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Antimicrobial Agents and Chemotherapy, September 2001, p. 2517-2523, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2517-2523.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Isolation of Rat Dihydrofolate Reductase Gene and
Characterization of Recombinant Enzyme
Yangzhou
Wang,1
Jeremy A.
Bruenn,1
Sherry F.
Queener,2 and
Vivian
Cody1,*
Structural Biology Department, Hauptman
Woodward Medical Research Institute, Buffalo, New York
14203,1 and Department of
Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 462022
Received 11 December 2000/Returned for modification 5 March
2001/Accepted 18 June 2001
 |
ABSTRACT |
While assays of many antifolate inhibitors for dihydrofolate
reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we
report the isolation of the rat DHFR gene through screening of a rat
liver cDNA library. The rat liver DHFR gene has an open reading frame
of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat
enzyme with those from other species indicate a high level of
conservation at the primary sequence level and more so for the amino
acid residues comprising the active site of the enzyme. Expression of
the rat DHFR gene in bacteria produced a recombinant protein with high
enzymatic activity. The recombinant protein also paralleled the human
enzyme with respect to the inhibition by most of the antifolates tested
with PT652 and PT653 showing a reversal in their patterns. Our results
indicated that rat DHFR can be used as a model to study antifolate
compounds as potential drug candidates. However, variations between rat
and human DHFR enzymes, coupled with unique features in the inhibitors,
could lead to the observed differences in enzyme sensitivity and selectivity.
| |
INTRODUCTION |
Dihydrofolate reductase (DHFR)
catalyzes the NADPH-dependent reduction of dihydrofolate to
tetrahydrofolate, which serves as a substrate for a number of
one-carbon transfer reactions in purine and pyrimidine synthesis,
including that of thymidylate. DHFR, along with other enzymes in the
folate metabolic pathway, is critical for the biosynthesis of DNA, RNA,
and certain amino acids (2-4, 51). Consequently,
inhibition of DHFR enzymatic activity depletes the tetrahydrofolate
pool inside the cell and inhibits DNA synthesis, subsequently leading
to cell death. For this reason, DHFR has been studied extensively and
many antifolates have been synthesized and tested as potential
candidates for drugs (5, 19-24, 28, 30, 35, 44-48, 52).
More recently, antifolates have been shown effective against such
opportunistic infectious agents as Pneumocystis carinii and
Toxoplasma gondii (1, 6, 9, 10). Since
immunosuppressed patients and those with AIDS are severly affected by
these pathogens, efforts have been focused on the design of antifolates
that are selective against P. carinii DHFR (pc DHFR) and
T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42,
44, 48). In most of these studies, antifolate selectivity
reported as a ratio of 50% inhibitory concentrations (IC50s) from two DHFR species was measured against crude
DHFR preparations from rat liver (44, 47, 48).
Evidence from sequence analysis and three-dimensional crystal
structures of DHFR from many species shows that there is a high degree
of conservation at the primary sequence and structural level among
DHFRs (8, 10-14, 17, 19, 29, 34, 36, 37, 41, 43, 50, 53,
55). However, kinetic and biochemical characterization
data reveal differences in the mechanism of action that result in
significant species specificity by selected inhibitors (15-17,
21, 25, 26, 31, 36, 39, 43). For
example, trimethoprim (TMP) [2,4-diamino-5-(3,4,5,-trimethoxybenzyl)-pyrimidine], which is used against bacterial infections, demonstrates a
significantly higher affinity for bacterial DHFR than for eukaryotic
DHFR. While a drug such as TMP also shows reasonable selectivity
against pc- and tgDHFR, it is not a particularly effective inhibitor,
as indicated by its high IC50s (2-4, 51).
Clearly, drugs with high potency as well as selectivity for effective
therapy are still in demand.
Despite the availability of crystal structures of DHFR from many
species and the extensive body of literature on the effects of
compounds against rat liver DHFR, neither the DNA sequence nor the
protein structure of the rat enzyme is known. Because of a high degree
of similarity in the N-terminal sequence of rat and human DHFR
(38, 54), it was assumed that the rat enzyme would be a
faithful representation of the human enzyme. However, only a detailed
comparison of its sequence, structure, and kinetics with the
human enzyme will provide the needed data. Therefore, we have
isolated the DHFR gene from a rat cDNA library and expressed an active
form of recombinant DHFR protein from Escherichia coli. Inhibitory data for select antifolates reveals the expected pattern of
homologies; however, one example shows significant differences in the
rat and human DHFR IC50s.
| |
MATERIALS AND METHODS |
Reagents.
