A potent antioxidant small molecule aimed at targeting metal-based oxidative stress in neurodegenerative disorders

Article abstract of DOI:10.1039/c2cc36808k

Metal-ion misregulation and oxidative stress have been linked to the progressive neurological decline associated with multiple neurodegenerative disorders. Transition metal-mediated oxidation of biomolecules via Fenton chemical reactions plays a role in d

Full text of DOI:10.1039/c2cc36808k

ChemComm
View Article Online
View Journal
COMMUNICATION
A potent antioxidant small molecule aimed at
targeting metal-based oxidative stress in
Cite this: DOI: 10.1039/c2cc36808k
neurodegenerative disorders†
Received 31st August 2012,
Kimberly M. Lincoln,a Paulina Gonzalez,a Timothy E. Richardson,b
Accepted 17th December 2012
David A. Julovich,b Ryker Saunders,a James W. Simpkinsb and Kayla N. Green*a
DOI: 10.1039/c2cc36808k
www.rsc.org/chemcomm
Metal-ion misregulation and oxidative stress have been linked to diseases to date which include: Huntington’s, Alzheimer’s (AD),
the progressive neurological decline associated with multiple Parkinson’s (PD) and Lou Gehrig’s disease.5,11 In AD increased levels
neurodegenerative disorders. Transition metal-mediated oxidation of Cu, Fe and Zn accumulate in the beta-amyloid plaque affected
of biomolecules via Fenton chemical reactions plays a role in regions of the brain, such as the hippocampus, and these regions
disease progression. Herein we report the synthesis, characterization display increased levels of oxidative injury.3,10 Furthermore, in PD
and antioxidant activity of 2; a pyclen derivative with enhanced total Fe levels are increased specifically in the substantia nigra, along
antioxidant character.
with a 30–40% decrease in glutathione concentration.10,11
Using the rationale that oxidative stress, in conjunction with
Oxygen plays a vital role in maintaining the energy metabolism metal ion misregulation are key factors in neurological disease
of living organisms.1 In fact, the brain comprises 2% of the development, current therapies for these disorders are aimed at
total body mass and consumes about 20% of total body oxygen. disrupting the aberrant metal ion chemical reactions that
Neurons and astrocytes are responsible for the brain’s enor- produce excess ROS.2,12,13 Treatment with Vitamin E and
mous use of oxygen, as it is utilized in cellular respiration and coenzyme Q10 in animal models of AD and PD, respectively,
for the biosynthesis of neurotransmitters.1,2 Due to its elevated were found to be neuroprotective but display no proven benefit
oxygen demand, the brain can become susceptible to oxidative in the clinical setting.1,10,14 These results are indicative of the
damage, induced by unregulated redox-active metals such as Fe need for a small molecule containing both ionophore and
and Cu.2–5 Reactions of free Cu or Fe with oxygen and a antioxidant activity.15,16 A small molecular system capable of
biological reductant such as ascorbate, can lead to an excess bimodal modulation of the metal-ions, as well as regulation of
production of reactive oxygen species (ROS) via Fenton ROS would prove useful in combating these disorders.
chemical reactions giving rise to oxidative stress.6,7
Previous studies have shown that tetraazamacrocycles are
Transition metal ions such as Cu, Fe and Zn are essential for capable of disrupting metal-induced beta-amyloid formation;
neuronal functions that involve free radical detoxification, electron in addition, we recently showed that 1, (Chart 1), possessed
transport, oxygen transport, neurotransmitter biosynthesis and antioxidant and protective capacity against ROS induced cell
neurotransmission.5,8,9 Disruptions or alterations in metal-ion death.5,12,14,17 Given this success we set-out to enhance the
regulatory pathways could contribute to an excessive build-up of antioxidant activity of 1, without affecting the metal ion
ROS; known to cause protein aggregation, free radical generation and chelation capability.18,19 The conversion from a pyridine to a
oxidative stress.10,11 Oxidative stress, coupled with transition metal pyridol, (2) was pursued as pyridols are known to react with
ion misregulation has been implicated in multiple neurological hydroxyl radicals via addition at the C3 or C5 position of the
pyridol ring with the products being reminiscent of tannins,
well known for their antioxidant power.3,18,20,21 Methods used
a Department of Chemistry, Texas Christian University, 2800 S. University, Ft.
