Lipoyl-Homotaurine Derivative (ADM-12) Reverts Oxaliplatin-Induced Neuropathy and Reduces Cancer Cells Malignancy by Inhibiting Carbonic Anhydrase IX (CAIX)

Article abstract of DOI:10.1021/acs.jmedchem.7b01237

Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM-12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells' aggressiveness.

Full text of DOI:10.1021/acs.jmedchem.7b01237

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Article
Lipoyl-Homotaurine Derivative (ADM_12) Reverts
Oxaliplatin-induced Neuropathy and Reduces Cancer Cells
Malignancy by Inhibiting Carbonic Anhydrase IX (CAIX).
Marco Fragai, Giuseppina Comito, Lorenzo Di Cesare Mannelli, Roberta Gualdani, Vito Calderone,
Alexandra Louka, Barbara Richichi, Oscar Francesconi, Andrea Angeli, Alessio Nocentini, Paola Gratteri,
Paola Chiarugi, Carla Ghelardini, Francesco Tadini-Buoninsegni, Claudiu T Supuran, and Cristina Nativi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01237 • Publication Date (Web): 19 Oct 2017
Downloaded from http://pubs.acs.org on October 22, 2017
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LipoylꢀHomotaurine Derivative (ADM_12) Reverts
Oxaliplatinꢀinduced Neuropathy and Reduces Cancer
Cells Malignancy by Inhibiting Carbonic Anhydrase IX
(CAIX).
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Marco Fragai, ¥, ∫ Giuseppina Comito,§ Lorenzo Di Cesare Mannelli,# Roberta Gualdani, ¥ Vito
Calderone, ¥,∫ Alexandra Louka,∫ Barbara Richichi, ¥ Oscar Francesconi, ¥ Andrea Angeli,
#Alessio Nocentini, # Paola Gratteri, # Paola Chiarugi, § Carla Ghelardini,d Francesco Tadiniꢀ
Buoninsegni, ¥ Claudiu T. Supuran#* and Cristina Nativi¥,^*
¥ Department of Chemistry, University of Florence, via della Lastruccia 3ꢀ13, 50019 Sesto F.no
(FI) Italy.
∫ CERM, University of Florence, via L. Sacconi, 6 50019 Sesto F.no (FI) Italy.
§ Department of Exp. and Clinical Biomed. Sciences, University of Florence, V.le Morgagni, 50
50134 Firenze, Italy.
# Department NEUROFARBA, University of Florence, V. le Pieraccini, 6 50134 Firenze, Italy;
^ FioGen, via L. Sacconi,6 50019 Sesto F.no (FI) Italy.
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ABSTRACT Oxaliplatin (OXA) is a valuable and largely used cancer drug, which induces a
serious and intractable neuropathy. The lipoylꢀhomotaurine derivative (ADM_12) reverts in vivo
OXAꢀinduced neuropathy and it is an effective antagonist of the nociceptive sensor channel
TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to
inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells
aggressiveness.
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INTRODUCTION
The number of cancer patients is increasing worldwide and a large majority of them receive
antitumoral chemotherapy. The development of new and effective anticancer drugs has extended
the survival of tumorꢀbearing hosts, conceivably raising the proportion of patients having
experienced chemotherapyꢀinduced neuropathy (CIN).1 CIN is considered one of the most
disabling and invalidating disease affecting cancer patients: CIN induces or worsen depression,
insomnia and distress, greatly affecting the quality of life and often becoming the treatmentꢀ
limiting issue in the cancer therapy.2,3 Therapeutically, CIN is still considered an intractable
problem. As a matter of fact, no pharmacological treatments have demonstrated efficacy in the
prevention or therapy of CIN. Mixtures of opioids and nonꢀopioids, including vitamins and
mineral salts, are generally administrated with scarce or no evidence of efficacy confirming CIN
the major concern for both cancer patients and oncologists.4 Manifestations and causes of CIN
can vary substantially from drug to drug. A distinct, and probably one of the most difficult to
treat, is the neuropathy induced by oxaliplatin (OXA). Mechanical and cold allodynic sensation
in hands and feet are the painful effects produced which, surprisingly, may worsen after OXA
administration has finished.2 Thus, the availability of safe and effective analgesic drugs for the
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treatment of chronic CIN is a task of primary importance as well as an urgent need. Acute and
chronic peripheral neuropathies associated with the use of OXA have extensively been reported5
and related to the remodeling and alteration in the expression of transient receptors potential
(TRP) Ca2+ channels. In particular, thermosensitive transient receptors potential Vanilloid 1
(TRPV1) and Ankyrin 1 (TRPA1) are up regulated and hyper sensitized, mediating OXAꢀevoked
cold and mechanical allodynia.6,7 We recently reported about the TRPA1 antagonist 1 (ADM_09)
(Figure 1),8 which effectively reverts neuropathic pain evoked by OXA, without eliciting the
negative sideꢀeffects generally observed in neuropathic pain treatment. Preserving the αꢀlipoic
portion, which characterizes 1, we have also reported a second lipoicꢀcontaining TRPA1
antagonist, namely 2 (ADM_12) (Figure 1),9 successfully assayed to treat in vivo another orphan
pain, that is the orofacial pain.9
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Figure 1. Structure of 1, 2, compounds 3, 4, 10 and acetazolamide (AAZ).
With respect to 1, 2 presents the lipoic acid portion linked to an homotaurine and is characterized
by a terminal sulfonic acid residue.9 In a recent paper describing the inhibitory activity of a
bromophenol sulfonic acid vs. human (h) carbonic anhydrases (hCAs, EC 4.2.1.1) implicated in
cancer progression,10 some of us proved the involvement of the sulfonic acid residue in the
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binding to the CA IX active site by anchoring to the zincꢀcoordinated water molecule. Keeping
this in mind and capitalizing on data we previously reported for 2, we reasoned that 2 might be an
excellent candidate for both the treatment of neuropathic pain induced by OXA and as a new
inhibitor of CAs. Hence, we report herein on the investigation of 2, and the corresponding
homologues 3 and 4 (Figure 1) in: a) the treatment of OXAꢀinduced neuropathy (OXAIN), b)
binding tests vs. CA IX and CA XII as well as c) in CA IXꢀmediated cancer cells motility tests.
