(Received for publication, April 11, 1995; and in revised form, June 29, 1995 )
From the
A protein with high affinity (K 12
nM) for the immunomodulatory compound A77 1726 has been
isolated from mouse spleen and identified as the mitochondrial enzyme
dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a
pI 9.6-9.8 and a subunit M
of 43,000.
Peptides derived from the mouse protein displayed high microsequence
similarity to human and rat dihydroorotate dehydrogenase with,
respectively, 35 and 39 out of 43 identified amino acids identical.
Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative
effects of A77 1726 are mediated by enzyme inhibition and can be
overcome by addition of exogenous uridine. The rank order of potency of
A77 1726 and its analogues in binding or enzyme inhibition was similar
to that for inhibition of the mouse delayed type hypersensitivity
response. It is proposed that inhibition of dihydroorotate
dehydrogenase is an in vivo mechanism of action of the A77
1726 class of compounds. This was confirmed using uridine to counteract
inhibition of the murine acute graft versus host response.
Leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,
HWA 486) is a novel immunomodulatory and anti-inflammatory compound
currently under evaluation in phase III clinical trials for the
treatment of rheumatoid arthritis. It has shown dose-dependent clinical
efficacy in a 6-month, double-blind, placebo-controlled study on
patients with long-standing active arthritis, 81% of whom had failed
previous disease modifying anti-rheumatic drug therapy(1) . In
addition it has been shown to be effective in controlling the
development of autoimmune disorders and delaying transplant rejection
in animals(2, 3, 4, 5) . Its primary
metabolite A77 1726 (Fig. Z1), which mediates the
immunosuppressive and disease-modifying effects of the parent drug,
inhibits proliferation of cell lines and mitogen- or
cytokine-stimulated lymphoid cells in vitro(6, 7) by inhibiting progression from the G to the S phase of the cell cycle(7) . Although the
biochemical mechanisms by which A77 1726 exerts its effects are
unknown, they have been shown to differ from those of other
immunosuppressive agents such as corticosteroids, cyclosporin A,
rapamycin, or mycophenolic acid(7) .
Figure Z1: Structure 1Chemical structure of A77 1726.
The results presented here describe the purification of a high affinity binding site for A77 1726 and its identification as the mitochondrial enzyme dihydroorotate dehydrogenase. Evidence is presented that identifies the enzyme as a mediator of the in vitro and in vivo effects of the compound (see Structures 1 and 2).
Figure Z2: Structure 2Chemical structure of RU35072.
For solubilization, the membranes were
diluted to 6 mg/ml protein and mixed with an equal volume of
homogenization buffer lacking sucrose but containing 1% nonyl
glucoside, 2 mM EDTA, and 2 mM EGTA. The mixture was
stirred for 1 h and then centrifuged at 120,000 g for
60 min. The supernatant was stored at -80 °C for use in
binding and preliminary purification studies or filtered through
Millipore AP25 prefilters immediately prior to use in full purification
studies.
The eluate fraction was thawed and dialyzed
against 1 mM sodium phosphate, 0.5% nonyl glucoside, pH 7.0,
over a 2-h period. The fraction was then applied at 1 ml/min to a 4
1.6-cm hydroxylapatite column previously equilibrated with
dialysis buffer. The column was washed (60 ml) and eluted at 0.5 ml/min
in dialysis buffer with a linear 0-15 mM MgCl
gradient (30 ml) and a 15 mM MgCl
plateau
(10 ml). Remaining proteins were removed by successive washes with 0
mM MgCl
(6 ml), 300 mM sodium phosphate,
pH 7.0 (15 ml), and 500 mM sodium phosphate, pH 7.0 (15 ml at
room temperature).
The peak of eluted A77 1726 binding activity (12
ml) was desalted on PD-10 columns into chromatofocusing elution buffer
(1:45 dilution Pharmalyte pH 8-10.5, 0.5% nonyl glucoside, pH
8.0). A 29 0.5-cm column of PBE118 polybuffer exchanger, topped
with 1 cm of Sephadex G-25 course, was equilibrated with 400-800
ml of 25 mM triethylamine-HCl, pH 11, followed by 100 ml of
the same buffer containing 0.5% nonyl glucoside. Following application
of 5 ml of elution buffer, the desalted sample was applied at 0.5
ml/min and eluted with 150 ml of elution buffer. Remaining protein was
eluted with a 1 M NaCl wash. Eluate fractions were stored at
-80 °C.
Of the 23 peptide peaks collected, 5 were dissolved in 40% acetonitrile and applied to Polybrene-treated fiberglass filters. However, no phenylthiohydantoin-derivatives were released during attempted sequencing. The remaining 18 samples were dissolved in 0.1% trifluoroacetic acid and then 10 mM Tris-HCl, 0.05% SDS and applied to polyvinylidene difluoride membranes (Prospin, Applied Biosystems) with sequential 80-µl washes of 10 mM Tris-HCl, 0.05% SDS, pH 7.5 (twice), and 20% methanol (three times). The peptide on each membrane was sequenced directly in a model 473A microsequencer cartridge (Applied Biosystems).
