Disulfide Reductases – Current Developments
A. Irmler, A. Bechthold, E. Davioud-Charvet§, V. Hofmann, R. Réau#, S. Gromer*, R.
H. Schirmer* and K. Becker
Interdisciplinary Research Center, Giessen University, D-35392 Giessen, Germany
§
Institut de Biologie de Lille, CNRS-University, 59021 Lille cedex, France
#
Institute of Chemistry, UMR 6509, 35042 Rennes cedex, France
*Biochemistry Center, Heidelberg University, D-69120 Heidelberg, Germany
Introduction
The thioredoxin and the glutathione systems represent the two major antioxidant and
redox-regulatory principles in cells. Central players of these systems are the NADPHdependent flavoenzymes thioredoxin reductase (TrxR) and glutathione reductase
(GR). Both proteins belong to the family of homodimeric pyridine nucleotidedisulfide oxidoreductases which further includes e.g. lipoamide dehydrogenase,
trypanothione reductase, and mercuric ion reductase. Based on their central function in
redox homeostasis, disulfide reductases are most promising candidates for structure
based drug development (1). Whereas the mechanism and the structure of lipoamide
dehydrogenase and glutathione reductase are very much alike, thioredoxin reductases
are distinct. The smaller TrxR (35 kDa per subunit) in prokaryotes, plants, and fungi
lacks the C-terminal interface domain and differs fundamentally in 3-dimensional
structure and catalytic mechanism from the 55 to 60 kDa TrxRs identified so far in
mammals, Caenorhabditis elegans, Drosophila melanogaster and in Plasmodium
falciparum (2). The low Mr TrxRs use the same modules, e.g. NADPH-binding and
FAD-binding domains, as the high Mr enzymes but the active site and subunit
interface exhibit a completely different design: catalysis is accompanied by domain
movements which occur twice in each cycle (3-5). The high Mr TrxRs are
characterized by an additional C-terminal redox center represented by a Cys-Sec
sequence in human TrxR, by a Cys-Cys pair in TrxR from D. melanogaster and a CysXXXX-Cys motif in the enzyme from the malarial parasite P. falciparum. The
chemical difference between the disulfide group in PfTrxR and the selenylsulfide in
the mammalian enzyme may be exploited for drug design (6).
However, heterologous production of selenocysteine-containing proteins is hampered
by the need of a species-specific translation machinery involving a secondary structure
SECIS element in the mRNA that directs selenocysteine insertion at the position of the
UGA codon. Fusion of the human TrxR open reading frame with the SECIS element
of the bacterial selenoprotein formate dehydrogenase H leads to a catalytic activity of
16 %, in comparison to the wildtype enzyme with 100 %, the Sec498Cys mutant with
5 %, and the negligible activity of a C-terminally truncated protein (7).
Co-expression of the rat TrxR gene fused to the SECIS element of formate
dehydrogenase H with selA, selB and selC genes (encoding selenocysteine synthase,
SELB, and Sec-tRNA) even increased the activity to 25 % of the wildtype enzyme (8).
The determination of the 3-dimensional structure of the Sec498Cys mutant of rat TrxR
in a complex with NADP+ by x-ray crystallography revealed a striking similarity to
GR, including conserved amino acid residues which directly interact with glutathione
disulfide in GR despite the failure of glutathione disulfide to serve as a substrate for
TrxR (9). The folding of the C-terminal extension, typical for mammalian TrxRs and
distinct from GR, can approach the active site disulfide (Cys59-Cys64) of the other
subunit in the dimer, extending the electron transfer to the surface of the enzyme
where the substrate Trx is reduced. The movement of the C-tail may prevent the
enzyme from acting as a GR by blocking the redox active disulfide and could also
explain the broad substrate specificity of high Mr TrxRs that includes low molecular
weight compounds as well as proteins. Mammalian TrxR seems to have evolved from
the GR scaffold rather than from bacterial TrxR. Interestingly, P. falciparum
thioredoxin (PfTrx) was found to be a better substrate of hTrxR (KM = 2 HM, kcat =
3300 min-1) than of PfTrxR (KM = 10.4 HM, kcat = 3100 min-1) which suggests that the
parasite might employ host cell enzymes for its own purposes. Furthermore, PfTrxR
was found to reduce glutathione disulfide in a non-enzymatic reaction, the rate
constant k2 being 0.039 HM-1 min-1 at 25 °C and pH 7.4 (10). In Drosophila, this
reaction is likely to drive glutathione metabolism since this organism - and probably
many other insects - lack a genuine GR (11).