Unprecedentedly, our data showed the double role of 2 as analgesic but also as modulator in
cancer cells invasiveness.
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RESULTS
Synthesis. Following the synthetic strategy reported for 2,9 its homologous 3 and 4 were obtained
in two steps reacting the commercially available (±) αꢀlipoic acid with Nꢀhydroxysuccinimide in
tetrahydrofuran (THF) as solvent and in the presence of dicyclohexylcarbodiimide (DCC) to give
the activated lipoic derivative 5 (Scheme 1). The unreacted DCC and the dicyclohexyl urea
formed during the reaction, were removed by crystallization and pure 5 was treated with taurine 6
or with the amino sulfonic acid 7 in H2O/DMF to afford, after purification, 3 (60%) or 4 (80%)
respectively. As reported for 3 and 4 (see Scheme 1), the lipoicꢀcontaining alcohol 9, used as
control (see below), was obtained by reacting 5 with the amino alcohol 8 (Supporting
Information). After purification, 9 was isolated as a yellow waxy solid in 60% yield.
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Scheme 1. Synthesis of lipoicꢀcontaining derivatives 3, 4 and 9
Antioxidative properties. As widely reported,11,12 acute and chronic neurotoxicities associated
with the use of OXA are related to the oxidative stress caused by this cancer drug. Therefore, the
antioxidant profiles of 2 and compounds 3 and 4 were evaluated. We measured the oxidation in
vitro of nitro blue tetrazolium (NBT) after 30 min, in the absence and in the presence of an
.ꢀ
increasing concentration of 2 (Table S2, Supporting Information). The superoxide anion (O2 )
generated by the hypoxanthine–xanthine oxidase system increased the oxidized NBT level from
100 (basal) to about 4000 A.U. In the presence of 2 in the reaction mixture, the oxidation of NBT
was inhibited in a concentrationꢀdependent manner taking effect starting from 10 µM. The
highest concentration (1000 µM) allowed to obtain a complete prevention of oxidation. Similar
results were observed testing 3 and 4 but, differently from 2, they were not able to fully prevent
the oxidation (Supporting Information). The antioxidative properties were also tested in cell
culture of primary rat cortical astrocytes, a cell type strongly involved in OXAꢀdependent
neuropathy.13,14 A 4 h long cell treatment with 100ꢁM OXA induced a significant, SODꢀ
inhibitable, superoxide anion increase as evaluated by the cytochrome C assay (Figure 2A). The
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.ꢀ
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coꢀincubation with 100ꢁM 2 or 3 or 4 inhibited the O2 formation by about 75% in comparison to
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the untreated culture.
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oxaliplatin +
oxaliplatin +
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30 mg kgꢀ1
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vehicle + vehicle
oxaliplatin + vehicle
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control
oxaliplatin 100 ꢀM
oxaliplatin ꢀM + SOD 300 U/mL
**
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^^
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100 ꢀM
100 ꢀM
100 ꢀM
Time (min)
.ꢀ
Figure 2. (A) SODꢀinhibitable O2 levels in astrocyte cell lines. Astrocytes (5 × 105 cells/well, 2
control wells and 2 wells preꢀtreated with 2, 3 or 4) were exposed to 100 ꢁM OXA for 4h. The
.ꢀ
effect of 100 ꢁM 2 coꢀincubation on SODꢀinhibitable superoxide anion O2 on levels are
depicted. The nonꢀspecific absorbance measured in the presence of SOD was subtracted to the
total value. Values are expressed as mean ± s.e.m. of 3 experiments. **P < 0.01 vs. control
and ∧∧P < 0.01 vs OXA treatment. (B) OXAꢀinduced neuropathic pain: antiꢀhypersensitive effect
of 2 in comparison to 3 and 4. Rats were daily treated i.p. with 2.4 mg kgꢀ1 OXA (in 0.5%
glucose solution). The response to a noxious mechanical stimulus was evaluated on day 21 by the
pawꢀpressure test. Vehicle (1% CMC) or 30 mg kgꢀ1 2, 3 or 4 were administered p.o.,
measurements were performed over time. Each value represents the mean ± S.E.M. of 12 rats per
group, performed in two different experimental sets. *^P<0.01 vs. vehicle + vehicle; ^^P<0.01 vs
OXA + vehicle
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Antihyperalgesic properties. To evaluate the painꢀrelieving property of the tested compounds, a
painful neuropathic condition was reproduced in rats with a repeated treatment with OXA (2.4
mg kgꢀ1, i.p., daily). On day 21, the pain threshold was measured by the paw pressure test
applying a noxious mechanical stimulus. Oxaliplatinꢀtreated animals showed (Figure 2B) a
decreased threshold reported as a reduced weight tolerated on the paw (44.7 ±2.3 g in comparison
to 70.6 ± 2.5 g of control animals). Compound 2, acutely administered p.o. (30 mg kgꢀ1)
counteracted the OXAꢀinduced hypersensitivity taking effect 15 min after treatment. A complete
reversion of pain lasted up to 75 min. A similar profile was observed for compounds 3 and 4
(Figure 2B) even though a complete reversion of the noxious effect is observed only with 2.
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Synergistic effect of OXA and 2. The soundness data obtained with 2 in reverting OXAIN in
vivo, suggested to investigate a possible synergistic effect of 2 and OXA. The activity profile of 2
against tumor cells was thus evaluated in human colon cancer cell lines HTꢀ29. The incubation
with increasing concentrations of 2 (1ꢀ300 µM) for 24 h induced a progressive decreasing of cell
viability as evaluated by MTT test, reaching 20% of mortality (Table S3). Moreover, 2 was also
analyzed in the presence of the neurotoxic anticancer drug OXA. As shown in Table S4, OXA in
HTꢀ29 cells is active at 30, 100 and 300 µM (24 h). In the presence of 2 (100 µM) OXA activity
was not decreased; on the contrary, 2 increased OXA potency, decreasing cell viability starting
out at 1 µM reaching significant differences at 100 and 300 µM (Table 1). The analysis of
synergism performed by the ChouꢀTalalay method revealed a combination index (CI) 0.5
considered as “synergism”.
