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Antimicrobial Agents and Chemotherapy, December 2001, p. 3293-3303, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3293-3303.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Dicyclic and Tricyclic Diaminopyrimidine
Derivatives as Potent Inhibitors of Cryptosporidium
parvum Dihydrofolate Reductase: Structure-Activity and
Structure-Selectivity Correlations
Richard G.
Nelson1 and
Andre
Rosowsky2,*
Division of Infectious Diseases, Department
of Medicine, University of California, San Francisco, California
94143,1 and Dana-Farber Cancer Institute
and Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts
021152
Received 20 April 2001/Returned for modification 8 August
2001/Accepted 28 August 2001
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ABSTRACT |
A structurally diverse library of 93 lipophilic di- and tricyclic
diaminopyrimidine derivatives was tested for the ability to inhibit
recombinant dihydrofolate reductase (DHFR) cloned from human and bovine
isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In
parallel, the library was also tested against human DHFR and, for
comparison, the enzyme from Escherichia coli. Fifty
percent inhibitory concentrations (IC50s) were determined
by means of a standard spectrophotometric assay of DHFR activity with
dihydrofolate and NADPH as the cosubstrates. Of the compounds tested,
25 had IC50s in the 1 to 10 µM range against one or both
C. parvum enzymes and thus were not substantially different from trimethoprim (IC50s, ca. 4 µM). Another 25 compounds had IC50s of <1.0 µM, and 9 of these had
IC50s of <0.1 µM and thus were at least 40 times more
potent than trimethoprim. The remaining 42 compounds were weak
inhibitors (IC50s, >10 µM) and thus were not considered
to be of interest as drugs useful against this organism. A good
correlation was generally obtained between the results of the
spectrophotometric enzyme inhibition assays and those obtained recently
in a yeast complementation assay (V. H. Brophy et al., Antimicrob.
Agents Chemother. 44:1019-1028, 2000; H. Lau et al., Antimicrob.
Agents Chemother. 45:187-195, 2001). Although many of the compounds in
the library were more potent than trimethoprim, none had the degree of
selectivity of trimethoprim for C. parvum versus human
DHFR. Collectively, the results of these assays comprise the largest
available database of lipophilic antifolates as potential
anticryptosporidial agents. The compounds in the library were also
tested as inhibitors of the proliferation of intracellular C.
parvum oocysts in canine kidney epithelial cells cultured in
folate-free medium containing thymidine (10 µM) and hypoxanthine (100 µM). After 72 h of drug exposure, the number of parasites inside
the cells was quantitated by indirect immunofluorescence microscopy.
Sixteen compounds had IC50s of <3 µM, and five of these
had IC50s of <0.3 µM and thus were comparable in potency
to trimetrexate. The finding that submicromolar concentrations of
several of the compounds in the library could inhibit in vitro growth
of C. parvum in host cells in the presence of thymidine
(dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors,
in combination with leucovorin, may find use in the treatment of
intractable C. parvum infections.
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INTRODUCTION |
Diaminopyrimidine inhibitors of
dihydrofolate reductase (DHFR) such as trimethoprim, pyrimethamine,
trimetrexate, and piritrexim (compounds 1 to 4, respectively, in Fig.
1) are used in the prophylaxis and
treatment of opportunistic infections in patients whose immune systems
are impaired as a result of human immunodeficiency virus infection or
immunosuppressive chemotherapy (14, 38). Typically, these
agents are coadministered with a sulfonamide or sulfone inhibitor of
dihydropteroate synthetase (44, 49), and in the case of
trimetrexate or piritrexim, with leucovorin to minimize the toxic side
effects of the antifolate (13, 74). Although other
microbial parasites are known to cause life-threatening secondary
infections in AIDS patients in other parts of the world, the organisms
most frequently found to cause opportunistic disease in patients in the
United States and other industrialized countries, sometimes at the same
time, are Pneumocystis carinii and Toxoplasma gondii. The rationale for combining sulfa drugs with DHFR
inhibitors is that P. carinii and T. gondii, but
not mammalian cells, can synthesize essential tetrahydrofolate
cofactors de novo with the help of dihydropteroate synthetase
(39). Because of the lack of this mechanism in humans,
sulfa drugs are selective in that they do not affect the folate status
of the patient's cells. In the case of DHFR inhibitors that are more
potent than trimethoprim, such as trimetrexate or piritrexim,
leucovorin can be added to the regimen as a rescue agent to prevent
dose-limiting hematological toxicity. In this scenario, the selectivity
of the host-protective effect results from the fact that the parasites
lack an active transport pathway for reduced folates and thus are
impervious to rescue (39). Although combinations of DHFR
inhibitors with sulfa drugs have a sound theoretical rationale and,
indeed, are effective in a significant number of patients, a subset of
patients experience severe cutaneous allergy to the sulfa drug and thus have to discontinue this type of treatment (73).