Various mutational analyses proved the strict dependence of mammalian TrxR activity
on selenium. A Sec498Cys mutant of rat TrxR exhibited a catalytic activity in
reduction of thioredoxin with a 100-fold lower kcat and a 10-fold higher KM compared
with the wildtype rat enzyme and a total loss of hydrogen peroxidase activity. No
activity could be detected in the C-terminally truncated enzyme and in the product of a
SECIS element deletion mutant (12). The electron transport ensembles in hTrxR have
been confirmed by Lee et al. (13), who demonstrated that the C-terminal 497SH/498SeH pair of the reduced enzyme is converted by excess TrxS2 or H2O2 to a
thioselenide while the dithiol pair Cys 59/Cys64 is oxidized to give a disulfide. The
kinetic properties and inhibitor studies of specific active site mutants of rat TrxR
provided evidence that both Cys and Sec residues at the C-terminal redox center are
essential for the reduction of thioredoxin and that these residues function coordinately
with the active site sulfhydryls of the other subunit (14). Stopped-flow kinetics and
dithionite titrations on the reductive half-reaction of hTrxR as well as the
susceptibility of the NADPH reduced enzyme to trypsin digestion have supported the
hypothesis of redox communication between the flexible C-tail and the active site
dithiol (4, 15). Taken together, the data obtained with kinetic studies, mutant analyses
and spectral characterization now represent a model describing the catalytic
mechanism of mammalian TrxR.
Whether the active site residues of P. falciparum TrxR are provided by one or by both
subunits, as in the mammalian TrxR, has been studied by co-expression of the double
mutant PfTrxR-C88AC535A with PfTrxR wildtype which generates an inactive
heterodimer. This indicates that the C-terminal cysteines of one subunit of P.
falciparum TrxR are in communication with the active site thiol(ate)s of the other
subunit of the dimer, which means that the interaction of the two subunits at both
active sites is obligatory for catalysis (16, 17).
Three types of mammalian TrxRs are known, with TrxR1 being a cytosolic enzyme
and TrxR2 a mitochondrial enzyme. Both are ubiquitously expressed and exhibit 52 %
– 53 % homology. TrxR3, which has not been further characterized, is present in testis
and shows 70 % homology to TrxR1 and TrxR2 (18, 19).
Recently, a new type of pyridine nucleotide disulfide oxidoreductase has been
detected in mouse and Schistosoma mansoni, the selenocysteine-containing Trx and
GSSG reductase (TGR). This enzyme displays TrxR, GR and glutaredoxin activities
and seems to completely replace TrxR and GR in adult worms of Schistosoma. The
proposed electron flow within TGR includes several redox centers and the flexible Ctail as in hTrxR: NADPH
FAD
dithiol/disulfide center
C-terminal Seccontaining center
Cys residues within the Grx domain
downstream substrate
(20, 21).
Apart from the characterized mammalian homodimeric TrxR1 and TrxR2 with 55 - 57
kDa subunits, multiple forms of these enzymes exist; particularly, a 67 kDa form of
TrxR1 was detected. Homology analyses revealed three distinct isoforms of mouse
and rat TrxR1 mRNA which differed in 5' sequences that result from alternative use of
the first three exons but had common downstream sequences. These multiple
transcription start sites may be cell type specific or may have regulatory function (22).