A rat (6-month-old Sprague
Dawley male) liver lambda cDNA library for isolation of rat liver
DHFR cDNA was purchased from Stratagene (Cedar Creek, Tex.). Gateway
plasmids pDEST-15 and pDONR201 for the construction of expression
clones as well as the required clonase enzymes were obtained from Life
Technology (Bethesda, Md.). The pCRII vector and the TA cloning kit for
the cloning of PCR products were from Invitrogen (Carlsbad, Calif.). Different E. coli strains were used for DNA manipulations
and for protein expression: DH5
[supE44
lacU169 (
80 lacZM15)
hsdR71 recA1 endA1 gyrA96 thi-1 relA1] and DB3.1
[F
gyrA462 endA
(Sr1-recA) mcrB mrr
hsdS20(rB
mB) supE44 ara-14 galK2 lacY1
proA2 rpsL20(Smr) xyl5 
leu mtl1] were from Life Technology, and BL21(DE3)
[hsdS gal (cIts857 ind1 S am7
nin5 lacUV5-T7 gene 1] were from Novagen (Madison, Wis.). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Beverly, Mass.), Taq DNA
polymerase was from Sigma (St. Louis, Mo.) and Life Technology, and
pCRII vector was from Stratagene. Radioactive isotope
[-32P]dATP used for Southern hybridization was
obtained from Pharmacia-Amersham (Piscataway, N.J.). The Random Priming
DNA Synthesis kit for making radioactive DNA probes was purchased from
Stratagene. Nylon dot blot membranes were obtained from Osmonics
(Minnetonka, Mont.). Positive cDNA lambda clones were visualized using
a PhosphorImager scanner (Pharmacia-Amersham). All other chemicals and
reagents were obtained from Sigma. All chromatography columns were from Pharmacia-Amersham, and the BIOCAD 700E Perfusion Chromatography workstation was from PE-Biosystems (Norwalk, Conn.).
Library screening.
A mouse DHFR cDNA fragment was initially
generated from mouse liver total mRNA through reverse
transcription-PCR. First-strand cDNA was synthesized with a reverse
primer complementary to a 3'-untranslated region (UTR) of mouse DHFR:
5'-CGG GAT CCC CTC TCT AAA GAA AGA ATA ACT CAT AGA TCT AAA GCC-3'.
It was subsequently amplified with a PCR primer pair, 5'-CGG
GAT CCA TGG TTC GAC CGC TGA ACT GCA TCG-3' and 5'-CGG GAT
CCA AGT CCC ATG GTC TTG TAA AAA TGC-3'. The resulting 677-bp
fragment containing the full-length mouse DHFR open reading frame (ORF)
was cloned into a pCRII vector (Invitrogen) using the TA cloning
kit and sequenced to confirm the identity of the mouse DHFR sequence.
This 677-bp fragment was then used as a probe to screen a rat liver
cDNA lambda library (Stratagene) with a titer of ~1.75 × 107 PFU/µl. Approximately 1,000,000 plaques of the
library were screened using 20 150-mm-diameter NZY agar plates.
Briefly, 20 sets of 600 µl of freshly grown E. coli
XL1-Blue MRF' [(mcrA) 183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1
gyrA96 relA1 lac [F' proAB lac1q
ZM15 Tn10 (Tet)] cells at an
optical density at 600 nm (OD600) of 0.5 per plate were
each infected with 
1 × 105 plaques from cDNA
library and incubated at 37°C for about 8 h. Plaque lifts were made
in duplicate immediately and lysed in situ. Phage DNA was denatured and
fixed on the membrane through baking in a vacuum oven for 2 h at
80°C. Membranes were then hybridized to a
32P-radiolabeled probe from a mouse DHFR PCR fragment to
detect cross-reacting plaques by using the random priming kit from
Stratagene (49). Positive plaques were diluted and
repeatedly hybridized in duplicate until individual homogeneous clones
could be isolated. After obtaining the positive plaque stocks, clones
were incubated with the ExAssist helper phage and then introduced into
a nonsuppressing E. coli SOLR
{e14 (McrA)
(mcrCB-hsdSMR-mrr) 171 sbcC recB recJ uvrC
umuC::Tn5(Kanr) lac
gyrA96 relA1 thi-1 endA1R [F' proAB
lac1q ZM15]
Su} strain for the excision of pBlueScript
phagemid containing the positive cDNA insert.