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FIG. 1.
Structures of trimethoprim (compound 1), pyrimethamine
(compound 2), trimetrexate (compound 3), and piritrexim (compound 4).
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A vigorous program of chemical synthesis was launched by several groups
in the early 1990s with the goal of discovering new inhibitors of
P. carinii and/or T. gondii DHFR that might be
potent enough not to require coadministration of a sulfa drug while
being selective enough not to require leucovorin rescue. Hundreds of lipophilic condensed diaminopyrimidine antifolates, embodying a rich
spectrum of chemical diversity (see references in Table 1), were tested as inhibitors of DHFR in
cell-free assays (8, 10) and, in some cases, as inhibitors
of the growth of the intact organisms in vitro or in laboratory animals
(4, 51). However, with the exception of the older agents
trimethoprim and pyrimethamine, the only newer antifolates tested in
large, controlled clinical trials in AIDS patients suffering from, or
at risk of developing, P. carinii pneumonia and/or
toxoplasmosis have been trimetrexate (74) and piritrexim
(13).
Among the opportunistic pathogens other than P. carinii and
T. gondii that are known to pose a significant risk in the
management of AIDS is the intestinal parasite Cryptosporidium
parvum, which can be life threatening because of its ability to
bring on repetitive episodes of violent diarrhea and, in the most
extreme cases, massive exfoliation of the intestinal mucosa (33,
75). Cryptosporidiosis is typically acquired from unsterilized
drinking water and is usually self-limiting, except in immunosuppressed
individuals. Some success has been reported with pyrimethamine and
trimethoprim-sulfamethoxazole in patients infected with Isospora
belli, another opportunistic intestinal parasite whose clinical
effects include severe and unremitting diarrhea and thus resemble those
produced by C. parvum (12, 77). Several other
experimental treatments using non-antifolate drugs have also been
reported, some of which are likely to act primarily on the host cell
rather than the parasite (1, 5, 6, 32, 34, 37, 42, 43, 47,
78). The efficacy of these regimens is, at best, marginal. Thus,
until a consistently safe and effective drug against acute
cryptosporidiosis in AIDS patients is found, the main treatment of this
disease remains palliation with antidiarrheal drugs such as octeotride
(35).
With the availability of a large and structurally diverse library of
lipophilic DHFR inhibitors generated by our synthetic effort over the
years, the opportunity presented itself to test these compounds as
inhibitors of the DHFR activity of the difunctional C. parvum DHFR-thymidylate synthase (TS) enzyme, which was recently cloned and sequenced (76). A number of the compounds in
our library, along with others from the archives of the National Cancer Institute and the Walter Reed Army Institute of Research, were recently
found to be active in a yeast complementation assay using Saccharomyces cerevisiae in which the native yeast gene was
replaced with either the entire difunctional DHFR-TS gene or the
discrete DHFR domain of C. parvum (7, 40). In
the present paper, we report data on 93 compounds from the archival
collection of the Dana-Farber Cancer Institute (DFCI) as in vitro
inhibitors of recombinant C. parvum and human DHFR activity
in a spectrophotometric assay. To our knowledge, this constitutes the
largest published database on lipophilic polycyclic diaminopyrimidines
as inhibitors of these enzymes. Also reported for comparison are the
50% inhibitory concentrations (IC50s) of the
same compounds against Escherichia coli DHFR. Although
E. coli infection is not among the more dangerous complications of AIDS, drug-resistant E. coli strains are
gradually becoming more prevalent in the food supply and would almost
certainly pose a greater risk to an AIDS patient than to an individual
whose immune defense system is not impaired.
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MATERIALS AND METHODS |
Test compounds.
Data were obtained for a total of 93 compounds, comprising the nine structural families shown in Fig.
2 to 4.
Symmetrically 5,6-disubstituted
pteridines I.1 to I.8 were synthesized
from tetraaminopyrimidine and 1,2-diketones (52).
Pteridines II.1 to II.5 and pyrido[2,3-d]pyrimidine II.6
were obtained by N-alkylation of secondary amines with
2,4-diamino-6-bromomethylpteridine (56) or
2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine
(60). Pyrido[3,2-d]pyrimidines III.1 to III.6
were prepared from
2,4-diamino-6-bromomethylpyrido[3,2-d]pyrimidine and
anilines or N-methylanilines (58). Quinazolines
IV.1 to IV.3 were obtained from the corresponding anthranilonitriles
and chloroformamidine hydrochloride (62). Quinazolines
IV.4 to IV.7 were obtained from 2,4-diamino-5-iodoquinazoline and an
alkene or alkyne via a palladium-catalyzed Heck reaction, followed by catalytic hydrogenation in the case of IV.6 and IV.7 (65).