The gene organisation of the human and mouse trxr1 gene is highly conserved,
however, in contrast to mouse, for the human trxr1 gene, six mRNA isoforms were
detected which differ at the 5' end and encode putative proteins of different molecular
mass (23). Other studies showed that the promoters of mouse and human trxr1 genes
share some conserved sequences and that the human TrxR1 promoter fulfills the
typical criteria for a housekeeping gene (24). In Drosophila a single gene, Trxr1,
codes for two forms of the enzyme, a cytoplasmic and a mitochondrial one. The two
Drosophila trxr1 variants differ in their N-terminal sequences. Both TrxR1 forms are
functionally distinct in vivo and are essential for viability (25).
To facilitate drug screening programs on large TrxRs, analogues of 5,5'-dithiobis(2nitrobenzoate) (DTNB) have been developed and tested. These alternative substrates,
5,5'-dithiobis(2-nitrobenzamides) have >20-fold lower KM values than DTNB and are
therefore less likely to interfere with inhibitor testing (26). Recently, methylseleninate,
the precursor of the key metabolite methylselenol, which had previously been reported
to inhibit TrxRs, was found to serve as an excellent substrate instead (27).
Due to its central role in cell metabolism, the thioredoxin system is also involved in
many pathological conditions and provides potential targets for therapeutic
approaches. The organic gold compounds auranofin and aurothioglucose which are
widely used in the treatment of rheumatoid arthritis, inhibit the NADPH-reduced form
of hTrxR specifically with a Ki of 4 nM. GR and the selenoenzyme glutathione
peroxidase are inhibited at a 1000-fold higher concentration of these drugs (28).
Tumour cells have been observed to express several fold increased TrxR levels and
some potent antineoplastic agents such as carmustine, fotemustine and cisplatin are
effective inhibitors of mammalian TrxRs. (2,2´:6´,2´´-terpyridine) platinum(II)
complexes have been identified as stoichiometric and irreversible inhibitors of
NADPH-reduced hTrxR (29). For the most potent agent, N,S-bis(2,2´:6´,2´´terpyridine) platinum(II)-thioacetimine trinitrate, the Ki is 4 nM toward hTrxR
whereas GR is inhibited 1000-fold less which indicates a very high specificity for
hTrxR. The platinum complexes were found to suppress the proliferation of three
different glioblastoma cell lines as well as of two different head-and-neck squamous
carcinoma cell lines (29).
Cisplatin had been considered to inhibit hTrxR directly. Now, Arnér et al. (30) have
shown that the major metabolic product of cisplatin, a glutathione-platinum II
complex, is able to inhibit mammalian TrxR as well as glutaredoxin, the latter protein
being unaffected by the parent drug.
Further, water-soluble organotellurium compounds were described to inhibit TrxR and
the growth of human cancer cells at the low micromolar level, but the hydrophilicity
seems to restrict cellular uptake (31).
Novel inhibitors of the Trx/TrxR system are naphthoquinone (NQ) derivatives with
IC50 values down to 350 nM. These compounds were found to inhibit growth of two
breast cancer cell lines at the low micromolar level (32).
As a novel class of potential antimalarial agents, double headed prodrugs were
developed and successfully tested in vitro and in vivo (33). The most potent drug is
composed of a 4-anilinoquinoline (a chloroquine derivative) linked via a hydrolasesensitive labile ester bond to a 1,4-naphthoquinone acting as a potent PfGR inhibitor.
This compound was found to be equally effective against chloroquine-sensitive and
chloroquine-resistant P. falciparum strains, the EC50 value being below 30 nM.
Consistently, treatment with this drug led to a highly significant increase of survival
time in P. berghei infected mice.
Physiologically, peroxynitrite contributes to the control of malarial parasites in man
and mosquitoes. ONOO- inhibits hGR and PfGR in the micromolar range, with PfGR
being more sensitive than human GR (34). In contrast to other NO-containing
compounds, peroxynitrite does not lead to irreversible modification of active site
thiols. The crystal structure of ONOO--modified hGR at 1.9 Å resolution revealed that
inhibition is due to the exclusive nitration of 2 tyrosine residues (Tyr 106 and Tyr 114)
at the glutathione disulfide binding site.
Recombinant PfGR was successfully crystallized in our laboratory. The structure of
this important target molecule has recently been solved by Andrew Karplus and his
team at Oregon State University in Corvallis and opens novel avenues for antiparasitic
drug development.