Table 1. Viability cell test (MTT) run 24 h after the treatment with OXA and 2. The % of
viable cells with respect to the control is reported.
OXA 10µM
OXA 100µM OXA 300µM
OXA
OXA
1µM
30µM
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100 ± 2.2
2 (100 µM) 81.3 ± 3.1* 74.6 ± 3.2** 78.6 ± 0.8**
Control
71.4 ±
1.1***
67.4 ±
3.6***^
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**p<0.01 and ***p<0.001 vs control; ^p<0.05 and ^^^p<0.01 vs. 2 alone
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Inhibition of TRPA1 activated with OXA. As reported,9 2 can inhibit TRPA1 methanolꢀ and
AITCꢀevoked current with a low micromolar constant. Thus, using the patch clamp technique, we
investigated the effect of 2 on TRPA1 evoked current by OXA. After perfusion of OXA 50 ꢀM
we observed the activation of TRPA1 channel, which is rapidly and completely reverted in the
presence of 2, 30 ꢀM. The corresponding time courses (Figure 3A) and IV curves (Figure S2,
Supporting Information) are reported (see Table S1 and Figure S1 for details about compounds 3
and 4).
A
B
Figure 3. (A) Time course obtained from voltage ramps measured at +50 mV (black circles) and
ꢀ50 mV (grey circles). OXA (50 ꢀM) and 2 30 ꢀM, were applied at the time indicated. CHO cells
were transfected with wildꢀtype TRPA1 channel. (B) Summarized data of TRPA1 wholeꢀcell
currents evoked by OXA 50 ꢀM and OXA + 2 30 ꢀM. Each column represents mean ± SEM of n
= 5 cells. *** p < 0.0001 (unpaired Student’s t test).
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Compound 2 inhibits carbonic anhydrases IX and XII. Carbonic anhydrases (CAs, EC
4.2.1.1) are zinc metalloꢀenzymes that catalyze the hydration of carbonic dioxide, producing
bicarbonate and protons.15,16 CA IX is a transꢀmembrane enzyme upꢀregulated in many cancer
types and playing an important role in the acidification of the outer microenvironment under
hypoxia, a hallmark of many tumors.17 The involvement of CA IX to acidification of the tumor
environment has been correlated with the acquisition of metastatic phenotypes and is related to a
decrease in the success of anticancer drugs.18 Recently, some of us reported on the nanomolar
hCA IX and hCA XII inhibitor 10 (see Figure 1), presenting a sulfonic acid residue involved in
the binding to the CA IX active site by anchoring to the zincꢀcoordinated water molecule.10 Of
note, currently a sulfonamide CA IX inhibitor completed phase I clinical trials for the treatment
of advanced stage solid tumors and is scheduled for Phase II trials.19 Keeping in mind the specific
CA inhibition mechanism of 10, we investigated 2 as a possible ligand for CA IX and CA XII. In
fact, even though the properties of 2, 3 and 4 as TRPA1 inhibitors or to revert OXAIPN are rather
similar, overall the pharmacological profile of 2 is the most interesting. For this reason, all the
following tests have been performed on 2 selected as a lead molecule. We investigated the CA
inhibitory activity of 2 by applying the stopped flow carbon dioxide hydrase assay (Supporting
Information), in comparison to acetazolamide (AAZ, see Figure 1) as standard CA inhibitor
against the tumorꢀassociated isoforms hCA IX and XII (Table 2; see Table S5 for inhibition data
vs. other hCAs and Figure S3 for dose response curves). From the data shown in Table 2, 2
clearly arose as a good hCA IX and hCA XII inhibitor, with KI ranging in high nanomolar range
(respectively 653.7 and 727.5 nM). Such results ascribed to 2 are lower in efficacy than the
clinically used AAZ, but improved compared to derivative 10. Anyhow, it should be stressed that
the sulfonate derivative 10 originates from the CAꢀmediated hydrolysis of the corresponding
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sulfocoumarin, necessary to generate the active species from the original prodrug.10 Conversely,
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such activation was not necessary in the case of 2, which can directly bind the active site pocket.
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Table 2. Inhibition data of human CA isoforms hCA IX and XII with 2, compound 10 and the
standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
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KI (nM)a
hCA IX
hCA XII
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6830
25.2
727.5
4510
5.7
10b
AAZ
aMean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5ꢀ10 %
b
of the reported values). Ref. S7. Inhibitor concentration ranged from10ꢀ8 to 10ꢀ5 M (see also
Figure S3)
Molecular modeling. In order to fully explore the binding interaction of the sulfonate derivative
2 within CA active site, we undertook docking studies taking into consideration the tumorꢀ
associated isoform hCA IX. In fact, all the attempts to coꢀcrystalize 2 with CA failed and at
present, only one Xꢀray structure of a sulfonate compound within CA is available in Protein Data
Bank, that is the adduct of derivative 10 with a modified hCA II (PDB 4BCW).10 This structure,
also referred as hCA II/IX mimic, concerns an engineered protein in which two amino acid
mutations are present in the hCA II active site, namely A65S and N67Q, being serine and
glutamine the corresponding amino acids found in hCA IX. The sulfonate group of 10, which as
previously mentioned comes from the CAꢀmediated hydrolysis of the corresponding
sulfocoumarin,10 was found to anchor to the Znꢀcoordinated water molecule, differently from
compounds containing the sulfonamide moiety that instead directly coordinates to the Zn ion.20
Considering that 2 was assayed in vitro against hCA IX (KI = 653.7 nM), the target used in such
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experiments was prepared superposing 3IAI (hCA IX) and 4BCW (hCA II/IX mimic) through
the Zn ion and the backbone of H94, H96, H119 residues, then joining together only the enzyme
3D coordinates of 3IAI and the catalytic Zn coordinated OH/H2O group of 4BCW. We report
herein only the results obtained for the isomer R, as the stereoꢀcenter is located at the outer rim of
the aliphatic tail of the molecule and barely affects the poses arisen from the docking studies, thus
the highlighted binding mode. Taking as driving principle in the selection of the docking
solutions the position of the sulfonate group in the Xꢀray solved structure, three rather isoꢀ
energetic poses were selected, all characterized by not high values of the scoring function
(Figures 4 and 5).