N10-unsubstituted quinazolines IV.8 to IV.12 were
obtained from 2,4-diaminoquinazoline-5-carbonitrile and the appropriate
substituted anilines by reductive coupling in the presence of Raney
nickel (65). 5-Chloro-6-(arylaminoalkyl)quinazolines IV.13
to IV.16, IV.18 and IV.19 were derived from
2,4,6-triamino-5-chloroquinazoline and the appropriate substituted
aldehydes by similar reductive coupling (67). Compounds
IV.11, IV.12, and IV.17 were obtained from IV.8, IV.9, and IV.16,
respectively, by reaction with formaldehyde and sodium cyanoborohydride
(65, 67). Tetrahydroquinazolines V.1 to V.16 were
synthesized from 4-substituted cyclohexanones by heating with
cyanoguanidine (68). Thieno[2,3-d]pyrimidines VI.1 to VI.12 were derived from 5,6-substituted
2-aminothiophene-3-carbonitriles and chloroform-amidine hydrochloride
(64, 66), whereas analogs VI.13 to VI.18, with an
arylamine side chain, were synthesized in several steps from preformed
2,4-diamino-5-methylthieno[2,3-d]pyrimidine (69). Pyrido[4,3-d]-pyrimidines VII.1 to
VII.4 were made by alkylation of the parent amine (63).
9H-Indeno[2,1-d]pyrimidines VIII.1 to VIII.4
were synthesized from 3-cyano-2-alkoxy-1H-indenes and
guanidine (57).
5,6-Dihydrobenzo[f]quinazolines VIII.5 and VIII.6 were
prepared by heating 2-tetralones with cyanoguanidine (55).
Seven-membered ring analog VIII.10 was prepared similarly from
6,7-dichloro-2-benzosuberone (53). Fully aromatic
benzo[f]quinazolines VIII.7 to VIII.9 were prepared by
thermal cyclization of symmetrical N1,N5-diarylbiguanides,
by condensation of 2-aminonaphthalene-1-carbonitriles with guanidine,
or by oxidation of
1,3-diamino-5,6-dihydrobenzo[f]quinazolines with selenium
dioxide (54). Trimethoprim and trimetrexate were obtained
from the National Cancer Institute, Bethesda, Md. All compounds were of
recent or archival origin and were judged to be >95% pure at the time
of assay.
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FIG. 4.
Structures of di- and tricyclic diaminopyrimidines
tested as DHFR inhibitors (groups VIII and IX).
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Enzyme assays.
Recombinant C. parvum and human
enzymes were obtained and used as described earlier (7,
76). The C. parvum type 1 DHFR-TS allele was cloned
from an isolate obtained from an infected AIDS patient, and the
C. parvum type 2 allele was cloned from an isolate obtained
from an infected calf. The designations Cp-I and Cp-II will be used
here in order to conform to the nomenclature used earlier for the two
cloned proteins (76; for a discussion of C. parvum genotypes 1 and 2, see reference 46). The type
1 enzyme is found exclusively in humans, whereas the type 2 enzyme can infect either humans or animals. Molecular characterization of the
constructs SFGH-1 (from Cp-I) and NINC-1 (from Cp-I), the latter of
which was re-engineered to encode a 22-residue C-terminal TS domain of
Cp-I, was documented earlier (76). Briefly, all three
proteins were expressed from dhfr mutant E. coli
strain PA414 after transfection with the appropriate coding sequence and subcloning into expression plasmid pTrc99A, which contains a
promoter induced with IPTG
(isopropyl-
-D-thiogalactopyranoside). In the
case of the human enzyme, the E. coli mutant was transfected with previously described plasmid pDFR (50). All three
enzymes were purified to homogeneity from their respective bacterial
lysates by chromatography on a methotrexate-Sepharose affinity column. Assays of enzyme activity were performed at 37°C in a microtiter plate spectrophotometer by monitoring the change in UV absorbance at
340 nm in a solution containing 50 mM
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH
7.0), 1 mM EDTA, 75 µM 2-mercaptoethanol, 0.1% bovine serum albumin,
20 µM dihydrofolate, and 100 µM NADPH. Enzyme concentrations were
adjusted to give linear rates over the 5-min duration of the assay.
Reactions were initiated by adding 100 µl of 40 µM dihydrofolate in
assay buffer to an equal volume of assay buffer lacking dihydrofolate
and containing various concentrations of the inhibitor. Each titration
was performed twice, and the mean DHFR activity was plotted against the
inhibitor concentration to obtain the IC50.