DNA sequence analysis.
Multiple positive pBlueScript clones
containing rat liver DHFR cDNA inserts were sequenced using an ABI
PRISM automated sequencer from Roswell Park Cancer Institute Biopolymer
Facility (Buffalo, N.Y.). T7 forward and M13 reverse primers on the
vector were used to sequence and confirm the isolation of the
full-length rat liver DHFR cDNA. The rat liver DHFR cDNA sequence was
compared with known DHFR sequences from different species using BLAST
web services from the National Center of Biotechnology Information
(NCBI). Protein sequence alignment with other known DHFR species was
performed using the Wisconsin Package Version 10.0 (TRANSLATE, PILEUP,
REFORMAT), Genetics Computer Group, Madison, Wis.
Heterologous expression of rat liver DHFR gene.
The Gateway
cloning kit (Life Technology) was used to construct expression vectors
for the rat liver DHFR gene. The rat DHFR gene was PCR amplified with a
pair of oligonucleotide primers harboring the attB
bacteriophage recombination sites as well as the tobacco etch virus
(TEV) protease recognition site (ENLYQG). The PCR product was
introduced into the entry vector containing the attP
bacteriophage recombination site via the attB × attP recombination reaction. The resulting entry vector now contained an
attL recombination site that could be recombined into the
destination vector with the matching attR site. The
reactions, designated BP and LR reactions, respectively, were performed
according to the manufacturer's recommended conditions (Life
Technology). The resulting expression clone, pDest15-rDHFR, would
express a glutathione S-transferase rat DHFR
(GST-rDHFR) fusion protein in bacteria and would contain a TEV protease
recognition site between the GST and rat DHFR domains. The forward
primer contains the attB1 recombination site and the TEV
protease recognition site fused in frame with the N-terminal sequences
of rat DHFR (5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGA AAA
TCT GTA CTT CCA GGG GAT GGT TCG TCC GCT GAA CTG CAT CGT CGC C-3').
The reverse primer contained the attB2 recombination
site fused with a complementary region of the 3'-UTR of the rat DHFR
(5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG GAT CCA GCA GAA GTG
GTC TTA TAA AAT GC-3').
The pDEST15-rDHFR plasmid was sequenced to verify the presence and
integrity of the rat DHFR insert. The plasmid was subsequently introduced into the BL21(DE3) E. coli strain via chemical
transformation as previously described (49). Cells
containing the plasmids were grown at 30°C to an OD600 of
0.4 and were induced with 0.15 mM
isopropyl-
-D-thio-galactopyranoside (IPTG) for 4 h
at 30°C. Expression of the GST-rDHFR fusion protein was confirmed
through the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell lysate after the IPTG induction. The typical yield of soluble GST-rDHFR fusion protein was between 35 and 40 mg per
liter of cell culture after purification.
Purification of GST-rDHFR and rat DHFR proteins.
To purify
the GST-rDHFR fusion protein, a GST affinity column was used. After
induction with IPTG, cells were harvested, resuspended in lysis buffer
(50 mM KH2PO4 [pH 7.0] 50 mM KCl, and 1 mM
EDTA), and lysed with lysozyme (50 mg for 4 liters of cell culture) in the presence of protease inhibitors. To optimize lysis, cell suspension also went through a liquid nitrogen freeze-thaw cycle twice. Lysed cells were immediately centrifuged at 40,000 rpm for 45 min at 4°C (45 Ti rotor, L8 Beckman ultracentrifuge). The clear cell lysate
was filtered through a 0.45-µm-pore-size membrane and applied to the
GST affinity column on a BioCAD 700E perfusion chromatography workstation. Bound GST-rDHFR fusion protein was eluted with elution buffer (50 mM Tris-HCl [pH 8.0], 25 mM glutathione). The eluted fractions were then digested with TEV protease at 30°C overnight in
TEV digestion buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 5 mM DTT).
After digestion with TEV, one glycine residue from the TEV recognition
site should have been left at the N terminus of the rat DHFR protein.
