©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Prenylcysteines That Interact with P-glycoprotein and Inhibit Drug Transport in Tumor Cells (*)

(Received for publication, May 11, 1995; and in revised form, July 21, 1995)

Lili Zhang (1)(§) Clifford W. Sachs (2) Hua-Wen Fu (1) Robert L. Fine (2) Patrick J. Casey (1)(¶)

From the  (1)Department of Molecular Cancer Biology and Biochemistry and (2)Division of Hematology/Oncology, Departments of Medicine and Pharmacology, Durham Veterans Affairs Medical Center, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [^3H]vinblastine, [^3H]colchicine, and [^3H]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.


INTRODUCTION

Overexpression of a cell surface protein termed P-glycoprotein (Pgp) (^1)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.


EXPERIMENTAL PROCEDURES

Synthesis of Prenylcysteine Analogs

The synthesis of S-farnesylcysteine methyl ester (FCME), N-acetyl-S-farnesylcysteine (AFC), and N-acetyl-S-farnesylcysteine methyl ester (AFCME) have been described(22) . In a similar fashion, the sulfhydryl groups of Glu-Cys (Sigma) or Cys-Gly (Sigma) were alkylated with farnesyl bromide (Aldrich)(25) . The carboxyl groups of farnesylated Glu-Cys, Cys-Gly, and farnesylthioacetic acid (LC Laboratories, Woburn, MA) were converted to methyl esters by treatment with diazomethane(26) , prepared using the Mini Diazald kit (Aldrich) according to manufacture's instructions. For the synthesis of S-farnesylcysteine methyl amide (FCMA) and S-farnesylcysteine amide (FCA), the methyl ester group of FCME was converted to methyl amide or amide by reaction with methylamine (Aldrich) or ammonia hydroxide, respectively. Briefly, 1 mmol of methyl amine as a 40% aqueous solution (Aldrich) or ammonium hydroxide was mixed with 100 µmol of FCME dissolved in 0.5 ml of methanol, and the reactions were stirred at room temperature for 2 days. The yield of this reaction is 60-70% methylamide or amide and 30-40% free acid due to the hydrolysis of ester bond. N-Acetyl-S-farnesylcysteine amide (AFCA) and N-acetyl-S-farnesylcysteine methyl amide (AFCMA) were then prepared by acetylation of FCA and FCMA, respectively, as described(27) , except that 4-methylbenzyl chlorocarbonate was replaced by acetyl anhydride. All compounds were purified by C18 reverse-phase high performance liquid chromatography (HPLC) prior to use and the identities of the products confirmed by ^1H NMR or mass spectroscopy.

HPLC Analysis of Prenylcysteines

The relative hydrophobicity of prenylcysteines was determined by their retention times on reverse-phase HPLC column. Prenylcysteines were analyzed on a C18 HPLC column (Phenomenex, Torrance, CA) equilibrated with 30% acetonitrile containing 0.1% ammonia acetate/acetic acid, pH 6.0. The column was developed with a 50-min linear gradient to 100% acetonitrile in the same buffer system and then with 100% acetonitrile for additional 30 min. The elution of prenylcysteines was detected by their UV absorbance at 210 nm.

Cell Culture

Maintenance of Sf9 cells and their use in production of Pgp by infection with recombinant baculovirus containing the MDR1-V185 cDNA has been described(22) . The drug resistant human breast cancer cell line, MCF7/CL10.3 (hereafter designated as MCF7/MDR1) and its parental non-resistant cell line MCF7/WT, were obtained from Dr. Robert Clarke(28) . These cells were grown in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) containing 100 units/ml penicillin and 100 µg/ml streptomycin. The MCF7/MDR1 cells were originally established by stably transfecting the MCF7/WT cells with cDNA encoding human MDR1 gene (28) and were grown in drug-free medium throughout our studies. The expression level of Pgp in these cells was monitored by immunoblot analysis, and a fresh culture was started from frozen stock cells every 5 months.

Drug Uptake/Accumulation

For drug uptake and accumulation studies, MCF7/WT and MCF7/MDR1 cells were grown to 70-80% confluence in 12-well plates (Corning). The cell monolayers were washed with Dulbecco's phosphate-buffered saline (D-PBS, from Life Technologies, Inc.) and then incubated with serum-free IMDM medium supplemented with vehicle or prenylcysteine analogs and the respective radiolabeled drugs, either [^3H]vinblastine (Moravek Biochemicals, Inc., Brea, CA), [^3H]colchicine (DuPont NEN), or [^3H]taxol (Moravek Biochemicals). The final concentrations and specific activities for each radioactive drug were: [^3H]vinblastine, 10 nM at 9 Ci/mmol; [^3H]taxol, 12 nM at 7.4 Ci/mmol; [^3H]colchicine, 12 nM at 10 Ci/mmol. Following incubation at 37 °C for the indicated time periods, the cell monolayers were washed three times with ice-cold D-PBS and the cells were lysed with 20 mM Tris-HCl, pH 7.7, containing 0.2% SDS. Radioactivity in the cell lysates were determined by scintillation counting.