Assays using E. coli DHFR were performed under the same
conditions as those done with the human enzyme. Stock solutions of all
of the test compounds were made up in dimethyl sulfoxide, and
appropriate control experiments were done to ensure that the final
amount of dimethyl sulfoxide in the assay solution was low enough to
have no appreciable effect on the rate of the DHFR-catalyzed reaction
or the ability of cells to grow in culture.
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RESULTS AND DISCUSSION |
Binding of inhibitors to C. parvum DHFR versus their
binding to human DHFR.
The availability of the compounds shown in
Fig. 2 to 4 offered the opportunity to screen a structurally diverse
library of 93 lipophilic antifolates from the DFCI archive as
inhibitors and to determine whether any of them are selective for the
C. parvum enzyme. Yeast complementation assays had been
concurrently used to test a number of these compounds for the ability
to inhibit DHFRs from several different species, including C. parvum and humans (7, 40). Fifteen other compounds
provided by the National Cancer Institute were also tested by direct
spectrophotometric assay (7). One of these, trimethoprim,
proved to be >100 times more potent against the C. parvum
enzyme than against the human enzyme. Trimetrexate was considerably
more potent than trimethoprim but was completely nonselective. Three
other compounds were also more potent than trimethoprim against both
enzymes, but three of them contained a polar glutamic or aspartic acid
side chain and thus were not considered to be of the lipophilic type.
Trimethoprim emerged as the most selective member of this initial panel
and thus was considered the first promising lead in our search for anticryptosporidial drugs among lipophilic DHFR inhibitors. Although interesting patterns of activity were observed in the DHFR
complementation assays (7, 40), this method lacks the
quantitative power of a direct measurement of the kinetics of an enzyme
reaction. More importantly, compounds can only be assumed to be
equipotent as enzyme inhibitors by this method if they achieve the same
intracellular concentration over the time course of the assay, which
may not necessarily be the case, even for lipophilic antifolates. Thus, the yeast complementation assay should be viewed as only a preliminary screen. In the present study, the spectrophotometric assay was used to
assess the potency and selectivity of a larger group of lipophilic
antifolates from the DFCI collection with a view to uncovering
additional leads. Some of these compounds had already been tested in
the yeast assay, but many had not.
As shown in Table
2, the
IC
50s of trimethoprim against the Cp-I and Cp-II
enzymes were 4.0 and 3.8 µM, respectively, whereas
that against human
DHFR was 890 µM. Thus, as previously noted
(
7),
trimethoprim was equally active against the human and
bovine strains of
C. parvum DHFR and was remarkably selective
for these
enzymes relative to human DHFR. Of the 93 additional
diaminopyrimidine
antifolates that have now been tested against
the Cp-I and Cp-II DHFRs,
25 had IC
50s in the 1 to 10 µM range
against at
least one of the
C. parvum enzymes and thus were not
substantially different from trimethoprim. A second group of 25
compounds had IC
50s of <1.0 µM, and of these,
9 had IC
50s of <0.1
µM and thus were at least
40 times more potent than trimethoprim.
A third group, comprising 42 compounds, afforded <50% inhibition
at 10 µM, the concentration
arbitrarily chosen as a reasonable
upper limit for the screen, and thus
are merely footnoted in Table
2. The fact that roughly half of the
compounds in our library
were at least as potent as trimethoprim raises
doubt about a recent
comment in the literature that
C. parvum DHFR may be intrinsically
resistant to
2,4-diaminopyrimidine inhibitors (
11).
The best compound tested was IV.18, whose IC
50 of
0.0065 µM against the Cp-I enzyme approached the value obtained
earlier
for trimetrexate (
7). Like trimetrexate, however,
this compound
was nonselective. With the exception of I-2, which had
14-fold
selectivity for the Cp-I enzyme and 10-fold selectivity for the
Cp-II enzyme, almost all of the compounds were more like trimetrexate
than trimethoprim, in that they inhibited the
C. parvum and
human
DHFRs with about the same potency or were actually better
inhibitors
of the human enzyme. For the purpose of this discussion, we
have
arbitrarily chosen a 10-fold difference in
IC
50s as the minimum
threshold for selectivity.
This places I.1, IV.1, IV.2, and IX.4
in the nonselective category even
though they are, in fact, slightly
selective for the Cp-I enzyme. It is
of interest that five of
the nine compounds with
IC
50s of <0.1 µM against at least one
of the
C. parvum enzymes contained either the
3,4,5-trimethoxyphenyl
substitution of trimethoprim and trimetrexate
(cf. III.3, IV.14,
and IV.17) or the 2,5-dimethoxyphenyl substitution
of piritrexim
(cf. IV.13 and IV.18), while four others contained 3- and/or 4-chloro
substitutions on the phenyl ring (cf. III.5, III.6,
V.16, and
VIII.8). However, 10 of the other 13 compounds with a
3,4,5-trimethoxyphenyl
ring in the side chain and 5 of the other 9 compounds with a 2,5-dimethoxyphenyl
ring in the side chain had
IC
50s of >10 µM, indicating that these
substitutions are not necessarily favorable with every
diaminopyrimidine
ring
system.