The digestion mixture was loaded onto a G-25 desalt column to exchange
the buffer composition with that of the lysis buffer but at pH 8.0. Rat
DHFR protein was separated from GST and undigested GST-rDHFR fusion by
passage through a second GST affinity column. The rat DHFR protein was
collected as the flowthrough fraction. A final G-75 gel filtration
column removed any other potential impurities of different sizes.
Enzyme and inhibitor assay.
The activities of both the
recombinant rat liver DHFR and the GST-rDHFR fusion protein were
measured at room temperature spectrophotometrically at 340 nm for a
decrease in absorbance which occurs when NADPH and dihydrofolate are
converted to NADP+ and tetrahydrofolate. The assay was
performed essentially as described by Prendergast et al.
(43).
The spectrophotometric assay (micromolar IC
50 measurement)
used to measure the abilities of compounds to decrease the rate
of
enzymatic reduction of dihydrofolate to tetrahydrofolate in
the
presence of NADPH was performed at 37°C with saturating
concentrations
of substrate and cofactor, and with 150 mM KCl, as
previously
described (
6,
9,
44).
GenBank nucleotide sequence accession number.
The GenBank
accession number for full-length rat liver DHFR cDNA is AF318150.
| |
RESULTS AND DISCUSSION |
Isolation of rat DHFR gene.
The rat liver DHFR gene was
isolated through the screening of a rat liver cDNA library by using a
mouse DHFR probe. After multiple rounds of screening, one clone was
obtained that contained a full-length ORF. The nucleotide sequence of
the gene plus portions of the 5'- and 3'-UTRs are shown in Fig.
1.
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FIG. 1.
Shown is the DNA sequence and predicted amino acids
sequence of the rat DHFR gene, with portions of the 5' and 3' flanking
regions. The translation of the gene starts at position 88 and ends at
nucleotide 648. The polypeptide sequence in single-letter coding is
indicated below the DNA sequence. The GenBank accession number for the
rat DHFR gene is AF318150.
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|
The 561-bp ORF encodes a protein of 187 amino acids. Alignment of the
putative translated product of this gene with other
mammalian DHFR
proteins revealed significant homology, with 96%
primary sequence
identity with mouse and Chinese hamster DHFR
and 89% with human DHFR
(Fig.
2). Comparison of DHFR proteins
from opportunistic fungi revealed an identity of 41% to human-
P. carinii and rat-
P. carinii DHFR and 31% to a
protozoal parasite
T. gondii DHFR. The active enzymatic
sites between rat and other
DHFR proteins demonstrate a higher level of
conservation. Thirty-three
out of 35 amino acids within the primary
sequence that constitute
the active enzymatic site, as well as the
sites for substrate
and cofactor binding, are identical between human
and rat. Thirty-four
out of 35 are identical between rat and mouse and
Chinese hamster
(Fig.
2). Eleven amino acids (Fig.
2) that are
conserved among
all known DHFR proteins are also preserved in the rat
protein
isolated, which aligns perfectly with other known DHFRs.
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FIG. 2.
Protein sequence comparisons of rat DHFR with six other
known DHFR enzymes. Highlighted amino acid residues represent conserved
regions among different species. Light gray boxes indicate
identity among the seven DHFR proteins, and dark gray boxes represents
significantly similar residues.  and *, residues that surround the
active site of the DHFR enzyme; some residues (*) are conserved in
all known DHFR. Sequence alignment was performed using the Wisconsin
Package version 10.0 (TRANSLATE, PILEUP, REFORMAT), Genetic
Computer Group.
|
|
Comparison of rat liver DHFR protein with other known DHFRs indicated a
high level of conservation for DHFR proteins among
higher eukaryotes.
Functionally important residues within the
DHFR enzyme demonstrate an
even higher level of conservation.
The Phe
34,
Phe
31, Leu
22, Ile
7,
Ile
64, Leu
67, and Glu
30 residues
that are important for dihydrofolate and other ligand
binding
(
2) are conserved in rat DHFR. Lys
54, which
has been shown to be important for the association of
cofactors NADPH
and NADP (
26), are the same in rat DHFR. Overall,
the
organization of rat DHFR implies a structure similar to those
known for
other DHFR enzymes. X-ray crystallographic studies are
underway to
validate the structural homology of the rat DHFR structure
with
that of human
DHFR.
Expression and purification of recombinant rat DHFR protein.