Electrophoretic transfer onto Immobilon-P was carried out on a
Multiphor II/Novablot semidry system at a constant current of 2
mA/cm for 60 min in 48 mM Tris-HCl, 39 mM glycine, 0.0375% SDS, 20% methanol or 90 min in a CAPS buffer
system(13) .
The binding characteristics of a series of A77 1726 analogues were tested in competition binding studies. The rank order of potency of the compounds for affinity at the high affinity site was similar to that for the potency of inhibition in the mouse DTH assay (Table 1, part b). Compounds 2 and 6 appeared to have higher affinity than expected from their DTH potency; however the differences were small in view of the in vitro/in vivo nature of the comparison.
The high affinity binding site was solubilized with 0.5% nonyl glucoside (45% yield: Table 1, part a). The site showed maintained affinity for A77 1726. The moderate affinity site was not solubilized in a detectable form.
Figure 1:
Representative profiles of selected
fractions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
All samples were separated on discontinuous gels comprising a 5%
stacking gel and 10% resolving gel (except for the autoradiograph of
the SP-Sepharose eluate where a 4% stacking gel/12% resolving gel was
used). Chromatofocusing eluate, electrotransfered onto an Immobilon-P
membrane and Coomassie-stained (lane1).
Autoradiographs of photoaffinity-labeled soluble preparation (lane2), SP-Sepharose eluate (lane3),
hydroxylapatite eluate (lane4), and chromatofocusing
eluate (lane5). In each case, fractions for
photoaffinity labeling were incubated in the dark for 1 h with the
[I]iodoazido ligand (see Fig. Z2) prior
to photolysis (lanes2a, 3a, 4a,
and 5a). Parallel incubations, in which the photoaffinity
ligand was prevented from binding to the high affinity site by 10
µM HR325 (lane2b) or 1 µM A77 1726 (lanes3c, 4c, and 5c), were used to identify the site against a possible
background of low affinity sites. After photolysis the non-covalently
bound photolabel was removed by dilution with 3 mM HR325 and
PD10 desalting. The binding characteristics of the photoaffinity label
were examined in preliminary studies carried out in the dark using the
non-radioactive (
I) form of the compound. The
photoaffinity ligand had a K
of 29.1
nM for the mouse spleen high affinity site in a
[
H]A77 1726 competition binding study. The
reversible nature of the binding interaction before photolysis was
further established in an experiment in which membranes were incubated
for 2 h with 235 nM photoaffinity ligand. Although this
concentration should occupy >85% of the high affinity sites, more
than 98% of the original binding in the membranes was detected
following dilution of the membranes into a [
H]A77
1726 binding assay (5 nM final photoaffinity ligand
concentration). Irreversible binding of the radioactive photolabel to
mouse spleen membranes was not observed in the absence of photolysis
(data not shown). Molecular size markers are indicated in full for 1
and 2, by position for 3 and by position for M
45,000 and 36,000 markers only for 4 and 5. The M
43,000 bands in the different gels are
aligned.
An attempt at obtaining N-terminal sequence from the M 43,000 band was unsuccessful. In order to obtain
internal sequence, the purification procedure was scaled up 2-fold,
yielding 210 pmol of high affinity site, 1% of which produced a faint
single band of M
43,000 on SDS-PAGE and Coomassie
staining. The eluate was subjected to reverse phase high performance
liquid chromatography to remove detergent-derived non-protein
contaminants, which would prevent peptide detection, together with any
minor contaminating proteins not detected on the Coomassie-stained gel.
The M
43,000 protein was subjected to
endopeptidase digestion (Asp-N), with the resulting peptides separated
by an additional round of reverse phase chromatography. Sequence data
was obtained for 7 of the 18 peptides applied to polyvinylidene
difluoride membranes (Fig. 2). A search of the Swissprot data
bank revealed that only human dihydroorotate dehydrogenase (DHO-DH) had
amino acid sequence similarity with all the sequenced peptides. For the
five peptides that could be unambiguously aligned with the human
sequence, 35 out of 43 assigned amino acids were identical (Fig. 2). A greater degree of similarity was observed when the
peptides were compared with rat DHO-DH sequence (EMBL data base,
accession no. X80778) with 39 out of 43 residues identical. The
remaining two pentapeptides could be aligned with several possible
sequences within the human and rat proteins, with 3 of the 5 residues
being identical in each case.