Structural differences between pairs of analogs with widely divergent
potencies as DHFR inhibitors were, in some cases, astonishingly
small.
Among the pyrido[3,2-
d]pyrimidine analogs, for example,
methylation of the bridge nitrogen increased potency by approximately
1 order of magnitude (cf. III.2 and III.3) and replacement of
a
4-chlorophenyl or 3,4-dichlorophenyl group with a 3-chlorophenyl
group
in the side chain had a comparable effect (cf. IV.4 to IV.6).
However,
moving a substituent from the
para to the
meta
position
of the phenyl ring was not necessarily favorable and appeared
to depend on the nature of the heterocyclic moiety and the bridge
(cf.
V.6 and V.7 versus III.4 and III.5 and V.14 and V.15 versus
III.4 and
III.5). Insertion of an extra CH
2 group into the
bridge
of quinazoline analogs IV.13 and IV.14 caused potency to
decrease
by 2 orders of magnitude (cf. IV.15 and IV.16). In other
cases,
there was remarkably little variation in binding among compounds
with considerably different patterns of aromatic substitution
(cf. V.4
to V.15). Even though some of the individual structure-activity
correlations in Table
2 were intriguing, the limited data obtained
did
not yield any clear guidelines as to how selective inhibitors
of
C. parvum DHFR might be designed. Moreover, none of the
compounds
approached the dramatic selectivity reported earlier for
trimethoprim
as an inhibitor of the
C. parvum enzyme
(
7). A better understanding
of how an optimal combination
of high potency and high selectivity
might be designed into an
inhibitor of
C. parvum DHFR will no
doubt be easier to
obtain once the first three-dimensional structure
of a ternary complex
of the enzyme has been solved, e.g., by X-ray
crystallography or
nuclear magnetic resonance analysis. High-resolution
crystallographic
structural analysis of several such complexes
is in progress (R. G. Nelson, personal
communication).
Anticryptosporidial activities of DHFR inhibitors in culture.
In order to determine whether the potent inhibition of isolated DHFR
translates into antiparasitic activity in culture, three arbitrarily
chosen examples of the tetrahydroquinazoline type (V.4, V.10, and V.16)
were tested for the ability to block proliferation of the intracellular
forms of the organism in Madin-Darby canine kidney (MDCK) epithelial
cell monolayers grown in microscope chamber slides and infected with
C. parvum oocysts. To rule out the possibility that
inhibition of parasite growth was due to an antifolate effect on the
host cell, the medium was supplemented with 10 µM dThd and 100 µM
Hx. In some experiments, the medium could be supplemented effectively
with as little as 0.1 µM leucovorin alone, instead of dThd and Hx
(R. G. Nelson, results not shown). Parasites were visualized by an
indirect immunofluorescence assay in which the cells were fixed and
treated sequentially with rat polyclonal anti-C. parvum
serum, biotinylated anti-rat serum, and fluoresceinated streptavidin.
The fixed cells were also stained for DNA, and the doubly stained cells
were enumerated under the microscope. Photomicrographs of control cells
and of cells incubated for 48 h with trimethoprim, pyrimethamine,
trimetrexate, and tetrahydroquinazolines V.4, V.10, and V.16, all at
the same concentration (4 µM), are presented in Fig.
5 and 6.
As can be seen in these figures, trimethoprim (panels E and F) and
pyrimethamine (panels H and I) had little or no effect on the number of
red-stained parasite-containing vacuoles relative to those in untreated
controls (panels B and C), presumably because 4 µM is lower than the
optimal concentration of these drugs. In contrast, 4 µM trimetrexate
led to marked diminution of the number of parasitemic vacuoles (panels
K and L) and this was accompanied by aberrant nuclear morphology of the
parasites in these vacuoles (panels G and J) compared to that of
controls (panel A). Gratifyingly, compounds V.4 (panels M to O), V.10
(panels P to R), and V.16 (panels S to U) appeared to be equipotent
with trimetrexate in blocking parasitemia. A dose-response analysis was
then performed by repeating this experiment over a range of drug
concentrations of 0.001 to 30 µM. As shown in Fig. 6, trimethoprim at
30 µM decreased the number of intracellular parasites by 50% whereas
trimetrexate elicited the same result at 0.1 µM, in rough agreement
with the 1,000-fold tighter binding of the latter drug to DHFR. The
tetrahydroquinazoline analogs had IC50s in an
intermediate range (1 to 3 µM).