Worth, USA. E-mail: kayla.green@tcu.edu; Fax: +1 817 257 5851;
Tel: +1 817 257 6220
b Institute for Aging and Alzheimer’s Disease Research, Department of Pharmacology
& Neuroscience, University of North Texas Health Science Center, 3500 Camp
Bowie, Ft. Worth, USA. E-mail: james.simpkins@unthsc.edu;
Fax: +1 817 735 2091; Tel: +1 817 735 0498
† Electronic supplementary information (ESI) available: Synthesis and character-
ization of 2, assay protocols and supporting figures. CCDC 905445 and 911804.
For ESI and crystallographic data in CIF or other electronic format see DOI:
Chart 1
10.1039/c2cc36808k
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun.

View Article Online
Communication
ChemComm
Furthermore 1 and cyclen‡ lack the capability to reduced DPPH,
as these absorbance values were equivalent to negative control
(95% EtOH) levels.
Cyclic voltammetry was then performed to confirm that 2
helped mitigate the redox behaviour of CuII.22 Fig. S1 (ESI†)
compares data obtained for the perchlorate salts of Cu(1)ꢁCl
and Cu(2)ꢁCl, E1/2 = ꢀ602 and ꢀ651 mV, respectively. The
pyridol group results in a shift of the Cu(II/I) couple to a slightly
more negative value than Cu(1) indicating the presence of the
hydroxyl group is slightly electron donating. These potentials
are 400 mV more negative than those expected to redox couple
with biological reductants such as ascorbate.
Fig. 1 Thermal ellipsoid plots at 50% probability of (a) [Cu(2)ꢁCl]+ and (b) [Zn(2)ꢁCl]+.
The perchlorate ion is removed for clarity.
Under biological conditions oxygen in the presence of
ascorbate and copper is catalytically reduced to a hydroxyl
radical; which can be detected by co-incubation with CCA, to
produce the fluorescent hydroxy-CCA species. We performed
this reaction in situ and showed that this process was halted by
the addition of 2 along with structurally similar chelates as well
(Fig. S2, ESI†), presumably by chelation of the copper resulting
in a shifting of the CuII/I potential outside the ascorbate
window as shown by Faller and co-workers.7,23 These in situ
studies show that 2 can counter-act two components involved
in oxidative stress: radicals and metal-ion redox cycling.
Next, cellular studies were carried out to evaluate the intra-
cellular antioxidant efficacy of 2 compared to 1, using the
cell-permeable fluorophore 2,70-dichlorodihydrofluorescin
diacetate, (DCFH-DA), as an indicator for ROS. DCFH-DA
diffuses into cells and is deacetylated by cellular esterases to
non-fluorescent 20,70-dichlorodihydrofluorescin, (DCFH), which
is subsequently oxidized to the highly fluorescent compound
20,70-dichlorodihydrofluorescein (DCF), in the presence of ROS.
The fluorescence intensity is directly proportional to the amount
of ROS present in cell cytosol. FRDA cells (Friedreich’s Ataxia
Fibroblasts) were utilized in this experiment, since these cells
have higher levels of cytosolic ROS resulting from mitochondrial
malfunction; therefore, serve as a good model for studying metal
induced oxidative stress.24 BSO (2-amino-4-(butylsulfonimidoyl)-
butanoic acid) was used to induce oxidative stress in the cells, as
it inhibits the synthesis of glutathione in cells.
to produce 2 are detailed in Schemes S1–S3 (ESI†). Reaction of
2, at pH 7, in water with either Cu(ClO4ꢀ)2 or Zn(ClO4ꢀ)2 readily
yielded crystals suitable for X-ray diffraction analysis. As shown
in Fig. 1, the coordination environment is similar to the CuII
and ZnII congeners reported for 1; as the ligand bends
producing a square pyramidal coordination around the metal
cation, with a chloride ion coordinated in the fifth axial
position to the metal ion.22 The CuII and ZnII complexes are
bent 1121 and 1371 with respect to the planes formed by the
N-donors and metal-ions. These measurements along with
other bond lengths and angles are consistent with other
N-heterocyclic complexes with these ions. A full comparison
of the XRD data can be found in the supplemental section
(Tables S1 and S2, ESI†).
Redox active metal ions produce a variety of species known
to cause cellular damage including radicals and superoxide. As
such, the capability of 2 to reduce radicals was investigated
using the radical DPPH, (2,2-diphenyl-1-picrylhydrazyl). DPPH
is a stable radical in solution having an absorbance maximum
at 520 nm. As DPPH is reduced, the absorbance intensity of the
solution decreases, along with a change in color from purple to
yellow. As shown in Fig. 2, the pyridol congener of 1 displays
the ability to reduce free radicals in solution at concentrations
ranging from 1000–100 mM; meanwhile, displaying greater
radical reducing ability at higher concentrations. Based on
these results, we can attribute the radical reducing function-
ality of 2 to its hydroxyl group, as this feature is lacking in 1.