Figure 2: Amino acid sequence homology between the mouse spleen high affinity A77 1726 binding site and human and rat dihydroorotate dehydrogenase. Amino acid sequence of peptides isolated from Asp-N digestion of the purified high affinity binding site (see ``Experimental Procedures''). Two peptides from the mouse could not be lined up with human or rat sequence as they produced partial matches (3 out of 5 amino acids) with several sequences within the human and rat proteins. ?, no assignment possible; * highlights sequence differences; , T or P; +, L or V.
A77 1726 analogues displayed similar
rank order of potency for affinity at the high affinity site (K) and IC
for enzyme inhibition
(correlation coefficient 0.986 for compounds in Table 1with
defined IC
values).
Figure 3:
Effect of
uridine on antiproliferative activity of A77 1726. Tritiated thymidine
incorporation in mouse spleen cells stimulated with LPS and incubated
for 3 days in the presence of a range of A77 1726 concentrations in the
presence () or absence (
) of 30 µM uridine
(see ``Experimental Procedures'' for detail). Control cells
were cultured alone (177 cpm) or with LPS (59,510 cpm), LPS + 30
µM uridine (54,604 cpm), LPS + Me
SO
(54,300 cpm), and LPS + Me
SO + 30 µM uridine (47,661 cpm).
The aim of the current study was to isolate and characterize
the target protein that mediates the effects of A77 1726, the active
metabolite of leflunomide. A potential target protein was identified in
mouse spleen membranes using a radioligand binding approach (Table 1). Although the membranes carried at least three binding
sites, the high affinity site (K 12 nM)
was of most interest as its binding pharmacology for A77 1726 analogues
was qualitatively similar to that for the mouse DTH response in
vivo (Table 1). The DTH assay was used as a primary screen
to detect active compounds; as such, dose ranges were limited,
preventing quantitative correlation between binding and potency.
However, the qualitative relationship, which extended to over 70
compounds, (
)supported the role of the high affinity site in
drug action in vivo.
The purified site was unequivocally
identified as DHO-DH by a series of structural and functional criteria.
There was high microsequence identity between human enzyme (81%: (19) ) or rat enzyme (91%: EMBL data base) and pure peptides
derived from the high affinity site (Fig. 2). In addition, the
relative molecular weight of 43,000 for the site on SDS-PAGE (Fig. 1) was close to that of the bovine liver enzyme (M 42,000; (20) ) and that predicted for
human DHO-DH (43.0 kDa; (19) ). The weak silver staining of the
mouse protein is consistent with a protein lacking cysteine residues,
as is the case with the human enzyme(19) . A77 1726 binding
activity and dihydroorotate dehydrogenase activity co-purified through
all steps of the purification procedure. Finally, binding affinity of a
series of A77 1726 analogues correlated with enzyme inhibitory potency
of the compounds.
Dihydroorotate dehydrogenase (EC 1.3.3.1) catalyzes conversion of dihydroorotate to orotate, the fourth step in de novo biosynthesis of pyrimidine nucleotides(21) . Inhibition of dihydroorotate dehydrogenase activity by A77 1726 and its analogues would clearly have an anti-proliferative effect on a wide range of cells due to depletion of intracellular pools of UTP and CTP, as is the case for other known inhibitors of pyrimidine biosynthesis(22, 23) . That this is solely responsible for the anti-proliferative effects of the compound is indicated by maintained proliferation in LPS-stimulated mouse spleen cells receiving exogenous uridine during A77 1726 treatment (Fig. 3). Similar effects have been observed with other inhibitors of pyrimidine biosynthesis(22, 23) .
The ability of uridine to protect mice from inhibition of the acute graft versus host response (Table 3) directly demonstrates the role of DHO-DH as mediator of the in vivo effects of the A77 1726 class of compounds in this system. At least one other in vivo immunosuppressive effect of the compounds may be mediated by inhibition of de novo pyrimidine biosynthesis, since inhibition of the DTH response in mice shows a qualitative relationship with binding affinity and with inhibition of DHO-DH activity (Table 1).
The possibility that the A77 1726 class of compounds may have additional effects in vivo or in vitro cannot be ruled out by the current studies. However, some of the previously reported effects of long term treatment of cells with the compounds could be secondary to pyrimidine depletion. For example, inhibition of epidermal growth factor-dependent tyrosine kinase activity in intact cells (24) was demonstrated after a 4-day treatment with A77 1726, whereas a 30-min treatment had no effect(7) . However, proof that this effect reflects pyrimidine nucleotide depletion awaits uridine reversal studies.
In conclusion, the A77 1726 high affinity binding protein in murine spleen membranes, proposed as a putative target for A77 1726 action, has been isolated and identified as dihydroorotate dehydrogenase. The enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis and its inhibition accounts for the antiproliferative effects of the compounds in vitro and some of the in vivo effects of the compounds.