The putative rat DHFR gene was introduced into a Gateway expression
plasmid via two steps of recombination reactions. The resulting
expression clone produces a GST fusion protein in BL21(DE3) cells
upon induction by IPTG. The recombinant GST fusion protein, tentatively
named GST-rDHFR, was expressed as a 50.2-kDa protein with the GST and
rat DHFR moiety linked by a TEV protease recognition site. As shown in
Fig. 3, the target GST fusion protein was
separated from total cellular proteins by the affinity columns (lanes 1 and 2). A minor band with a molecular mass of approximately 29 kDa
below the dominant GST fusion band in lane 2 is a truncated translational product of the GST portion and can be removed by a second
round of GST affinity chromatography. Since no TEV protease recognition
site is present within the putative rat DHFR protein, it could be
separated from the GST portion after the digestion of TEV protease
(Fig. 3, lane 3). Digestion yielded the expected 21.6-kDa putative rat
DHFR protein and the 28.7-kDa GST protein. Subsequently, target rat
protein was purified through additional rounds of GST affinity
chromatography to remove the GST tags. After the final chromatographic
purification using a G-75 sizing column, a single band on the SDS-PAGE
gel (Fig. 3, lane 6) indicated the successful isolation of a highly
pure and homogeneous protein.
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FIG. 3.
Expression and purification of recombinant rat DHFR
enzyme in BL21(DE3) cells. Proteins were assayed on SDS-12% PAGE
gels. Lane 1, molecular mass markers, with sizes of standard fragments
(in kilodaltons) indicated on the left; lane 2, cell lysate with
overexpressed GST-rDHFR fusion; lane 3, peak fraction after the
GST affinity chromatography: lanes 4 and 5, peak fraction of GST
column digested with TEV protease; lane 6, purified rat DHFR
recombinant protein after the G-75 gel filtration column. The sizes of
the GST fusion protein, GST alone, and the pure rat DHFR
protein are on the right.
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|
All fractions during the purification process were assayed for the DHFR
enzymatic activity that reduces dihydrofolate to tetrahydrofolate
in
the presence of NADPH. Monitoring the decrease of absorbance
at 340 nm
spectrophotometrically due to the conversion of dihydrofolate
to
tetrahydrofolate revealed that both the GST fusion protein
and the pure
rat moiety released from the fusion demonstrate high
levels of DHFR
enzymatic activity. However, the pure rat protein
without the GST tag
is about 30-fold more catalytically active
than the GST fusion (data
not shown). Our results indicate that
we have isolated the DHFR gene
from rat liver and have successfully
expressed an active form of DHFR
recombinant
protein.
Inhibition of DHFR enzymatic activity with different
compounds.
Based upon similarities in N-terminal analysis of the
native rat and human proteins (38, 54), many inhibitor
binding assays of DHFR have been performed with rat liver
enzyme (44, 46). We tested the inhibition of recombinant
rat liver DHFR by several compounds and compared these results with
those obtained from the native DHFR preparations (Table
1).
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TABLE 1.
Comparison of micromolar IC50s of
seven known inhibitors against various forms of rat and human
DHFR and the selectivity ratio of those inhibitors against
pcDHFRa
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|
Seven antifolates which reflect a broad spectrum of inhibition and
structural features were used to compare potential differences
between
inhibition of the recombinant and native rat liver DHFR
enzyme (Table
1). Methotrexate (MTX) and MTXO represent classical
pteridine
analogues. PT652 and PT653 belong to a diaminopteridine
class with a
bulky planar tricyclic moiety, which were recently
synthesized and
tested for their potential potency and selectivity
against pcDHFR
(
46), and trimethoprim, pyrimethamine, and TAB
are
lipophilic 2,4-diaminopyrimidine
analogues.
A parallel inhibitory pattern of the compounds against the recombinant
rat liver DHFR and the GST-rDHFR fusion protein was
observed (Table
1).
The GST-rDHFR fusion protein can catalyze
the reduction of
dihydrofolate, but with less efficiency than
DHFR without the GST amino
terminus. The turnover value (moles
of product formed per
minute per mole of enzyme) for the GST-rDHFR
fusion is 300,000/min,
whereas the value for the unfused DHFR
is 10,000,000/min. Despite
the reduced enzymatic activity, the
inhibition profile of compounds
with different potency was clearly
reproduced with the GST fusion,
indicating that the active site
in the fusion protein is accessible
to those inhibitors and that
these inhibitors associate with the
enzyme with similar
kinetics.