The results of the DCFH-DA cell culture assay indicate that 1
is an effective antioxidant in the 1.25 nM–1.25 mM range. As
shown in Fig. 3, compound 2 is a potent antioxidant at each of
the concentrations tested from 1.25 nM to 1.25 mM. While 1 is
capable of quenching the ROS induced by BSO, 2 also inhibits
the naturally occurring ROS present when compared to
non-treated cells (media only). Fig. 3 shows at concentrations
of 1.25 nM, 12.5 nM, 125 nM and 1.25 mM for 2, there is a
decrease of 33%, 58%, 75%, and 87% [ROS], respectively,
compared to the control (media only). Again, we see that the
antioxidant activity of 2 increases with concentration, consis-
tent with the results from the DPPH study. Our studies indicate
that the binding of metals-ions to 2 is quite similar to 1,
therefore the enhanced antioxidant activity is attributed to
the pyridol component. Preliminary NMR data suggest –OH
addition onto the pyridol ring when 2 is incubated with H2O2.19
Currently we are working to fully characterize the product(s)
of 2 upon reaction with FeII and H2O2 (Fenton reaction).
Fig. 2 DPPH radical quenching assay showing the antioxidant character of 1
and 2 compared to the standard antioxidant BHT. n = 3. BHT = butylated hydroxyl
toluene (positive control).
c
Chem. Commun.
This journal is The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
of ROS. Cellular studies showed that 2 has a marked improve-
ment of preventing cellular death, induced by oxidative stress.
The authors are grateful for generous financial support from
TCU RCAF (to KG) and the Robert A. Welch Foundation (to KG,
P-1760), NIH (to JWS, P01 AG022550 and P01 AGAG027956) and
NIH training grant (to TER, T32 AG020494).
Notes and references
‡ Pyclen = 1,4,7,10-Tetraaza-2,6-pyridinophane, Cyclen = 1,4,7,10-Tetraaza-
cyclododecane, Hydroxylpyclen = 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca-
1(15),11,13-trien-13-ol, DCFH
= Dichlorodihydrofluorescein, BHT =
Fig. 3 DCFH-DA fluorescent response in FRDA cells after 12 hours exposure to
Butylhydroxytoluene, DPPH = 2,2-Diphenyl-1-picrylhydrazyl, BSO = Buthio-
nine sulphoximine, CCA = Coumarin-3-carboxylic acid, FRDA = Friedreich’s
ataxia.
BSO [1 mM] showing dose dependence with
1 and 2. n = 8. *Indicates
significance with respect to BSO control and a p value o 0.05.
1 S. Gandhi and A. Y. Abramov, Oxid. Med. Cell. Longevity, 2012,
2012, 428010.
2 J. K. Andersen, Nat. Med., 2004, 10(Suppl.), S18–S25.
3 K. Jomova, D. Vondrakova, M. Lawson and M. Valko, Mol. Cell.
Biochem., 2010, 345, 91–104.
4 A. I. Bush and R. E. Tanzi, Neurotherapeutics, 2008, 5, 421–432.
5 L. R. Perez and K. J. Franz, Dalton Trans., 2010, 39, 2177–2187.
6 S. Rivera-Mancia, I. Perez-Neri, C. Rios, L. Tristan-Lopez,
L. Rivera-Espinosa and S. Montes, Chem.–Biol. Interact., 2010, 186,
184–199.
7 L. Guilloreau, S. Combalbert, A. Sournia-Saquet, H. Mazarguil and
P. Faller, ChemBioChem, 2007, 8, 1317–1325.
8 L. Rossi, M. F. Lombardo, M. R. Ciriolo and G. Rotilio, Neurochem.
Res., 2004, 29, 493–504.
9 Y. H. Hung, A. I. Bush and R. A. Cherny, JBIC, J. Biol. Inorg. Chem.,
2010, 15, 61–76.
10 M. T. Fodero-Tavoletti, V. L. Villemagne, C. C. Rowe, C. L. Masters,
K. J. Barnham and R. Cappai, Int. J. Biochem. Cell Biol., 2011, 43,
1247–1251.