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FIG. 5.
Inhibition of the development and multiplication of
C. parvum intracellular life-cycle stages by lipophilic
antifolates. MDCK epithelial cells (2.5 × 104/cm2) were plated in eight-well microscope
chamber slides and grown to confluence for 48 h in
para-aminobenzoic acid and folate-free RPMI 1640 medium
(deficient medium) supplemented with 100 µM Hx and 10 µM dThd and
containing 5% dialyzed fetal calf serum (dFCS). The cells were
transferred to deficient medium-1% dFCS lacking Hx and dThd for
24 h and subsequently infected by incubation with C.
parvum oocysts (3.5 × 103/cm2)
for 3 h at 37°C. After three washes to remove unexcysted oocysts
and extracellular sporozoites, fresh deficient medium-1% dFCS
containing 4 µM drug (or no drug) was added. Drugs and media were
renewed at 24 h postinfection, and the experiment was terminated
at 48 h. After formaldehyde fixation, 1% Triton X-100 extraction,
and blockade in 0.5% bovine serum albumin, the infected monolayers
were processed for indirect immunofluorescence assay by consecutive
60-min incubations with 1:500 dilutions of rat polyclonal
anti-C. parvum serum, biotin-conjugated anti-rat serum,
and fluorophore CY3-conjugated streptavidin containing the DNA stain
4',6'-diamidino-2-phenylindole (DAPI) at 1 µM. Panels in the first
and second horizontal rows are photomicrographs (magnification, ca.
×1,000) of the same microscopic fields stained for nuclei with DAPI
and for parasite-containing vacuoles with the anti-C.
parvum polyclonal antibody, respectively. Panels in the third
horizontal row are lower-power photomicrographs (magnification, ca.
×400) demonstrating the overall level of C. parvum
infection and the inhibition of parasite multiplication by the
antifolates. Results are representative examples of a number of
experiments repeated on different days. Panels: A to C, controls (no
drug); D to F, trimethoprim; G to I, pyrimethamine; J to L,
trimetrexate; M to O, compound V.10; P to R, compound V.15; S to T,
compound V.16. All drugs were present at a concentration of 4 µM.
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FIG. 6.
Inhibition of the development and multiplication of
intracellular C. parvum parasites by lipophilic
antifolates. MDCK epithelial cells were plated, grown, and infected as
described in the legend to Fig. 5. Following postinfection washes to
remove unexcysted oocysts and extracellular sporozoites, deficient
medium-1% dFCS was added to each well of the chamber slide and 20 mM
stock solutions of the indicated antifolate drugs were serially diluted
fivefold across seven wells. The last well received no drug and served
as a control to quantify uninhibited growth. Drugs and media were
renewed at 24 h postinfection, the experiment was terminated at
48 h, and the monolayers were processed as described in the legend
to Fig. 5. The slides were microscopically examined for
epifluorescence, and the numbers of parasite-containing fluorescent
vacuoles were counted in each of five microscopic fields per well
(magnification, ca. ×400). Results are expressed as percentages of the
control determined as follows: (average number of parasite-containing
vacuoles in drug-treated cells  average number of vacuoles in
drug-free controls) × 100. Error bars indicate the standard
deviation at each drug concentration. Each drug was tested at least
twice with similar results. Control experiments using uninfected MDCK
cells showed that the drug concentrations used were not toxic to the
cells (data not shown). Symbols:  , trimetrexate;  , trimethoprim;
 , pyrimethamine; ×, compound V.10; *, compound V.15;  ,
compound V.16.
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On the basis of the pilot experiment whose results are depicted in Fig.
5 and
6, we reasoned that most of the compounds in
the DFCI library
would be effective somewhere in the 0.3 to 30
µM range, with
trimethoprim and pyrimethamine at one end of the
spectrum and
trimetrexate at the other. However, because we felt
that a compound
would not be worth studying further unless its
potency could be shown
to be at least 10-fold greater than that
of trimethoprim, we selected 3 µM as a reasonable upper drug concentration
limit for all subsequent
experiments. Of the 93 compounds tested,
the majority had
IC
50s of >3.0 µM and thus did not meet this
rather
stringent criterion. However, nine compounds with
IC
50s of
0.6
µM, i.e., the pteridine I.2, the
pyrido[3,2-
d]pyrimidines III.5
and III.6, the
5-chloroquinazolines IV.14, IV.17, and IV.18, the
tetrahydroquinazolines V.2 and V.5, and the
benzo[
f]quinazoline
VIII.8 appeared to be somewhat more
potent than V.4, V.10, or
V.16 and >50 times more potent than
trimethoprim. Because our
target endpoint was 50% inhibition, which
was 0.2 µM for several
of the compounds in the library,
concentrations of <0.2 µM were
not tested. As shown in Fig.
6, the
IC
50 of trimetrexate was ca.
0.1 µM. The four
compounds with the lowest IC
50s, the
pyrido[3,2-
d]pyrimidine
III.6, the quinazolines IV.17 and
IV.18, and the tetrahydroquinazolines
V.2 and V.5 approached
trimetrexate in potency. However, because
of their lack of enzyme
selectivity (Table
2), in vivo use of
these compounds would presumably
require coadministration of
leucovorin.
An interesting structure-activity correlation that becomes evident from
the results in Tables
2 and
3 is that a
low IC
50 in the spectrophotometric assay of DHFR
inhibition is not always
accompanied by proportionally high potency in
the
C. parvum growth
assay. For example, among the 20 compounds whose IC
50s in the
spectrophotometric
DHFR assay against the Cp-I enzyme were <0.6
µM, there were 8 (40%)
whose IC
50s in the growth assay were at
least
50-fold higher (i.e., >3.0 µM). Conversely, there were no
compounds
whose IC
50s in the growth assay were lower than
the
IC
50s in the enzyme assay. A reasonable
explanation for divergence
between DHFR inhibition and growth
inhibition is that the uptake
of some of the lipophilic
diaminopyrimidine antifolates into cells,
and ultimately into the
intracellular parasite, may occur via
a mechanism that is more complex
than simple diffusion. Evidence
has been presented that shows that
trimetrexate and piritrexim
are both substrates for the P-glycoprotein
efflux pump in mammalian
cells (
2). Because of the very
unusual nature of the intracellular
localization and mode of nutrient
uptake of
C. parvum within its
host cell (
11),
very little is known about how drugs actually
get into this organism at
different stages of its lifecycle. However,
it is easily conceivable
that
C. parvum may express some type
of multidrug resistance
pump whose affinity for lipophilic substrates
extends to nonclassical
antifolates. Whatever the exact details
of how these compounds actually
get into the parasite, it seems
clear that in designing and evaluating
lipophilic antifolates
as candidate drugs against cryptosporidiosis,
account must be
taken of their ability to reach the target enzyme.
The relatively low activities of V.9 and V.10
(IC
50s, >3.0 µM) against
C. parvum
in culture were of interest because, in a
standard biochemical assay
based on selective [
3H]uracil incorporation
into the nuclear DNA of
T. gondii tachyzoites
cocultured
with human embryonic lung cells in folate-containing
medium, these
compounds and the 3-trifluoromethoxybenzyl analog
V.14 had previously
been found to have IC
50s as low as 0.1 to
0.3 µM (
69). Compound V.10 was also active against
P. carinii trophozoites cocultured with rat embryonic lung
fibroblasts in
medium containing 10 µM folic acid, but complete
suppression of
growth was achieved only at a fairly high concentration
of 30
µM (
69). Thus, it appears that lipophilic
antifolates with potent
in vitro activity against
T. gondii
are not necessarily as potent
against either
C. parvum or
P. carinii. A compound with the same
potency against
T. gondii,
P. carinii, and
C. parvum
would potentially
be of therapeutic interest because AIDS patients are
sometimes
infected by two or more of these organisms. However, the
likelihood
of finding a single potent inhibitor that binds equally well
to
the DHFRs of all three species without also binding tightly to
mammalian DHFR seems
remote.
The enzyme inhibition data for 20 of the compounds in Table
2 were
correlated with the results of the yeast complementation
assay reported
earlier (
7). Seven compounds (IV.3, VI.3, VI.4,
VI.10,
VI.13, VI.14, and VIII.5) were inactive in the yeast assay,
and five of
these had IC
50s of >10 µM in the
spectrophotometric
assay against at least one of the
C. parvum enzymes. Compound
VI.4 was anomalous in that it was
ineffective in the yeast assay
even though its
IC
50 against both
C. parvum enzymes
was in the
1 to 5 µM range. Low activity in the yeast assay could
occur if
drug diffusion through the plate is slow or if drug
penetration
through the yeast cell wall is inefficient. Seven compounds
(VI.6,
VI.7, VII.1, and VII.6) had some activity in the yeast assay but
were less effective than trimetrexate, and these compounds also
had an
IC
50s of >10 µM in the enzyme assay. Five
compounds (IV.1,
IV.2, V.9, V.10, and VIII.8) were almost as effective
as trimetrexate
in the yeast assay (
7), and all of them
had IC
50s of <1 µM
in the enzyme assay. Thus,
with the exception of VI.4, which had
an IC
50 in
the 1 to 5 µM range against both
C. parvum enzymes
by
spectrophotometric assay but was inactive in the yeast assay,
there was
good agreement between the results of the two
methods.