When recombinant rat DHFR protein was used to test these inhibitors the
potencies of most compounds showed a similar pattern
to the
IC
50s previously reported for rat (Table
1). The difference
between the IC
50s of recombinant and native enzymes does
not exceed
threefold. The compounds that demonstrated the largest
differences
are PT652 and PT653 (Table
1).
A recombinant human DHFR enzyme was also used to determine the
comparative efficiency of inhibitors against the rat and human
enzymes. As shown in Table
1, the broad spectrum of potency in
rat was reproduced with human recombinant DHFR. The largest difference
between human and rat recombinant DHFR IC
50s is fivefold
for compound
PT652. The sensitivity of the human enzyme to PT652 is
approximately
ninefold less compared to that of the native rat enzyme
(Table
1). In the case of PT653, the IC
50s for recombinant
rat and human
DHFR are comparable. However, PT653 is more sensitive for
recombinant
rat enzyme whereas PT652 is more sensitive for the native
protein.
Other than PT652 and PT653, the selectivity ratios
between recombinant
rat-to-human DHFR and native rat-to-human
DHFR indicated a consistency
between the recombinant and the native
enzyme. All compounds tested,
however, demonstrated lower
IC
50s against recombinant rat DHFR
than the human
counterpart (Table
1).
For comparison, the inhibitory profiles of the seven compounds against
pcDHFR were also included and the selectivity ratios
against different
forms of rat and human DHFR are shown (Table
1, last three columns).
Again, most compounds demonstrate a similar
selectivity ratio against
pcDHFR for either the native or the
recombinant rat DHFR. PT652 and
PT653 had different sensitivities
for the rat enzyme, and the
largest variation was obtained with
PT653, where the selection ratio
was approximately 14.3-fold greater
against pcDHFR for the native rat
enzyme but was reduced to about
6.7-fold greater for recombinant
enzyme. For PT652, there was
no selectivity against pcDHFR with either
the native or the recombinant
rat DHFR. However, for the human enzyme,
a moderate selectivity
was observed (
44). For the rest of
the compounds, selectivity
against pcDHFR appears to be consistent with
all three forms of
mammalian DHFR, with the selectivity ratio of the
human enzyme
being higher than that of the rat
enzyme.
Summary.
Rat liver DHFR has been used as the in vivo model to
study hundreds of antifolates and related compounds against human DHFR for their potential drug implications, despite the fact that the sequence of neither the gene nor the protein was known. To better correlate the published literature for rat liver DHFR and human DHFR,
we have cloned and sequenced the DHFR gene from a rat liver cDNA
library. With a GST fusion tag, we expressed and purified an active
form of recombinant rat DHFR protein in large quantity in E. coli cells.
Seven inhibitors chosen to represent different categories of
antifolates with various selectivity against DHFR from the fungal
pathogen
P. carinii were tested against the recombinant rat
liver
enzyme. Similar inhibition profiles against both the rat and
human
enzyme were observed despite the fact that the IC
50s
of recombinant
rat DHFR were consistently lower than those of the
recombinant
human protein. The inhibitors also demonstrate consistent
pcDHFR
selectivity with both human and rat DHFR despite some variations
with certain compounds. As noted, compounds with large bulky
hydrophobic
groups as found in PT652 and PT653 showed inconsistencies
among
the various assays. Conceivably, while binding of these
antifolates
provide a reasonable portrayal of their interactions with
human
DHFR, structural variations between human and rat enzyme coupled
with unique features in the inhibitors could lead to differences
in
enzyme sensitivity and
selectivity.
| |
ACKNOWLEDGMENTS |
We thank Carol Yarborough for the assistance on the
chromatographic work.
This work was supported in part by funds from GM51670 (V.C.), the
Greater Buffalo Community Fund (V.C.), and the Wendt Foundation (J.A.B.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Structural
Biology Department, Hauptman Woodward Medical Research Institute, 73 High St., Buffalo, NY 14203. Phone: (716) 856-9600, ext. 322. Fax: (716) 852-6086. E-mail: cody{at}hwi.buffalo.edu.
| |
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Antimicrobial Agents and Chemotherapy, September 2001, p. 2517-2523, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2517-2523.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