Miscellaneous Methods

Membranes were prepared from Sf9 cells infected with MDR1 baculovirus as described(23) . Vanadate-sensitive ATPase activity was determined by measuring the released inorganic phosphate(22) . Photoaffinity labeling of Pgp in Sf9 membranes was performed as described previously(22) . The protein concentrations were determined by Amido Black protein assay (29) or a dye binding assay (Bio-Rad) using bovine serum albumin as standard(30) .


RESULTS

Influence of Carboxyl Group Modifications on Prenylcysteine Interaction with Pgp

The basic structure of a prenylcysteine consists of an isoprenoid attached to the sulfhydryl group of cysteine. Our previous study had indicated that both the isoprenoid and the cysteine with a methylated carboxyl group are required for specific interaction with Pgp(22) . The type of isoprenoid required for stimulating Pgp activity is rather flexible, as molecules containing either the 20-carbon geranylgeranyl or 15-carbon farnesyl are essentially equipotent(22) . To further explore the structure-activity features of prenylcysteines, we synthesized a series of farnesylcysteine analogs focusing on carboxyl and amino group modifications. An important finding from the earlier studies was that the methyl-esterified carboxyl group is a key component in prenylcysteine recognition by Pgp(22) . To assess the structural specificity of this modification, we replaced the methyl ester with methyl amide, producing FCMA, or simply with the amide, producing FCA. We also prepared farnesylated dipeptides in which the carboxyl group of the cysteine is involved in an amide bond with the amino group of glycine, F-Cys-Gly, and this dipeptide containing a methyl ester at carboxyl group, F-Cys-Gly-OMe. The structures of these and other compounds used in the studies herein are shown in Fig. 1.


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 alpha-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 Me(2)SO, and the final concentration of Me(2)SO was held constant at 1%. A, stimulation of Pgp ATPase activity by FCME (bullet), FCMA (up triangle), FC (circle), and FCA (). B, stimulation of Pgp ATPase activity by the farnesylated dipeptide containing a C-terminal glycine, F-Cys-Gly (up triangle), and the same dipeptide with its carboxyl group methyl-esterified, F-Cys-Gly-OMe ().



Influence of Amino Group Modifications on Prenylcysteine Interaction with Pgp

The presence of a free amino group in prenylcysteines is of particular interest, as it has been noted that many Pgp substrates contain a basic nitrogen atom(2) . Furthermore, we had noticed previously that acetylation of the amino group of FCME substantially reduced its ability to stimulate the ATPase activity of Pgp(22) . To assess the influence of the amino group of prenylcysteines in interaction with Pgp in more detail, we first made a prenylcysteine analog without an amino group, FTAME. In addition, we acetylated the amino groups of FC, FCME, FCMA, and FCA to produce AFC, AFCME, AFCMA, and AFCA, respectively. These latter compounds lack a cationic nitrogen under physiologic conditions. We also synthesized two farnesylated dipeptides containing a Glu residue attached to the cysteine amino group via an amide linker. In one of these farnesylated dipeptides, Glu-F-Cys, the free carboxyl group was retained, while in the other, Glu-F-Cys-OMe, it was subject to methylation. Thus, these dipeptides have lost the free amino group on cysteine but contain an additional amino group on Glu.

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 (circle), and AFC (up triangle), in comparison with FCME (bullet). B, Pgp ATPase activity in the presence of a dipeptide containing an amide-linked Glu (up triangle) and the same dipeptide after carboxyl group methylation ().



Relative Hydrophobicity of Prenylcysteine Analogs

To determine whether the ability of prenylcysteines to stimulate the Pgp ATPase activity reflects the hydrophobic nature of these compounds, we compared the relative hydrophobicity of prenylcysteines by their retention times on reverse-phase HPLC column. Prenylcysteines bind to the column by hydrophobic interactions. More hydrophobic molecules thus bind to the column more strongly, and are eluted at higher concentrations of acetonitrile. As shown in Table 1, the retention times of prenylcysteines were affected by modifications of the isoprenoid, carboxyl, or amino groups. The structure of isoprenoid is an important factor determining the hydrophobicity of prenylcysteines, in that 20-carbon compounds GGC and GGCME have significantly greater retention times (15 min) than the corresponding 15-carbon FC and FCME. The increase in hydrophobicity of geranylgeranyl compounds does not, however, influence their interactions with Pgp in that GGCME and FCME are equipotent in stimulating Pgp ATPase activity(22) . Furthermore, GGC, although more hydrophobic than the substrate-active analogs FCA and FCMA (Fig. 1), was inactive when interacting with Pgp(22) .



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.

Prenylcysteine Analogs as Inhibitors of Pgp

While conducting the initial experiments assessing the effect of AFCME on the ATPase activity of Pgp, we noted that concentrations of this particular prenylcysteine above 10 µM suppressed the ATPase activity of the transporter(22) . To determine whether this ability to inhibit ATPase activity of Pgp extended to drug-stimulated as well as basal ATPase activity, we assessed the effect of AFCME on the ATPase activity of Pgp in Sf9 membranes in the absence of any drug and in the presence of either verapamil or FCME, both of which stimulate Pgp ATPase activity. Pgp ATPase activity stimulated by either 10 µM verapamil or 20 µM FCME, the concentrations for maximum stimulation of ATP hydrolysis by these two compounds, were inhibited in a dose-dependent fashion by AFCME with an IC of 15 µM (Fig. 4). Similar results were also obtained with the analogs AFCMA and AFCA (results not shown).


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 (circle), 10 µM FCME (up triangle), or 10 µM verapamil (bullet). 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(m) of ATP but rather the effect was on V(max). 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, [^3H]azidopine. Pgp was the major protein radiolabeled by [^3H]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 [^3H]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 (bullet), 10 µM (circle), or 20 µM () AFCME as described in the legend to Fig. 2. B, effect of AFCME on [^3H]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 [^3H]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.[^3H]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 [^3H]azidopine binding to Pgp extended to Pgp-mediated drug transport, we measured [^3H]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 [^3H]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 [^3H]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 [^3H]vinblastine uptake in human breast cancer MCF7 cells. MCF7/WT () and MCF7/MDR1 cells (up triangle, bullet, circle) were grown to 70-80% confluence and incubated with 10 nM [^3H]vinblastine (9 Ci/mmol) and either vehicle (up triangle), 40 µM AFCME (bullet), or 20 µM verapamil (circle) 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 [^3H]vinblastine as compared to MCF7/WT cells (Fig. 7A, solidbars). Inclusion of AFCME in a dose-dependent fashion enhanced the accumulation of [^3H]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 [^3H]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 [^3H]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 [^3H]vinblastine (9Ci/mmol, panelsA and B), 14 nM [^3H]colchicine (10 Ci/mmol, panelC), or 12 nM [^3H]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 [^3H]vinblastine accumulation in MCF7/MDR1 cells. B, effect of prenylcysteine analogs on [^3H]vinblastine accumulation in MCF7/WT cells. C, effect of prenylcysteine analogs on [^3H]colchicine accumulation in MCF7/MDR1 cells. D, effect of prenylcysteine analogs on [^3H]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 [^3H]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 [^3H]colchicine and [^3H]taxol in MCF7/MDR1 versus MCF7/WT cells can also be abolished by those prenylcysteine analogs that exhibited efficacy in the [^3H]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.


DISCUSSION

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 [^3H]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.


FOOTNOTES

*
This work was supported by American Cancer Society Grant BE-117B and a Pardee Foundation grant (to P. J. C), and American Cancer Society Grant IRG 158J and a grant from Ladies Auxiliary to VFM (to R. L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from Leukemia Society of America.

Established Investigator of American Heart Association. To whom correspondence should be addressed: Dept. of Molecular Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8613; Fax: 919-613-8642.

(^1)
The abbreviations used are: Pgp, P-glycoprotein; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; FC, S-farnesylcysteine; FCME, carboxyl methyl ester of FC; FCA, carboxyl amide form of FC; FCMA, carboxyl methyl amide form of FC; F-Cys-Gly, S-farnesylated Cys-Gly; F-Cys-Gly-OMe, carboxyl methyl ester form of F-Cys-Gly; FTAME, methyl ester form of farnesylthioacetic acid; AFC, AFCME, AFCA, and AFCMA, amino-acetylated forms of FC, FCME, FCA, and FCMA, respectively; Glu-F-Cys, S-farnesylated Glu-Cys; Glu-F-Cys-OMe, carboxyl methylated form of Glu-F-Cys; GGC, S-geranylgeranylcysteine; GGCME, carboxyl methyl ester of GGC; HPLC, high performance liquid chromatography, IMDM, Iscove's modified Dulbecco's medium; D-PBS, Dulbecco's phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Drs. Ursula A. Germann and Michael M. Gottesman for providing MDR1 baculovirus, Dr. Robert Clarke for MCF7/MDR1 cell line, and Dr. Anthony A. Ribeiro at Duke Spectroscopy Center for NMR spectra of prenylcysteines. Instrumentation at the Center was established with funding from the National Institutes of Health, National Science Foundation, North Carolina Biotechnology Center, and Duke University. We also thank the Keck Foundation for support of the Levine Science Research Center at Duke University.


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