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A
B
S
S
O
HN
O
N
H
O
S
H
Thr199
O
H
O
O
O
H
Zn
His96
His94
His119
Figure 4. (A) Diagram of the Hꢀbond network the sulfonate moiety is involved in, within the
active site cavity. (B) Superimposition of the three orientations found for 2 within hCA IX active
site.
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Figure 5. Simulated binding modes of compound 2 within hCA IX active site (PDB ID 3IAI):
three diverse orientations are shown in panels (A), (B) and (C). The hydrogen bonds are shown as
yellow dashed lines.
In the three different poses the sulfonate group was always found to be involved in a wide
network of Hꢀbonds (Figure 4A). In particular, two oxygen atoms of the sulfonate moiety
accepted a Hꢀbond from the Znꢀbound water molecule, which in turn donates an additional one to
OG1 oxygen of T199 side chain. Furthermore, one of these sulfonate O atoms established a Hꢀ
bond with the backbone NH of T199, whereas the other one might be consider as “halfꢀ
coordinated” to the Zn ion. The three 2 poses are differentiated by the position of the lipoic tail
within the binding site cavity (Figure 4B) and each is characterized by hydrophobic contacts
involving the propylenic linker and the lipoic moiety which respectively interact with L198,
P201, P202 (Figure 5A), V121, L91, V131, Q92 (Figure 5B) and V121, W5, H64, N62 (Figure
5C). Moreover, the amidic carbonyl of the conformers in Figure 5B and Figure 5C is at H bond
distance with Q92.
Compound 2 reduces CA IX-mediated acidosis. Recently, in order to analyze the role of CA
IX in cancer cells motility, we investigated the extracellular pH in normoxia in extracted human
prostate fibroblasts (HPFs) from healthy individuals affected by prostate hyperplasia, cancerꢀ
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associated fibroblasts (CAFs) and in prostate cancer (PCa) cells after the treatment with
conditioned medium (CM) from HPFs or CAFs.21 The contact of CAFs with PCa cells gives rise
to an increase in extracellular acidity ascribed to CA IX and suggesting that the conditioning loop
among stromal and cancer cells involved in cancer cells malignancy and aggressiveness also
takes account for acidification of tumor microenvironment. In Figure 6 we report on the effect of
2 on alkalinization of extracellular pH in HPFs and CAFs. Fibroblasts were grown to subꢀ
confluence and serum starved for 48h in the presence of 2 (100 µM); at the end of incubation, the
pH of culture medium was immediately measured by pHꢀmeter. We observed that the
administration of 2 unambiguously reduces extracellular acidification confirming a key role of
CA IX in this phenomenon.
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7.8
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Figure 6. Effect of 2 on alkalinization of extracellular pH in HPF and CAF. Cells were serum
starved and treated with 100 µM of 2 for 48h. *p < 0,05 vs. CAF.
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Figure 7. A) HPF and CAF were treated with 100 µM of
2 for 48 h and CM were
harvested. PCa cells were then treated with serum free medium or different CM for 72 h,
and then were photographed (B) Left: evaluation of Eꢀcadherin and Vimentin in the same
experimental setting described in A. (B) Right: actin immunoblot was used for
normalization. Cadherin expression was shown in bar graph, a.u.= arbitrary units.
Compound 2 positively interferes in the epithelial-mesenchymal transition (EMT)
and invasiveness in PCa cells. A specific role is played by CA IX in the regulation of
EMT of cancer cells, a key epigenetic program associated with increased motility,
survival and stemness.21 In particular, we demonstrated that PCa cells undergo EMT in
response to their contact with stromal fibroblasts expressing CA IX.21 Here, we report on
the effect of
2
on CAFꢀmediated EMT. PCa cells were treated for 72h with conditioned
(100 µM). We
medium (CM) obtained from HPFs and CAFs in the presence or not of
2
observed that the use of the compound is active in impairing EMT in cancer cells due to
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CAFs contact (Figure 7A), preventing the elongation of the cells, as well as the expression
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of known EMT markers, as Eꢀcadherin decrease and upꢀregulation of Vimentin (Figure
7B).
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Figure 8. Left: invasion analysis of Pca cells after treatment for 24h with different CM
obtained from HPF or CAF (control or in presence of
2, 100 µM). Right: bar graph
represents the mean of invading cells. *** < 0.001 vs. untreated CAF.
In keeping rounded morphological features,
2
clearly affects invasiveness of cancer cells.
and assayed
We finally treated cells with CM from fibroblasts in the presence or not of
2
for their ability to invade through reconstituted matrigel barrier. Our data revealed that the
ability of CAFs to elicit a proꢀinvasive behaviour of cancer cells is severely impaired
during the administration of
2
(Figure 8).
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Compound 2 is a MMPs-inhibitor. As above discussed, stromal CA IX leads to extracellular
acidification. Since the sensitivity of MMPs to acidity is acknowledged and the key role played
by CA IX in MMPꢀ2 and MMPꢀ9’s secretion by CAFs is documented,21 we reasoned that, given
the similar activeꢀsite topology of MMPs and CAs, it would be worthy to investigate the binding
properties of 2 vs. MMPs. The intrinsic high similarity of human MMPs’ active site, including
MMPꢀ2, MMPꢀ9 and MMPꢀ12, is well documented,22,23 therefore, the catalytic domain of human
MMPꢀ12 has been used as representative model of the whole protein family to analyze the
structural basis for the interaction of MMPs with the investigated scaffold. To determine the
binding affinity of 2 for the catalytic domain of MMPꢀ12, we analyzed the alteration of the
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chemical shifts observed in 2D Hꢀ15N HSQC spectra of the protein upon the titration with the
lipoic acid derivative. Several residues forming the S1’ cavity, and two zinc binding histidines
experience a sizable chemical shift variation at submillimolar concentration of the ligand (see
Figure 9). The fit of the experimental data provided a dissociation constant of 875 ± 78 µM.24
The uncommon structure of 2 as MMP inhibitor deserved a more accurate investigation. Thus, a
detailed structural characterization of the complex MMPꢀ12ꢀ2 by Xꢀray diffraction was carried
out to investigate the binding mode and the involvement of the catalytic zinc in ligand binding.