Fig. 4 Calcein AM viability assay of FRDA cells after 48 hour exposure to BSO
[1 mM] followed by addition of 2. n = 8 for each sample. *Indicates significance
with respect to BSO control and a p value o 0.05
11 E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008, 108,
1517–1549.
12 J. J. Braymer, A. S. DeToma, J. S. Choi, K. S. Ko and M.-H. Lim, Int. J.
Alzheimer’s Dis., 2011, 1–9.
13 F. Kielar, M. E. Helsel, Q. Wang and K. J. Franz, Metallomics, 2012, 4,
899–909.
14 L. E. Scott and C. Orvig, Chem. Rev., 2009, 109, 4885–4910.
15 E. Crabb and E. A. Moore, Metals and Life, Chelation Therapy,
Springer, 2009.
Preliminary 13C NMR data suggest electrophilic –OH addition
onto the pyridol ring, when 2 is incubated with H2O2; consis-
tent with similar compounds reported in literature.18–20
Finally, cell viability studies using live-cell penetrating
Calcein AM as a fluorophore show that 2 protects cells against
BSO insult ranging from 1.25 nM–12.5 mM, with an EC50 31.46 ꢂ
4.96 nM (Fig. 4). This dose response is visualized in the 16 P. A. Adlard, R. A. Cherny, D. I. Finkelstein, E. Gautier, E. Robb,
M. Cortes, I. Volitakis, X. Liu, J. P. Smith and K. Perez, et al., Neuron,
fluorescent cell images, Fig. S3 (ESI†), which show that cell
viability increases with an increasing concentration of 2. An
2008, 59, 43–55.
17 K. M. Lincoln, T. E. Richardson, L. Rutter, P. Gonzalez,
interesting feature to be noted from these cellular studies is the
fact that both 1 and 2 are capable of entering cells and do not
appear to interrupt the vital functions of cytosolic metalloen-
zymes, presumably via metal ion extraction.
Finally, the macrocyclic compound 2 is a chelator capable of
preventing metal-induced amyloid formation as well as disag-
J. W. Simpkins and K. N. Green, ACS Chem. Neurosci., 2012, 3,
919–927.
18 D. E. Green, M. L. Bowen, L. E. Scott, T. Storr, M. Merkel,
K. Bohmerle, K. H. Thompson, B. O. Patrick, H. J. Schugar and
C. Orvig, Dalton Trans., 2010, 39, 1604–1615.
19 J. A. Zazo, J. A. Casas, A. F. Mohedano, M. A. Gilarranz and
J. J. Rodriguez, Environ. Sci. Technol., 2005, 39, 9295–9302.
20 S. Steenken and P. Oneill, J. Phys. Chem., 1979, 83, 2407–2412.
gregation as evidenced by Turbidity and Tyr-10 fluorescence 21 V. Koleckar, K. Kubikova, Z. Rehakova, K. Kuca, D. Jun, L. Jahodar
and L. Opletal, Mini–Rev. Med. Chem., 2008, 8, 436–447.
studies which largely parallel the activity observed with 1
´
22 V. Felix, J. Costa, R. Delgado, M. G. B. Drew, M. T. Duarte and
(Fig. S4–S6, ESI†). These studies are consistent with the reactivity
of former chelating systems reported by others as well.25–27
In conclusion, a new small molecule 2 has shown potential
as a metal-binding and potent antioxidant agent. The antiox-
idant capacity of 1 was enhanced dramatically via conversion of
the pyridine backbone to a pyridol, 2. We have shown that this
compound is capable of reducing stable radicals, such as DPPH
and halts CuII/I redox cycling responsible for the production
C. Resende, Dalton Trans., 2001, 1462–1471.
23 C. Hureau and P. Faller, Biochimie, 2009, 91, 1212–1217.
24 T. E. Richardson, S. H. Yang, Y. Wen and J. W. Simpkins, Endocrinology,
2011, 152, 2742–2749.
25 L. E. Scott, B. D. G. Page, B. O. Patrick and C. Orvig, Dalton Trans.,
2008, 6364–6367.
26 W. Chen, X. Wang, Y. He, C. Zhang, Z. Wu, K. Liao, J. Wang and
Z. Guo, Inorg. Chem., 2009, 48, 5801–5809.
27 P. Faller, C. Hureau, P. Dorlet, P. Hellwig, Y. Coppel, F. Collin and
B. Alies, Coord. Chem. Rev., 2012, 256, 2381–2396.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun.