(Received for publication, May 11, 1995; and in revised form, July 21, 1995)
From the
Prenylcysteine methyl esters that represent the C-terminal
structures of prenylated proteins demonstrate specific substrate-like
interactions with P-glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L.,
and Casey, P. J.(1994) J. Biol. Chem. 269, 15973-15976).
The simplicity of these compounds provides a unique system for probing
the structural specificity of P-glycoprotein substrates. We have
further assessed the structural elements of prenylcysteines involved in
the interaction with P-glycoprotein. Carboxyl group methylation, a
modification in many prenylated proteins, plays an essential role of
blocking the negative charge at the free carboxylate. Substitution of
the methyl ester with a methyl amide or simple amide does not change
the ability of the molecule to stimulate P-glycoprotein ATPase
activity, but substitution with a glycine is not tolerated unless the
carboxyl group of glycine is methylated. The presence of a nitrogen
atom, which is found in many P-glycoprotein substrates and modifiers,
is also essential for prenylcysteines to interact with P-glycoprotein.
The structure at the nitrogen atom can, however, influence the type of
interaction. Acetylation of the free amino group of prenylcysteine
results in a significant loss in the ability of prenylcysteines to
stimulate P-glycoprotein ATPase activity. Instead, certain acetylated
prenylcysteines behave as inhibitors of this activity. In studies using
MDR1-transfected human breast cancer cells, the acetylated
prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport
and enhance the steady-state accumulation of
[H]vinblastine,
[
H]colchicine, and
[
H]taxol. These inhibitors do not, however,
affect drug accumulation in parental cells. These studies provide a
novel approach for designing P-glycoprotein inhibitors that could prove
effective in reversing the phenotype of multidrug resistance in tumor
cells.
Overexpression of a cell surface protein termed P-glycoprotein
(Pgp) ()in many cancer cells causes these cells to develop
cross-resistance to many natural product therapeutic drugs, a
phenomenon known as multidrug resistance or
MDR(1, 2) . Acquisition of multidrug resistance by
certain types of cancer cells poses a serious challenge to
chemotherapy. Pgp antagonists, also termed MDR modifiers, are of
clinical interest for use in overcoming Pgp-mediated drug resistance
and improving the efficacy of chemotherapeutic treatments. Several
distinct types of MDR modifiers have been identified, including calcium
channel blockers, hormonal agents, calmodulin antagonists, and
immunosuppressants(3) . When co-administered with anticancer
drugs, MDR modifiers can suppress Pgp-mediated drug efflux and restore
the drug sensitivity to MDR cells. However, these MDR modifiers can
also interfere with other cellular processes in addition to their
effects on drug transport, leading to significant side effects of these
compounds in vivo, which seriously limits their clinical uses.
Discovery of new agents with greater selectivity toward Pgp would be
facilitated by additional information concerning the essential
structural determinants of molecules recognized by Pgp.
In MDR cells, Pgp functions as an energy-driven efflux pump that can extrude many structurally unrelated cytotoxic agents from the cells. Mammalian Pgps that confer drug resistance are members of the MDR1 gene family, which includes one gene in humans and two in mice and hamsters(2) . Characterized substrates for these transporters, in addition to cytotoxic drugs noted above, also include hydrophobic peptides and ionophores(4, 5, 6) . Disruption of the MDR1a gene in mice reveals a phenotype of increased drug sensitivity and a potential function of Pgp at the blood-brain barrier (7) ; however, the precise physiologic role(s) of this transporter in normal cells still eludes investigators. Pgp belongs to a superfamily of membrane transport proteins designated as ABC (for ATP binding cassette) transporters(8) . Mammalian ABC transporters that are closely related to Pgp include the MDR2 gene product, a phosphatidylcholine translocase(9, 10) , and the multidrug resistance-associated protein (MRP), which was first identified in a drug-resistant human small cell lung cancer cell line(11) . MRP has recently been implicated in transport of glutathione S-conjugates from cells(12, 13) .