In this paper we describe two new series of potent irreversible TrxR inhibitors,
naphthazarin derivatives and Pd complexes of phospholes, as well as their inhibitory
characteristics.
Materials and Methods
The Trx and the TrxR genes were obtained by PCR amplification from a human cDNA
library with sequence derived primers. Site directed mutagenesis (QuikChange,
Stratagene) performed on the respective genes was used for the change of cysteine 72
into serine in Trx (Trx-C72S) and for the replacement of selenocysteine by cysteine in
hTrxR (TrxR-Sec498Cys). Expression of the genes was carried out in the pQE30/M15 system (Quiagen) and the produced His-tagged proteins were purified to
homogeneity via a Ni-NTA column. Non-modified hTrxR for the inhibitor studies was
obtained by the purification procedure from placenta as previously described (28). The
enzyme assays were principally performed as published (29). Briefly, the assays were
conducted at 22°C in a total volume of 1 ml. For determining TrxR activity, the
enzyme was added to a buffer consisting of 100 mM potassium phosphate, 2 mM
EDTA, pH 7.4, and 3 mM DTNB or 20 HM Trx-C72S. The reaction was started by
adding 200 HM NADPH (for the DTNB-assay) or 100 HM NADPH (for the Trxassay) and the change in absorbance at 412 nm and 340 nm, respectively, was
monitored. The GR assay was performed in a buffer containing 47 mM potassium
phosphate, 1 mM EDTA, 200 mM KCl, pH 6.9, and 100 HM NADPH. The assay was
started with 1 mM GSSG and the consumption of NADPH was detected by the
decrease in absorbance at 340 nm. The inhibitory compounds were preincubated with
the enzyme for 2 min - 10 min before starting the assay.
Results and Discussion
Inhibition of human TrxR by naphthazarin and derivatives
Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) is known to display antiplasmodial
and anticancer activities and to inhibit both glutathione reductase and thioredoxin
reductase. The attack of the quinone moiety by glutathione is involved in cytotoxic
properties of naphthazarin, therefore derivatives were designed that were alkylated at
carbons 2 and 3 to prevent formation of thiol conjugates and to allow introduction of
structural diversity (35). The synthesized 2,3-dimethylnaphthazarin (JD155) was
further modified by bromination into the 2-bromomethyl derivative (JD159), or the 6bromomethyl derivative (JD141), or the dibromomethyl derivative (JD145), and by
bromine substitution into the 2-acetoxymethyl derivative (JD231) as depicted in Table
1.
In comparison to the unmodified naphthazarin (NZ) with an IC50 of 650 nM toward
hTrxR in the DTNB assay, the presence of the methyl groups in JD155 decreases the
IC50 value to 200 nM. Bromination of the aromatic ring, however, results in an
inhibition at almost stoichiometric concentrations of the compounds JD141 (IC50 = 5
nM) and JD145 (IC50 = 8 nM/5 nM) with DTNB as well as with human Trx-C72S as
substrate. The inhibitory potential of the acetylated derivative JD231 (IC50 = 150 nM)
is not as strong as that of the brominated structures but also reduces the IC50 when
compared with NZ and JD155.
Competition with DTNB and NADPH
When representing the inhibitory data of NZ in a Dixon-plot, an initial competitive
character of the inhibition became evident (Fig. 1). The Ki value was calculated to be
2 HM. For the compounds JD155, JD159, JD145, JD141, and JD231, the type of
inhibition seems to be complex with a superposition of different inhibition types. As
described below, the initial competition seems to be followed by an irreversible
enzyme inhibition. The inhibitors JD141 and JD231 were also tested for their
competition with NADPH but neither of the compounds was able to significantly
increase the KM for NADPH.
Inhibition of P. falciparum TrxR by naphthazarin and derivatives
The inhibitory effect of NZ on PfTrxR (IC50 = 600 nM) was found to be comparable
to that of hTrxR in the DTNB assay (Table 2). The naphthazarin derivatives were able
to inhibit the PfTrxR activity with IC50 values between 0.3 HM (JD145, JD141) and 6
HM (JD155). This indicates that the inhibitory potential toward PfTrxR was in
principle lower than toward hTrxR. In comparison with hTrxR, it was easier to
measure the initial competitive component of the inhibition on PfTrxR.
Inhibitor
IC50 on hTrxR
in the DTNB-Assay
Structure
OH
O
650 nM
NZ
OH
O
OH
O
200 nM
JD 155
OH
O
OH
O
Br
JD 159
OH
OH
JD 145
O
Br
OH
JD 141
O
Br
OH
80 nM
8 nM
(Trx-Assay: 5 nM)
O
O
5 nM
(Trx-Assay: 5 nM)
Br
OH
O
OH
O
OAc
JD 231
OH
150 nM
O
Table 1: Inhibition of hTrxR by naphthazarin and derivatives.
The reversibility of the inhibition of hTrxR and PfTrxR was tested in detail for JD231.
Exhaustive dialysis of the inhibited enzyme against TrxR assay buffer led to a
recovery of enzymatic activity of no more than 25 % when compared with controls.
This indicates that the inhibition of TrxRs by naphthazarin derivatives is very stable.
The inhibition was furthermore found to be time-dependent. A previously reported
mechanism for bioreductive alkylation of thiol proteins by 2-halogenomethyl-1,4-NQ
involves the formation of a NQ methide intermediate followed by Michael addition of
a nucleophile (36).
4
1/V
[ml/U]
100 M
3
200 M
2
300 M
400 M
1
500 M
0
-4
-2
0
2
4
6
8
NZ [ M]
Fig.1: Dixon-plot for the inhibition of hTrxR by naphthazarin (NZ). The competitive
component of inhibition becomes evident at DTNB concentrations in the KM range.
Inhibition of hTrxR by phospholes
Phospholes are phosphacyclopentadienes possessing a weak aromatic character and a
nucleophilic phosphorus atom in one ring allowing chemical modification (37).
Because of the structural similarity to the platinum-complexes they are interesting
candidates for inhibitor studies on disulfide reductases. The effect of different
phosphole compounds (P1–P8) on hTrxR activity was tested with the substrate DTNB
as well as with hTrx-C72S. As shown in Table 3, 4 HM and 5 HM of the compounds
P1, P3, P4 and P5 led to an inhibition of 50 % of hTrxR activity when the substrate
hTrx-C72S was used. The inhibitory effect of P2 was much lower. Surprisingly, with
the substrate DTNB, P1 and P2 seemed to activate the enzyme activity, but did not
serve as substrates themselves, whereas P3, P4 and P5 inhibited the enzyme in the
lower micromolar range (IC50 = 5 HM–20 HM).
The presence of palladium in the compounds P6, P7, and P8, however, drastically
increased the inhibitory potential to stoichiometric concentrations. With both
substrates, DTNB and hTrx-C72S, a 1000-fold higher inhibition (IC50 = 5 nM) of
hTrxR activity could be observed, as shown in Table 4. The determination of the
inhibitory types with DTNB, as well as with NADPH and hTrx-C72S as substrates
yielded ambiguous results indicative of a complex mechanism.
Inhibitor
Structure
OH
IC50
OH
O
OH
O
JD 155
OH
O
OH
O
Br
JD 159
JD 141
OH
O
OH
O
Br
Br
OH
O
OH
O
Br
OH
O
OH
O
OAc
JD 231
OH
Ki
O
NZ
JD 145
Type of inhibition
(initial)
competitive
1.8 HM
600 nM
(with the disulfide
substrate)
6 HM
complex
1.2 HM
competitive
5 HM
300 nM
competitive
0.5 HM
300 nM
competitive
0.5 HM
1 HM
competitive
5 HM
O
Table 2: Inhibition of PfTrxR by naphthazarin and derivatives. Characterization of the
inhibitors by the IC50- and Ki-values and the inhibitor type.
Inhibition of hGR by phospholes
To test the specificity of hTrxR inhibition by the phosphole compounds (in the
presence of 3 mM DTNB), the inhibitory effects on the closely related enzyme hGR
(in the presence of 100 HM GSSG) was determined. Table 5 shows the IC50 values of
the phospholes P3–P8 on hGR and, for comparison, the values on hTrxR. The
compounds P3–P5 inhibit hGR slightly stronger in the micromolar range than hTrxR,
suggesting a similiar kind of binding to both enzymes.
Inhibitor
Structure
P1
P2
N
P
N
P
P3
S
P4
S
P5
S
P
S
N
S
S
S
P
W(CO)5
P
Se
S
IC50 on hTrxR
with 3 mM DTNB
activation!
IC50 on hTrxR
with 20 /M hTrxC72S
~ 5 HM
(methanol)
activation!
35% inhibition
by 40 HM P2
(THF)
~ 5 HM
~ 5 HM
~ 6 HM
~ 5 HM
20 HM
~ 4 HM
Table 3: Effect of the phospholes P1-P5 on hTrxR activity with DTNB or hTrx-C72S
as substrates and characterization of the inhibitory potential by the IC50-values.
W(CO)5 is an inorganic Lewis acid. If not otherwise stated, the inhibitors were
dissolved in DMSO.
The palladium-containing compounds P6–P8, however, have more than 30-fold higher
IC50 values for hGR than for hTrxR. This indicates, as in the case of hTrxR-inhibition
by auranofin and the (2,2':6',2''-terpyridine)platinum(II) complexes (28, 29), that the
C-terminal selenocysteine of hTrxR is likely to be targeted by the inhibitors.
Inhibitor
P6
Structure
N
Pd
Cl Cl
P7
P8
P
P
Pd
Cl
Cl
N
P
Pd
H3C Cl
N
N
S
N
IC50 on hTrxR
with 3 mM DTNB
IC50 on hTrxR
with 20 /M hTrx
5 nM
5 nM
5 nM
5 nM
5 nM
5 nM
Table 4: Effect of the palladium containing phospholes P6-P8 on hTrxR activity with
DTNB or hTrx-C72S as substrates and characterization of the inhibitory potential by
the IC50-values. If not otherwise stated, the inhibitors were dissolved in DMSO.
Inhibitor
IC50 on hGR
IC50 on hTrxR
dissolved in
P3
1.5 HM
5 HM
DMSO
P4
1 HM
6 HM
DMSO
P5
2 HM
20 HM
DMSO
P6
1.4 HM
8.5 nM
Ethanol
P7
350 nM
10 nM
Ethanol
P8
320 nM
10 nM
Ethanol
Table 5: Inhibition of hGR (in the presence of 100 HM GSSG) by the phospholes in
comparison to hTrxR (in the presence of 3 mM DTNB). The DMSO concentrations in
the inhibition assays were too low to initiate conformational changes in hGR by this
co-solvent (38).
Inhibition of hTrxR and of the mutant hTrxR-Sec498Cys by (2,2':6',2''terpyridine)platinum(II) complexes
The hypothesis of the penultimate selenocysteine of hTrxR being the main target of
the inhibitor attack was supported when comparing the inhibition of hTrxR and its
mutant hTrxR-Sec498Cys. The specific activity of the mutant enzyme was nearly 100fold lower (0.4 U/mg for the mutant, 30 U/mg for the wildtype) as previously
described (39). We now tested the inhibition of wildtype and mutant TrxR by three
different (2,2':6',2''-terpyridine)platinum(II) complexes using Trx-C72S and DTNB,
respectively, as substrates. Incubation of the wildtype enzyme for 2 min with the
compounds resulted in IC50 values from 8 nM to 70 nM. This is in accordance with
previously reported data (29). Incubation of the hTrxR-Sec498Cys mutant, however,
resulted in nearly 1000-fold increased IC50 values (10 HM to 26 HM). These data
strongly indicate that the selenocysteine residue of hTrxR is the site of action of the
platinum-complexes tested.
Acknowledgements
The authors wish to thank Prof. Gordon Lowe, Oxford, for kindly placing inhibitors at
our disposal. The study was supported by the Deutsche Forschungsgemeinschaft (Be
1540/6-2 within the Research Focus on Selenoproteins).
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