To confirm the structural data obtained, compound 9 (see Scheme 1) was synthesized and Xꢀray
diffraction of the complex MMPꢀ12ꢀ9 performed. Compound 9 preserves the lipoic portion but
presents a propyl alcohol replacing the propyl sulfonate residue of 2 (see Scheme 1).
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Figure 9. Hꢀ15N HSQC spectrum of the catalytic domain of MMPꢀ12 in the absence (red) and
presence of 2 (3.3 mM) (blue), with the surface model of MMPꢀ12 showing in red the residues
experiencing the largest chemical shifts variation.
Compound 2 and 9 are non-zinc binding ligands of MMP-12. In both structures, AHA
(hydroxamic acid, see Supporting Information) is not displaced by 2 or 9 and shows its typical
binding to the catalytic zinc at full occupancy:25 it chelates zinc with the two oxygens and
establishes hydrogen bond interactions with the carbonyl amide oxygen of A182 and with a
carboxylate oxygen of the catalytic E219. As consequence, the two lipoicꢀcontaining ligands do
not interact directly with the catalytic zinc ion. As a matter of fact, the sulfonate residue (2)
points outside the MMPꢀ12’s site and is placed at the very entrance of the substrate groove, in
addition, one of the sulfonate oxygens is at 3.1 Å distant from the NH moiety of Y240. A second
sulfonate oxygen is 2.7 Å far from the amidic carbonyl oxygen of G179. (Figure 10).
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Figure 10. AHA and 2 placed into MMPꢀ12 site. Hydrogen binding interactions are indicated as
yellow dotted lines.
Likewise, the hydroxyl group of ligand 9 is located in the proximity of the groove entrance and it
does not interact with any protein residue (Figure S6). Both ligands bend similarly upon binding
to the protein in such a way that the heterocyclic portion is accommodated into the S1’ pocket
(Figure 11), while the chain occupies the groove normally occupied by the substrate before to be
hydrolyzed. The electron density of the lipoicꢀcontaining ligands appears of good quality in both
cases supporting their full occupancy; in detail, it shows that the heterocyclic ring of both, 2 and
7, containing a disulfide bridge, remains closed witnessing that the SꢀS function is not reduced. In
the case of the sulfonate ligand, 2, the two sulfur atoms of the ring into the S1’ pocket are placed
at 3.6 Å from the carbonyl amide oxygens of V235 and F237. In the case of the OH ligand
(compound 9) the ring is slightly tilted so that the two sulfur atoms are at 3.6 Å from the carbonyl
oxygens of T239 and K241. Moreover, the carbonyl amide oxygen present in the middle of the
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ligands points towards AHA whereas the hydroxyl group of 9 and the sulfonate of 2, protrude
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outside the molecule towards the solvent (Figure 11).
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Figure 11. View of the interaction of 2 (left panel, pdb code 5N5J) and 9 (right panel, pdb code
5N5K) with MMPꢀ12. The two ligands and AHA are represented as green sticks, the protein as
transparent surface and the catalytic zinc as yellow sphere.
DISCUSSION AND CONCLUSION
So far, OXA is the cancer drug commonly used to treat colonꢀrectal and resistant prostate cancer.
However, the oxidative stress triggered by OXA induces a neuropathy which is considered the
neuropathic pain the most difficult to treat. In this manuscript, we showed the properties of 2 to
revert OXAIN in an experimental animal model and we proved 2 effective in reducing cancer
cells invasiveness by inhibiting CA IX in vitro. Compound 2, formed by a lipoic acid residue
linked to the homotaurine, has a remarkable safe profile in terms of cells viability and
cardiotoxicity.9 In order to exclude a possible effect of the distance between the cyclic disulfide
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and the sulfonic acid tail on the biological properties of 2, the lower homologue 3 and the higher
homologue 4 were synthesized The lipoic portion of 2 reasonably accounts for the antioxidative
properties we assessed in vitro (NBT test, Table S2 and astrocytes cell culture Figure 2). In vivo,
2, administered to rat previously treated with OXA, completely counteracted the OXAꢀinduced
hypersensitivity (see Figure 2B). The oxidative stress caused by OXA is also known to trigger the
activation of TRPA1 channel evoking a noxious stimolous;7 we confirmed 2 an efficient inhibitor
of TRPA1,9 able to completely revert the OXAꢀinduced activation of the channel (Figure 3).
Compound 2 and the two homologues, namely 3 and 4, showed similar antioxidative properties,
close effects as TRPA1 inhibitors as well as in in vivo tests. This allows to exclude any effect due
to the distance between the disulfide bridge and the sulfonic acid portion.
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The functional contribution of CA IX to specific biological processes critical for cancer
progression, including pH regulation, migration and invasion, provide the rational for its use as
relevant therapeutic target.18 In 2015 it was published the first example of CA IX inhibitor
characterized by a sulfonate residue (see compound 10, Figure 1). Considering the structural
features of 2, we investigated its binding affinity vs. CA IX and CA XII, both involved in cancer
progression. Tests of inhibition vs. hCA IX and hCA XII clearly showed a high nanomolar
affinity of 2 for both the CAs (Table 1). The binding interactions of the sulfonate 2 with the CA
active site, explored running computational studies, allowed to properly rationalize the good
inhibition constants assessed. We confirmed these inhibition properties by testing 2 in CA IXꢀ
mediated biochemical processes. We observed that the administration of 2 reduces extracellular
acidification and clearly impairs EMT in cancer cells (Figure 7), preventing the expression of
ETM markers and reducing invasiveness of cancer cells (Figure 8). These results were boosted by
the synergist effect observed for 2 and OXA when used to treat HTꢀ29 cancer cells (Table 1).
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These preliminary data suggest that the use of OXA and 2 in combination might allow to
decrease OXA concentration in therapy reducing the painful effects produced. The binding
properties of 2 vs. MMPs were also examined. The chemical shifts recorded by 2D 1Hꢀ15N HSQC
spectra of MMPꢀ12 (model for the MMPs family, including the acidity sensitive MMPꢀ2 and
MMPꢀ9) upon the titration with 2 provided a high micro molar Kd value. Xꢀray diffraction,
carried out to investigate the binding mode of 2, showed that it is a nonꢀzinc binding ligand of
MMPꢀ12. Concluding, the simple structure of 2 collects unique biological properties: i) it blocks
OXAꢀinduced TRPA1 current, ii) reverts in vivo OXAIN, iii) has a synergistic effect with OXA,
iv) inhibits CA IX and CA XII, two CAs involved in tumor progression and v) impairs EMT and
invasiveness in cancer cells. Even though the inhibition of CAs should be confirmed in vivo, the
unique properties of 2 and the soundness of the data reported could represent a glimmer in the
dark scenario exposed to cancer patients suffering from the side effects related to the chronic
administration of chemotherapy and from the intractable neuropathic pain induced by
chemotherapy.
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EXPERIMENTAL SECTION
Materials and methods. Patchꢀclamp studies were carried out in Chinese Hamster Ovary (CHO)
cells transiently expressing TRPA1 channel. For heterologous protein expression, cells were
plated in 6ꢀwells cell culture dishes with 2 ml growth medium, 24 h before transfection. Cells
were transiently transfected using XꢀtremeGENE 9 transfection reagent (Roche), according to the
protocol supplied by the manufacturer. EGFP fluorescence was used as marker of successful
transfection. Electrophysiology studies were performed 48ꢀ72 h after transfection. Purity (≥ 95%)
of compounds 3, 4 and 9 was determined by HRMS.
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Male SpragueꢀDawley (SD) rats (Harlan, Varese, Italy) weighing approximately 200 to 250 g at
the beginning of the experimental procedure were used. Pirc (F344/NTacꢀApcam1137) rats,
originally obtained from Taconic (Taconic Farms, Inc. USA) were bred in CESAL (Centro
Stabulazione Animali da Laboratorio, University of Florence, Italy) in accordance with the
Commission for Animal Experimentation of the Italian Ministry of Health. Animals were housed
in CeSAL and used at least 1 week after their arrival.
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2-(5-(1,2-Dithiolan-3-yl)pentammidoethanesulfonic acid (3) and 4-(5-(1,2-dithiolan-3-
yl)pentamido)butane-1-sulfonic acid (4). General procedure. The activated lipoic acid 5 in
DMF/H2O as solvent was treated at room temperature with the aminosulfonic derivative 626 or 7
to afford after purification by column chromatography on silica gel the derivative 3 (60%) or 4
(80%) respectively, obtained as sodium salt. Compound 3: Column chromatography on silica gel
1
(eluent: CH2Cl2/MeOH 4/1; 5/2). Glassy solid; HꢀNMR (500 MHz, D2O): δ 3.67ꢀ3.62 (m, 1H,
3
CHꢀ3), 3.53ꢀ3.50 (t, J(H,H)=6.8 Hz, 2H, CH2ꢀ10), 3.22ꢀ3.12 (m, 2H, CH2ꢀ1), 3.06ꢀ3.02 (t,
3J(H,H)=6.8 Hz, 2H, CH2ꢀ9), 2.46ꢀ2.41 (m, 1H, CHꢀ2’), 2.23ꢀ2.20 (m, 2H, CH2ꢀ7), 1.95ꢀ1.91 (m,
1H, CHꢀ2), 1.73ꢀ1.56 (m, 3H), 1.40ꢀ1.36 ppm (m, 3H). 13CꢀNMR (125 MHz, D2O): δ 176.8,
ꢀ
56.5, 49.8, 40.3, 38.1, 35.5, 35.0, 33.7, 27.9, 24.9 ppm. HRMS calculated for C10H18NO4S3 [Mꢀ
H]ꢀ : 312.03980; found: 312.03923. Elemental Anal. (C10H18NNaO4S3) calculated: C, 35.81; H,
5.41; N, 4.18; found: C, 35.78; H, 5.54; N, 4.46. Compound 4: Column chromatography on silica
1
gel (eluent: CH2Cl2/MeOH 4/1; 5/2). Glassy solid; HꢀNMR (500 MHz, D2O): δ 3.68ꢀ3.63 (m,
1H, CHꢀ3), 3.20ꢀ3.12 (m, 4H, CH2ꢀ1, CH2ꢀ12), 2.88ꢀ2.85 (m, 2H, CH2ꢀ9), 2.46ꢀ2.42 (m, 1H, CHꢀ
2), 2.21ꢀ2.18 (m, 2H, CH2ꢀ7), 1.96ꢀ1.92 (m, 1H, CHꢀ2’), 1.72ꢀ1.67 (m, 3H), 1.59ꢀ1.57 (m, 5H),
13
1.40ꢀ1.35 (m, 2H). CꢀNMR (125 MHz, D2O): δ 176.8, 56.5, 50.6, 40.3, 38.8, 38.1, 35.6, 33.7,
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ꢀ
27.8, 27.4, 25.1, 21.6 ppm. HRMS cald for C12H22NO4S3 [MꢀH]ꢀ : 340.07110; found: 340.07008.
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Elemental Anal. (C12H22NNaO4S3) calculated: C, 39.65; H, 6.10; N, 3.85; found: C, 39.58; H,
6.32; N, 4.05.
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5-(1,2-Dithiolan-3-yl)-N-(3-hydroxypropyl)pentanamide (9). A solution of the amino alcohol
8 (390 mg, 5.19 mmol) in DMF/H2O was treated at room temperature with solid NaHCO3 (5.19
mmol) then cooled to 0 °C. A solution of 5 (1.5 g, 4.94 mmol) in DMF was added and the
reaction mixture warmed to room temperature and stirred overnight. Water (100 mL) is then
added to the mixture and the organic layer extracted with EtOAc (2x). The crude (1.4 g) was
purified by column chromatography on silica gel (eluent MeOH/DCMꢀ8/1) to afford 9 as waxy
solid (770 mg, 60%). Column chromatography on silica gel (eluent: CH2Cl2 with 8% MeOH).
Waxy solid; 1HꢀNMR (500 MHz, CDCl3): δ 6.30 (bs, 1H, NH), 3.64ꢀ3.61 (m, 2H, CH2ꢀ11), 3.59ꢀ
3.54 (m, 1H, CHꢀ3), 3.41ꢀ3.38 (m, 2H, CH2ꢀ9), 3.27 (bs, 1H, OH), 3.20ꢀ3.09 (m, 2H, CH2ꢀ1),
2.49ꢀ2.43 (m, 1H, CHꢀ2), 2.24ꢀ2.18 (m, 2H, CH2ꢀ7), 1.49ꢀ1.87 (m, 1H, CHꢀ2’), 1.75ꢀ1.60 (m,
13
6H), 1.52ꢀ1.42 ppm (m, 2H). CꢀNMR (125 MHz, CDCl3): δ 174.1, 59.4, 56.4, 40.3, 38.5, 36.4,
36.4, 34.6, 32.2, 28.9, 25.5 ppm. HRMS cald for C11H22NO2S2 [M+H]+: 264.10920; found:
+
264.10903. Elemental Anal. (C11H21NO2S2) calculated: C, 50.16; H, 8.04; N, 5.32; found: C,
50.08; H, 8.30; N, 5.02.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publication website at DOI:
xxx.
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1H and 13C spectra for compounds 3, 4 and 8, in vitro and in vivo methods, hCAs inhibition,
NMR spectroscopy, Xꢀray data (PDF). Molecular formula strings (CSV).
Authors will release the atomic coordinates and experimental data upon article publication. PDB
codes: 5N5J for 2 and 5N5K for 9.
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AUTHOR INFORMATION
Corresponding Authors
*For C.N. eꢀmail: cristina.nativi@unifi.it Phone (+39)0554573540;
*For C.L. eꢀmail: claudiu.supuran@unifi.it
ORCID:
Marco Fragai: 0000ꢀ0002ꢀ8440ꢀ1690
Giuseppina Comito: 0000ꢀ0002ꢀ4614ꢀ067X
Lorenzo Di Cesare Mannelli: 0000ꢀ0001ꢀ8374ꢀ4432
Roberta Gualdani: 0000ꢀ0003ꢀ3617ꢀ4953
Vito Calderone: 0000ꢀ0002ꢀ7963ꢀ6241
Alexandra Louka: 0000ꢀ0001ꢀ5054ꢀ6480
Barbara Richichi: 0000ꢀ0001ꢀ7093ꢀ9513
Oscar Francesconi: 0000ꢀ0002ꢀ6155ꢀ4926
Andrea Angeli: 0000ꢀ0002ꢀ1470ꢀ7192
Alessio Nocentini: 0000ꢀ0003ꢀ3342ꢀ702X
Paola Gratteri: 0000ꢀ0002ꢀ9137ꢀ2509
Carla Ghelardini: 0000ꢀ0001ꢀ9856ꢀ8144
Francesco Tadini Buoninsegni: 0000ꢀ0001ꢀ5594ꢀ2554
Claudiu Supuran: 0000ꢀ0003ꢀ4262ꢀ0323
Cristina Nativi: 0000ꢀ0002ꢀ6312ꢀ3230
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Notes
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The authors declare no competing financial interest.
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Funding Sources
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Fondazione CRF ꢀ 2014.0163, Banca CR Firenze ꢀ 2016 and Istituto Toscano Tumori, ITT15.
ACKNOWLEDGEMENTS
Authors are grateful to Prof. Claudio Luchinat (University of Florence) and Dr. Stefano Roelens
(INSTM, Florence) for useful discussions. We thank Prof. Doug Golenbock (University of
Massachusetts Medical School, Boston, USA) for providing pcDNA3ꢀEGFP (Addgene plasmid
n.13031).
ABBREVIATIONS USED
AAZ, acetazolamide; AHA, hydroxamic acid; CA, carbonic anhydrase; CAF, cancer associated
fibroblasts; CIN, chemotherapyꢀinduced neuropathy; CM, conditioned medium; EMT, epithelialꢀ
mesenchymal transition; MMP, matrix metalloproteinase; NBT, nitro blue tetrazolium; NHS, Nꢀ
hydroxysuccinimide; OXA, oxaliplatin; OXAIN, OXAꢀinduced neuropathic pain; SOD,
superoxide dismutase; TRP, transient receptors potential.
REFERENCES
(1) Woolf, C.J.; Mannion, R.J. Neuropathic pain: aetiology, symptoms, mechanisms and
management. Lancet 1999, 353, 1959ꢀ1964.
(2) Majithia, N.; Loprinzi, C.L.; Smith, T.J. New practical approaches to chemotherapyꢀinduced
neuropathic pain: prevention, assessment and treatment. Oncology 2016, 30, 1020ꢀ1029.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 29
1
2
3
4
5
6
(3) Grothey, A. Clinical management of oxaliplatinꢀassociated neurotoxicity. Clin. Colorectal
Cancer 2005, 1, S38ꢀ46.
7
8
9
(4) Hoshino, H.; Hida, K.; Ganeko, R.; Sakai, Y. Goshajinkigan for reducing chemotherapy
induced periphereal neuropathy: protocol for a systematic review and metaꢀanalysis. Int. J.
Colorectal Dis. 2016, 32, 737ꢀ740.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5) Argyriou, A.A.; Bruna, J.; Marmiroli, P.; Cavalletti, G. Chemotherapyꢀinduced peripheral
neuropathy: an update. Crit. Rev. Oncol. Hematol. 2012, 82, 51ꢀ77.
(6) Zhao, M.; Isami, K.; Nakamura, S.; Shirakawa, H.; Nakagawa, T.; Kaneko, S. Acute cold
hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced
responsiveness of TRPA1 in mice. Mol. Pain 2012, 8, 55.
(7) Nassini, R.; Gees, M.; Harrison, S.; De Siena, G.; Materazzi, S.; Moretto, N.; Failli, P.; Preti,
D.; Marchetti, N.; Cavazzini, A.; Manicini, F.; Pedretti, P.; Nilius, B.; Patacchini, R.; Geppetti, P.
Oxaliplatin elicits mechanical and cold allodynia in rodends via TRPA1 receptors stimulation.
Pain 2011, 152, 1621ꢀ1631.