Studies on a yeast Pgp homolog, the Ste6 transporter, hint at a potential function of Pgp as a transporter of lipidated peptides. Pgp shares significant sequence and structural homology with Ste6; the latter protein is dedicated to the export of a specific mating peptide, termed a-factor, from yeast cells producing the peptide(14, 15) . a-Factor is subject to an important post-translational processing event termed protein prenylation (16, 17) that results in the mature peptide containing a C-terminal cysteine with a 15-carbon isoprenoid on the sulfhydryl group and a methyl ester on the carboxyl group(18) . The modified C-terminal cysteine is a key structural feature recognized by Ste6, as yeast strains defective in this processing are unable to produce exportable a-factor peptide (19, 20) . The finding that expression of Pgp in yeast lacking Ste6 can complement the defect and, at least partially, restore mating activity suggests that the similarities between Pgp and Ste6 extend to function as well as structure(21) . It further implies that the modified C terminus of a-factor, which is recognized by Ste6, is also included in its specific interaction with Pgp. If so, understanding the mechanism involved in this process may lead to important insight about drug transport and physiologic functions of Pgp.
We reported previously that prenylcysteine methyl esters corresponding to the C-terminal structures of prenylated proteins demonstrate characteristic substrate-like interaction with Pgp(22) . The potencies of these compounds were similar to those of the well studied compounds verapamil and N-acetyl-leucyl-leucyl-norleucinal, significantly higher than that of a hydrophobic peptide gramicidin, but lower than that of the highly potent immunosuppressive agent cyclosporin A(5, 6, 23) . The structural features required for the specific interaction between prenylcysteine methyl esters and Pgp were strikingly similar to those required for interaction of a-factor with Ste6. In both cases, carboxyl methylation of the prenylcysteine was essential(20, 22) , whereas neither transporter appeared to distinguish between a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid on the sulfhydryl group of cysteine(22, 24) . In addition to providing intriguing evidence for a potential physiologic link between Pgp and prenylation products in mammalian cells, the simplicity of prenylcysteine structure also serves as an unique system to probe the essential elements recognized by Pgp. In the current study, we have extended analysis of the structural specificity of prenylcysteines in their interaction with Pgp. By synthesizing a series of prenylcysteine analogs, we determined the respective roles of carboxyl and amino groups on the molecule. Several of the identified compounds function as inhibitors of Pgp, and the effects of these compounds on accumulation of cytotoxic drugs in MDR cells are presented.
Figure 1: Chemical structures of prenylcysteine analogs.
The interaction of the prenylcysteine analogs with
Pgp was assessed by determining their effect on Pgp-catalyzed ATP
hydrolysis using membranes isolated from Sf9 cells producing Pgp as a
source of the transporter(22) . Replacement of the methyl ester
of FCME with either the methyl amide or simply the amide did not affect
the ability of prenylcysteine to stimulate ATPase of Pgp, as both FCMA
and FCA activated the ATP hydrolysis in a fashion essentially
indistinguishable from that of FCME (Fig. 2A). The
maximum stimulation of ATPase activity by FCME, FCMA, and FCA was
3-4-fold at concentrations of 20-30 µM. At
concentrations > 30 µM these compounds showed
inhibitory effect, a phenomenon also characteristic of other Pgp
substrates(23) . Replacement of the methyl ester with a glycine
residue to produce the farnesylated dipeptide F-Cys-Gly resulted a
compound that was inactive in the assay (Fig. 2B);
however, this compound still has a free carboxyl group provided by the
-carboxyl of glycine. Methylation of that carboxyl group to
produce F-Cys-Gly-OMe restored the ability of prenylcysteine analog to
stimulate the ATPase activity to essentially the same potency as the
parent compound (Fig. 2B). These data indicate that it
is the absence of negative charge on the carboxyl side of
farnesylcysteine, rather than the presence of a methyl group, that is
critical for the interaction of prenylcysteines with Pgp.
Figure 2:
Effect of carboxyl group modification on
prenylcysteine stimulation of Pgp ATPase activity. Membranes were
isolated from Sf9 cells infected with MDR1 baculovirus 3 days
post-infection. Vanadate-sensitive ATPase activity of 5-10 µg
of membrane protein was determined in the presence of the indicated
concentrations of prenylcysteine compounds as described under
``Experimental Procedures.'' Prenylcysteine compounds were
dissolved in MeSO, and the final concentration of
Me
SO was held constant at 1%. A, stimulation of
Pgp ATPase activity by FCME (
), FCMA (
), FC (
), and
FCA (
). B, stimulation of Pgp ATPase activity by the
farnesylated dipeptide containing a C-terminal glycine, F-Cys-Gly
(
), and the same dipeptide with its carboxyl group
methyl-esterified, F-Cys-Gly-OMe (
).
Consistent with previous observations, acetylation of FCME to
produce AFCME altered its ability to stimulate the Pgp ATPase activity
in that AFCME produced only a modest stimulation of about 2-fold ((22) , Fig. 3A). Similar behavior was seen
with AFCMA and AFCA (data not shown). The prenylcysteine analog lacking
a nitrogen atom, FTAME, had little effect on Pgp ATPase activity (Fig. 3A). Interestingly, when the acetyl group was
replaced with Glu, which itself possesses a free amino group, the
dose-response profile of the farnesylated dipeptide
(
Glu-F-Cys-OMe) was virtually the same as that seen with FCME (Fig. 3B). Both AFC (Fig. 3A) and the
dipeptide with free carboxylate,
Glu-F-Cys (Fig. 3B), were inactive, supporting the notion that
removal of the negative charge of the carboxylate is essential for
interaction with Pgp. Taken together, these observations suggest that,
although the presence of a nitrogen atom is essential for
prenylcysteines to functionally interact with Pgp, the structure of
amino group can influence the nature of their interaction with this
transporter.
Figure 3:
Effect of amino group modification on
prenylcysteine stimulation of Pgp ATPase activity. The experiments were
performed as described in the legend to Fig. 2. A, Pgp
ATPase activity in the presence of the acetylated prenylcysteine
analogs AFCME (), FTAME (
), and AFC (
), in comparison
with FCME (
). B, Pgp ATPase activity in the presence of
a dipeptide containing an amide-linked
Glu (
) and the same
dipeptide after carboxyl group methylation
(
).
Carboxyl group modifications also lead to a significant change of the hydrophobic nature of prenylcysteines. It is interesting to note that methyl esterification of carboxyl group on FC had more impact on overall hydrophobicity of the molecule than switching the isoprenoid moiety from 15-carbon farnesyl to 20-carbon geranylgeranyl group (compare retention times of FCME and GGC). These observations are consistent with the hydrophobicity parameters of these compounds determined by computational methods, which revealed that GGC is two log(P) units more hydrophobic than FC, while methyl esterification of the carboxyl group increases log(P) by 2.34 units(31) . In addition, although carboxyl group modifications of FC by the amide, methyl amide, or methyl ester resulted in a substantial range in retention times (compare FCA, FCMA, and FCME in Table 1), the abilities of these compounds to stimulate ATPase activity were nearly identical (Fig. 2A).
Acetylation of amino groups on FCA, FCMA, and FCME resulted in a slight decrease of retention times compared to their non-acetylated forms. It is unlikely, however, that it is this slight difference of hydrophobicity that leads to their altered abilities to interact with Pgp (Fig. 3A), as the retention times of these compounds were in the similar range as the molecules that were capable of eliciting stimulation of Pgp ATPase activity. Furthermore, the prenylcysteine analog without the amino group, FTAME, displayed a significantly greater retention time than the majority of the prenylcysteines, yet was inactive in interacting with Pgp. Taken together, these data strongly support the notion that it is the structural features of the carboxyl and amino groups of prenylcysteines, rather than the overall hydrophobicity of these molecules, that determine their specific interaction with Pgp.
Figure 4:
Effect of AFCME on basal and
drug-stimulated ATPase activities of Pgp. Vanadate-sensitive Pgp ATPase
activity was determined in the presence of the indicated concentrations
of AFCME and either vehicle alone (), 10 µM FCME
(
), or 10 µM verapamil (
). See
``Experimental Procedures'' for assay
conditions.
The possibility that AFCME inhibition of drug-stimulated Pgp ATPase
was mediated by direct interaction at the ATP binding sites was
examined through a kinetic approach. The results, as shown in Fig. 5A, indicate that AFCME inhibition of ATPase was
non-competitive with respect to ATP, i.e. AFCME did not alter
the K of ATP but rather the effect was on V
. The mechanism of inhibition of
drug-stimulated Pgp ATPase by AFCME was then further assessed by
examining photoaffinity labeling of Pgp with a photoactive substrate,
[
H]azidopine. Pgp was the major protein
radiolabeled by [
H]azidopine in membranes
isolated from MDR1-infected Sf9 cells, and no such labeling band was
seen in membranes isolated from mock-infected membranes (Fig. 5B, lanes1 and 2).
The presence of AFCME in the reaction inhibited
[
H]azidopine labeling of Pgp with a IC
25 µM, while AFC, which is inactive in
stimulating Pgp ATPase (Fig. 3B), had little effect.
These results provide strong evidence that the mechanism of inhibition
by acetylated prenylcysteines is through a competition for drug binding
to the transporter.
Figure 5:
Inhibition of Pgp by AFCME. A,
Lineweaver-Burk plot of Pgp ATPase activity determined in the presence
of increasing concentrations of AFCME. Vanadate-sensitive Pgp ATPase
activity was measured at the specified ATP concentrations and either 0
(), 10 µM (
), or 20 µM (
)
AFCME as described in the legend to Fig. 2. B, effect
of AFCME on [
H]azidopine labeling of Pgp
expressed in Sf9 cells. Membranes from mock-infected Sf9 cells (M, lane1) and MDR1-infected Sf9 cells (lanes 2-11) were photolabeled with
[
H]azidopine in the presence of vehicle (lanes 1 and 2), vinblastine (lane3), AFCME (lanes 4-7), or AFC (lanes
8-11). Membranes containing 50-60 µg of protein
were incubated in the presence of either the specified competitors or
vehicle for 30 min at room temperature in 10 mM Tris-HCl, pH
7.5, containing 0.25 M
sucrose.[
H]Azidopine was then added to a final
concentration of 250 nM, and the samples were incubated for an
additional 20 min, followed by UV irradiation for 10 min. Proteins were
separated by 7.5% SDS-PAGE gel, and radiolabeled bands were detected by
fluorography.
To determine if the inhibitory effect of AFCME
on drug-stimulated Pgp ATPase and [H]azidopine
binding to Pgp extended to Pgp-mediated drug transport, we measured
[
H]vinblastine uptake in human breast cancer
cells overexpressing MDR1 gene (the MCF7/MDR1 cells). As a
result of elevated efflux mediated by Pgp, MCF7/MDR1 cells exhibited
much lower net uptake of radiolabeled vinblastine compared to parent
cell line, MCF7/WT (Fig. 6). Inclusion of 40 µM AFCME in the medium enhanced the uptake of
[
H]vinblastine to a level similar to that seen
when verapamil, a well characterized MDR modifier, was included (Fig. 6). Neither AFCME nor verapamil affected the
[
H]vinblastine uptake in the parental MCF7/WT
cells (data not shown), indicating that the increase in uptake elicited
by the compounds is due to an effect on a Pgp-mediated process.
Figure 6:
Effect of AFCME on
[H]vinblastine uptake in human breast cancer MCF7
cells. MCF7/WT (
) and MCF7/MDR1 cells (
,
,
)
were grown to 70-80% confluence and incubated with 10 nM [
H]vinblastine (9 Ci/mmol) and either
vehicle (
), 40 µM AFCME (
), or 20 µM verapamil (
) as indicated. After the specified time period,
cells were washed and radioactivity in cell lysates were determined
under ``Experimental
Procedures.''
