 1 and Hsp990
that are active starting from 0.1 M, compound 8 showed cytotoxic activity starting from 1 M. By
contrast, compounds 23 and 24 induced apoptosis only in HCT116 cell exposed to 10 M, polyamine 19
induced minimal and not dose-dependent apoptosis.
Figure 3. Percentage of apoptosis in HCT116 cells exposed for 24 hours to the indicated concentrations
of new and reference compounds.
These results suggest that the isoxazole diamide scaffold, important to obtain active inhibitors of the
Hsp90 chaperone family, can be easily modified in order to insert a cationic or a strongly basic group.
These appendages are well known as mitochondrial targeting tools and are potentially useful to improve
selectivity of therapeutic agents. We identified derivatized compounds in the mitochondrial fractions of
1
8
tumor cells. Notably, compared to other known compounds , the guanidine 8 showed an interesting
biological activity being able to inhibit TRAP1 ATPase in the nanomolar range and cell viability in the
low micromolar range and to induce apoptosis. Despite sharing the same pharmacophoric core as 8, other
synthesized derivatives appear to be less active, both concerning cytotoxicity and TRAP1 ATPase
inhibition. The guanidine 13, with a longer linker than 8, is the closest in the overall comparison of
activities, being about six times less potent in both TRAP1 ATPase and MTT assays. Moreover, while the
pyridinium 23 and the phosphonium analogue 24 have a similar cell proliferation IC as 13, and a higher
5
0

apoptotic effect at 10 M, their TRAP1 ATPase inhibition is lower. On the contrary, polyamine 19
demonstrated a negligible cell proliferation IC50 coupled with a fair ATPase activity. These findings
suggest that other mechanisms, e.g. a poorer kinetics of cell membrane crossing, might influence their
biological activity. Compound 8 was indeed our best identified inhibitor: it entered the mitochondria,
while reference compound 1 did not, and maintained similar TRAP1 inhibitory potency. The recalculated
IC50 value of 21 nM of this compound lies well within the values obtained for closely related 3,4-
isoxazolediamides, but lacking the guanidine appendage and indicates that introduction of the latter did
not produce any major change in the affinity for the Hsp90 protein. Compound 8 also featured high
affinities for both Hsp90 and TRAP1 proteins, with K s 1.5 and 10 nM, respectively. The slightly higher
d
-
affinity for the first protein corresponds to a G° difference for compound/protein binding of 4.7 kJ mol
1
,
only 10% of the G° values. Future studies will address the issue of how increase the specificity of
these novel mitochondria-directed agents toward TRAP1. Indeed, these novel compounds, besides having
been selected based on the prediction to interfere with TRAP1 ATPase domain, are still dual
Hsp90/TRAP1 inhibitors. Thus, in the perspective to design agents suitable for clinical use, it is
mandatory that future inhibitors improve their selectivity toward single paralogs to enhance the anticancer
activity and reduce the off-target toxicity. In this direction, the recent description of TRAP1 crystal
2
0
structure represents an important advancement for future drug design strategies.
In summary, these preliminary results showed that different appendages can be easily bound to a Hsp90
inhibitor core with the aim to obtain a mitochondrial drug accumulation. Initial biological results suggest
that guanidine-based compounds are the most interesting potential hit structures, being able to interfere
with TRAP1 function and to inhibit proliferation of colon carcinoma cells. However, further studies are
needed to improve selectivity toward TRAP1.
Aknowledgments.
This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (Grant IG13128
to ML and FE)
A. Supplementary data. Supplementary data associated with this article can be found, in the online
version, at
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3

Highlights
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


Analogues of known Hsp90 inhibitors with a permanent or ionizable cationic head.
Inhibition of TRAP1 ATPase activity and detection in mitochondrial cell fraction.
Best apoptotic and growth inhibition activities of guanidine bearing derivative.
Similar affinities of most active compound for Hsp90 and TRAP1 binding.

*Graphical Abstract
N
CONHEt
O
O
TRAP1 ATPase IC50
00 nm
N
CONHEt
O
5
O
HN
OH
HO
HCT116 MTT IC50
.3 µM
HN
1
SPACER
Charge(s)
HO
OH
NH
O
NH2
HN