(8) Nativi, C.; Gualdani, R.; Dragoni, E.; Di Cesare Manneli, L.; Sostegni, S.; Norcini, M.;
Gabrielli, G.; la Marca, G.; Richichi, B.; Francesconi, O.; Moncelli, M.R.; Ghelardini C.;
Roelens, S. A TRPA1 antagonist reverts oxaliplatinꢀinduced neuropathic pain. Sci. Rep. 2005, 3,
2005.
(9) Gualdani, R.; Ceruti, S.; Magni, G.; Merli, D.; Di Cesare Manneli, L.; Francesconi, O.;
Richichi, B.; Francesconi, O.; la Marca, G.; Ghelardini, C.; Moncelli, M.R.; Nativi C. Lipoicꢀ
based TRPA1/TRPV1 antagonist to treat orofacial pain. ACS Chem. Neurosci. 2015, 6, 380ꢀ385.
ACS Paragon Plus Environment

Page 27 of 29
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(10) Grandane, A.; Tanc, M.; Di Cesare Mannelli, L.; Carta, F.; Ghelardini, C.; Zalubovskis R.;
Supuran, C. 6ꢀSubstituted sulfocumarins are selective carbonic anhydrase IX and XII inhibitors
with significant cytotoxicity against colorectal cancer cells. J. Med. Chem. 2015, 58, 3975ꢀ3983.
(11) Gauchan, P.; Andoh, T.; Kato A.; Kuraishi, Y. Involvement of increased expression of
transient receptor potential melastin 8 in oxaliplatinꢀinduced cold allodynia in mice. Neurosci.
Lett. 2009, 458, 93ꢀ95.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12) Descoeur, J.; Pereira, V.; Pizzoccaro, A.; Francois, A.; Ling, B.; Maffre, V.; Couette, B.;
Busserolles, J.; Courteix, C.; Noel, J.; Lazdunski, M.; Eschalier, A.; Authier N.; Bourinet, E.
Oxaliplatinꢀinduced cold hypersensivity is due to remodeling of ion channel expression in
nociceptors. EMBO Mol. Med. 2011, 3, 266ꢀ178.
(13) Di Cesare Mannelli, L.; Pacini, A.; Bonaccini, L.; Zanardelli, M.; Mello T.; Ghelardini, C.
Morphologic features and glial activation in rat oxaliplatinꢀdependent neuropathic pain. J.
Pain 2013, 14, 1585ꢀ1600.
(14) Di Cesare Mannelli, L.; Pacini, A.; Micheli, L.; Tani, A.; Zanardelli, M.; Ghelardini, C.
Glial role in oxaliplatinꢀinduced neuropathic pain. Exp. Neurol. 2014, 261, 22ꢀ33.
(15) Supuran, C.T. Carbonic Anhydrase: novel therapeutic applications for inhibitors and
activators. Nature Rev. Drug Discovery 2008, 7, 168ꢀ181.
(16) Neri D.; Supuran, C.T. Interfering with pH regulation in tumor as a therapeutic strategy.
Nature Rev. Drug Discovery 2011, 10, 767ꢀ777.
(17) Swietach, P.; Hulikova, A.; VanghanꢀJones, R.D.; Harries, A.L. New insight into the
physiological role of carbonic anhydrase IX in tumor pH regulation. Oncogene 2010, 29, 6509ꢀ
6521.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 29
1
2
3
4
5
6
7
8
9
(18) Tafreshi, N.K.; Lloyd, M.C.; Bui, M.M.; Gillies R.J.; Morse, D.L. Carbonic anhydrase IX as
an imaging and therapeutic target for tumors and metastases. Subcell Biochem., 2014, 75, 221ꢀ
254.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Pacchiano, F.; Carta, F.; McDonald, P.C.; Lou, Y.; Vullo, D.; Scozzafava, A.; Dedhar, S.;
Supuran, C.T. Ureidoꢀsubstituted benzenesulfonamides potently inhibit carbonic anhydrase IX
and show antimetastatic activity in a model of breast cancer metastasis J. Med. Chem. 2011, 54,
1896−1902.
(20) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C.T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti S.M.; De Simone, G. Crystal
structure of the catalytic domain of the tumorꢀassociated human carbonic anhydrase IX. Proc.
Natl. Acad. Sci. U.S.A. 2009, 106, 16233−16238.
(21) Fiaschi, T.; Giannoni, E.; Taddei, M.L.; Cirri, P.; Marini, A.; Pintus, G.; Nativi, C.; Richichi,
B.; Scozzafava, A.; Carta, F.; Torre, E.; Supuran C.T.; Chiarugi, P. Carbonic Anhydrase IX from
cancerꢀassociated fibriblastes drives epitelialꢀmesenchymal transition in prostate carcinoma cells.
Cell Cycle 2013, 12, 1791ꢀ1801.
(22) Bertini, I.; Calderone, V.; Cosenza, M.; Fragai, M.; Lee, Y.M.; Luchinat, C.; Mangani, S.;
Terni, B.; Turano, P. Conformational variability of matrix metalloproteinases: beyond a single
3D structure. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 334ꢀ339.
(23) Rao, B.G. Recent developments in the design of specific Matrix Metalloproteinase inhibitors
aided by structural and computational studies. Curr. Pharm. Des. 2005, 11, 295ꢀ322.
(24) Winter, A.; Sigurdardottir, A.G.; Di Cara, D.; Valenti, G.; Blundell T.L.; Gherardi, E.
Developing antagonists for theMet/HGF/SF proteinꢀprotein interaction using a fragmentꢀbased
approach. Chem. Sci. 2015, 6, 6147ꢀ6157.
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Page 29 of 29
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7
8
9
(25) Udi, Y.; Fragai, M.; Grossman, M.; Mitternacht, S.; AradꢀYellin, R.; Calderone, V.;
Melikian, M.; Toccafondi, M.; Berezovsky, I.N.; Luchinat, C.; Sagi, I. Unraveling hidden
regulatory sites in structurally homologous metalloproteinases. J. Mol Biol. 2013, 425, 2330ꢀ
2346.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(26) Deng, H.; Liu, X.; Xie, J.; Yin, R.; Huang, N.; Gu Y.; Zhao, J. Quantitative and siteꢀdirected
chemical modification of hypocrellins toward direct drug delivery and effective photodynamic
activity. J. Med. Chem. 2012, 55, 1910ꢀ1919.
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