While there are, to date, no reported clinical attempts to use
trimetrexate and leucovorin to treat severe
C. parvum
infection,
it is conceivable that this approach might be worth trying
when
the disease fails to respond to other forms of treatment. Our
finding that several compounds in the test library inhibited the
in
vitro growth of
C. parvum in mammalian cells at
physiologically
reasonable low micromolar concentrations comparable to
those at
which trimetrexate was similarly effective suggests that
lipophilic
DHFR inhibitors deserve to be more extensively tested in
combination
with leucovorin for the treatment of the most difficult
cases
of
cryptosporidiosis.
It is important to note that the purpose of this study was simply to
explore, at a molecular level (i.e., in a cell-free assay),
whether any
particular structural class among the diaminopyrimidine
DHFR inhibitors
we tested might serve as a starting point for
systematic structural
modifications aimed at the eventual discovery
of a drug whose potency
and selectivity profile is superior to
that of trimethoprim or
pyrimethamine. However, it should be remembered
that DHFR inhibition
data, by themselves, are only an early step
in predicting whether an
antifolate will be active in an infected
cell or a whole animal;
indeed, even screening for antifolate
activity in culture can provide
only a preliminary estimate of
whether a drug will be an effective
anticryptosporidial agent
in patients. Thus, even though trimethoprim
was found in this
study to be very selective in its binding affinity
for
C. parvum DHFR versus mammalian DHFR and was also found
to suppress parasite
growth in a cell assay, as others have previously
noted (
79),
the ability of this agent to control
cryptosporidial diarrhea
in AIDS patients, even in combination with
sulfamethoxazole, has
actually been disappointing. It is axiomatic that
the activity
of a drug in a whole animal is determined by a host of
factors,
only one of which is binding to the drug's target. Thus,
while
we consider the database reported in this paper to be of interest
from the standpoint of structure-activity correlation at a molecular
level, our results are not intended to be seen as anything more
than a
preliminary indication of therapeutic
potential.
Binding of inhibitors to E. coli DHFR.
As shown
in Table 4, the
IC50 of trimethoprim against E. coli
DHFR, determined under the same assay conditions as were used with the
C. parvum and human enzymes, was 0.012 µM, in good
agreement with previously published results (3, 9). Among
the 86 out of 93 compounds tested against the E. coli
enzyme, 31 (36%) were found to be better inhibitors than trimethoprim.
Moreover, 12 of these (II.2, III.3, III.5, IV.14, IV.18, V.2, V.8,
V.10, V.13, V.14, V.16, and VI.5) had IC50s in
the 0.1 to 1.0 nM range and 4 others (II.3, III.6, IV.13, and IV.17)
had IC50s that could not be accurately determined
because they were <0.1 nM. However, because the most active compounds
against E. coli DHFR also proved to be very potent against
the human enzyme (cf. Table 2), they did not match the remarkable
selectivity of trimethoprim.
The preferential affinity of trimethoprim for
E. coli DHFR
versus human DHFR has been tentatively ascribed to the fact that,
in
the case of the bacterial enzyme (at least in the binary complex
without NADPH present), the trimethoxybenzyl group can interact
with
two different hydrophobic pockets at the active site, called
the upper
and lower clefts, whereas in the human enzyme the trimethoxybenzyl
group binds only to the upper cleft (
45). The decreased
ability
of our compounds to discriminate between the
E. coli
and human
DHFRs, relative to that of trimethoprim, may be due to the
increased
separation between the aryl group of the side chain and the
diaminopyrimidine
moiety, as well as to the fact that the
diaminopyrimidine moiety
is part of a di- or tricyclic ring system.
This combination of
features apparently causes the diaminopyrimidine
derivatives to
lose the ability to interact only with the upper cleft
of the
human enzyme. It may be noted that trimethoprim displays much
more selectivity for the
E. coli enzyme than for the
C. parvum enzyme under identical assay conditions. Thus, we
believe that
finding di- or tricyclic DHFR inhibitors with selectivity
for
the
C. parvum enzyme comparable to that of trimethoprim
for the
E. coli enzyme may prove very difficult and that
greater success
might be achieved by seeking to increase the potency of
trimethoprim
rather than to increase the selectivity of trimetrexate or
piritrexim.
| |
ACKNOWLEDGMENTS |
Support of these studies was provided in part by grant RO-AI29904
to A.R., contract UO1-AI40319 to R.G.N., and contract NO1-AI35171 to
S.F.Q. from the National Institute of Allergy and Infectious Diseases.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dana-Farber
Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617)
632-3117. Fax: (617) 632-2410. E-mail: andre_rosowsky{at}dfci.harvard.edu.
| |
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Antimicrobial Agents and Chemotherapy, December 2001, p. 3293-3303, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3293-3303.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