We
next examined the structural requirements of the various prenylcysteine
analogs in their abilities to restore cytotoxic drug accumulation in
MCF7/MDR1 cells under steady-state conditions, i.e. a 2-h
treatment with drugs. Again, overexpression of Pgp in MCF7/MDR1 cells
resulted in substantially reduced accumulation of
[H]vinblastine as compared to MCF7/WT cells (Fig. 7A, solidbars). Inclusion of
AFCME in a dose-dependent fashion enhanced the accumulation of
[
H]vinblastine in MCF7/MDR1 cells to a level
essentially the same as that in MCF7/WT cells. The EC
for
AFCME enhanced accumulation was
30 µM, making it
slightly less potent than verapamil in this regard. Accumulation of
[
H]vinblastine in MCF7/MDR1 cells was not
affected by AFC or FCME, but the two other prenylcysteine analogs
structurally related to AFCME, namely AFCA and AFCMA, both restored
drug accumulation in the cells in an essentially identical fashion as
AFCME. This finding is consistent with the abilities of these three
compounds to inhibit the drug-stimulated ATPase activity of Pgp noted
earlier. Additionally, the AFCME analog lacking the acetylated amino
group, FTAME, did not affect the drug accumulation in the same
experiment (data not shown). None of the prenylcysteines tested
affected [
H]vinblastine accumulation in MCF7/WT
cells when included at the highest concentration used, i.e. 50
µM (Fig. 7B).
Figure 7:
Effect of prenylcysteine analogs on
radiolabeled drug accumulation in MCF7 cells. MCF7/WT and MCF7/MDR1
cells, grown to 70% confluence, were washed with D-PBS and
incubated with serum-free IMDM medium including 10 nM [
H]vinblastine (9Ci/mmol, panelsA and B), 14 nM [
H]colchicine (10 Ci/mmol, panelC), or 12 nM [
H]taxol
(7.4 Ci/mmol, panelD). Following incubation at 37
°C for 2 h, the cells were washed with ice-cold D-PBS and the
radioactivity retained in the cells was determined as described under
``Experimental Procedures.'' Drug accumulation under each
treatment was determined in triplicate wells. A, effect of
prenylcysteine analogs on [
H]vinblastine
accumulation in MCF7/MDR1 cells. B, effect of
prenylcysteine analogs on [
H]vinblastine
accumulation in MCF7/WT cells. C, effect of prenylcysteine
analogs on [
H]colchicine accumulation in
MCF7/MDR1 cells. D, effect of prenylcysteine analogs on
[
H]taxol accumulation in MCF7/MDR1 cells. The
data shown are from one experiment and are representative of at least
three independent experiments for each
treatment.
The results obtained in
the [H]vinblastine accumulation experiments
prompted us to examine the effect of the prenylcysteines on additional
cytotoxic drugs known to be substrates for Pgp. The results, shown in Fig. 7(C and D), show that the reduced
accumulation of both [
H]colchicine and
[
H]taxol in MCF7/MDR1 versus MCF7/WT
cells can also be abolished by those prenylcysteine analogs that
exhibited efficacy in the [
H]vinblastine
accumulation experiments, namely AFCME, AFCMA, and AFCA. Taken
together, these data indicate that AFCME, AFCA, and AFCMA are specific
inhibitors of Pgp-mediated drug transport, and that amino group
acetylation is a key feature in their inhibitory interaction with Pgp.
In this report we detail the structural features of prenylcysteines involved in their specific interaction with Pgp. Our results indicate that, in addition to the isoprenoid, both carboxyl and amino groups are involved at some level in the interaction with Pgp. The two groups, however, do display distinct flexibilities toward structural modifications. In addition, we demonstrate that modification at the amino group to eliminate its charge characteristics results in prenylcysteine compounds that by themselves inhibit Pgp ATPase and interrupt functional interaction of other substrates with Pgp; these compounds can thus be viewed as inhibitors of the transporter.
Prenylcysteines containing carboxyl derivatives such as methyl
ester, methyl amide, amide, and even a bulky methylated glycine residue
can all activate the ATPase activity of Pgp in a similar fashion. This
flexibility of structures recognized by Pgp is not as surprising as may
first seem when one considers the broad spectrum of drugs transported
by Pgp(2) . The finding that simply amidating the carboxyl
group to produce FCA results in an prenylcysteine equipotent with FCME
indicates that the methyl group itself is not recognized by Pgp.
Methylation more likely plays an indirect role in the interaction by
blocking the negative charge on free carboxylate, which otherwise
prevents binding of prenylcysteine to Pgp. This notion is supported by
the observation that when the carboxyl derivative is a glycine, which
possess a free carboxylate itself, the compound is inactive with Pgp
unless the carboxylate of the attached glycine is methylated. It is
interesting to note that the structural characteristics required for
interaction with Pgp is extended to a dipeptide, Glu-F-Cys, in
that only in its carboxyl-methylated form can it activate the Pgp
ATPase. The structure of this farnesylated dipeptide is strikingly
similar to glutathione S-conjugates, the potential physiologic
substrates of the related ABC transporter
MRP(12, 13) . However, the glutathione S-conjugates transported by MRP such as leukotrienes are
anionic molecules with free carboxylate at cysteine residue. Our
results thus suggest that distinct structural features of substrates
are recognized by these two related multidrug transporters.
Consistent with studies on many other Pgp substrates and modifiers (32, 33, 34) , our results point to an
essential role of the nitrogen atom for prenylcysteines to interact
with Pgp. Furthermore, our data indicate that the free amino group of a
prenylcysteine is crucial for optimal substrate-like interaction with
Pgp, as assessed by their ability to stimulate the ATPase activity of
the transporter. In addition, and somewhat surprisingly, elimination of
the charge characteristics of the free amino group by acetylation
promoted inhibitory interaction of prenylcysteine with Pgp. This
counteracting substrate binding effect may also exist for other drugs
transported by Pgp and may explain the diverse profiles of their
ability to stimulate ATPase activity(23) . Both FCME and AFCME
can prevent the interaction of substrates with Pgp as assessed by
[H]azidopine labeling experiments(22) .
It is still not clear from our experiments how the differences in the
abilities of prenylcysteines to activate or inhibit ATP hydrolysis by
Pgp are reflected in their capacities to be transported by Pgp.
However, it is clear that the ability of prenylcysteines to act in
these regards does not reflect the overall hydrophobicity of these
molecules (see Table 1). Prenylcysteine analogs thus provide an
unique system for structure-activity studies, and further experiments
with radiolabeled prenylcysteines in an in vitro transport
system should provide important information about the mechanism of
Pgp-mediated transport processes.
Another interesting finding in
these structure-activity studies on prenylcysteines is that the
position of the free amino group required for optimal substrate
activity is rather flexible, in that the dipeptide Glu-F-Cys-OMe
is as potent as the parent compound FCME. This observation is not only
consistent with the known diversity of substrates handled by Pgp, it
also indicates that the specific interaction between prenylcysteines
and Pgp may be extended to short peptides. Possibilities here include
endogenous prenylated peptides involved in signaling functions
(mammalian homologs of yeast a-factor?) and degradation products
of prenylated proteins.
The inhibitory interaction of AFCME, AFCMA, and AFCA with Pgp resulting from acetylation of their substrate-active precursors is also manifest in the abilities of these molecules to interrupt interaction of cytotoxic drugs with the transporter. While the mechanism of inhibition of Pgp ATPase activity by these acetylated prenylcysteines is not completely defined, there is a strong correlation between their ability to inhibit drug-stimulated ATPase activity of the transporter and their ability to inhibit Pgp-mediated drug transport. In this regard, the action of the acetylated prenylcysteines on Pgp is similar to that seen with cyclosporin A(5, 6, 35) . All three acetylated prenylcysteines that inhibited drug-stimulated ATPase activity of Pgp are capable of potentiating accumulation of taxol, colchicine, and vinblastine in MDR cells while not affecting the accumulation of any of these drugs in non-MDR cells. It is somewhat surprising that FCME, which also exhibits specific interaction with Pgp, is not an active molecule in the drug accumulation studies, as it might be expected to compete for drug transport as an alternate substrate. A likely explanation for the lack of effect in intact cells is that the positive charge on the amino group prevents it from crossing the membrane bilayer. This cannot be a general phenomenon, however, since many cell-active inhibitors of Pgp contain cationic amines(2) . Production of radiolabeled prenylcysteines for use in assessing cellular uptake of these molecules shall resolve this problem. Nonetheless, our studies indicate that the structure of amino group plays a critical role for the potency of some MDR modifiers, as has been suggested in studies on phenothiazines(33) . Further studies on this issue could provide insight for designing new lead compounds targeting Pgp.