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Fate of a Bioactive Fluorescent Wortmannin Derivative in Cells Katie R. Barnes,†,§ Joseph Blois,†,§ Adam Smith,† Hushan Yuan,† Fred Reynolds,† Ralph Weissleder,† Lewis C. Cantley,‡ and Lee Josephson*,† Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, 149, 13th Street, Charlestown, Massachusetts 02129, and Department of Systems Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, 77 Avenue, Louis Pasteur, Boston, Massachusetts 02115. Received June 19, 2007; Revised Manuscript Received September 13, 2007
Here, we report on NBD-Wm, a fluorescent wortmannin (Wm) probe that maintains the bioactivity of Wm as an inhibitor of PI3 kinase and as an antiproliferative agent. The attachment of the NBD fluorochrome permits NBDWm in cells to be monitored by NBD fluorescence-based methods such as FACS or fluorescence microscopy or with an anti-NBD antibody. The fluorescence of NBD-Wm treated cells reached a peak at 1.5 h and then decreased because of the extrusion of a fluorescent compound into the culture media. Cells accumulated NBD-Wm to levels about 30-fold higher than those in the media. NBD-Wm modified five major proteins, with the modification of the catalytic subunit of PI3 kinase being a minor band. The bioactivity of NBD-Wm, coupled with a variety of techniques available for determining its disposition, suggest that NBD-Wm can be a useful tool in understanding the mechanism of action of viridins.
Table 1. Biological Activity of Wm and NBD-Wma
INTRODUCTION Wm is a fungal product that covalently reacts with a lysine residue in the active site of the p110 catalytic subunit of PI3K and is a nonisotype specific inhibitor of these enzymes, which are important regulators of a wide variety of biological processes. Wm has been widely used to define the PI3 kinase dependence of biological phenomena, with some 4700 Medline citations (1995–2007) after the 1994 observation (1) that it was an inhibitor of this enzyme. The central role played by PI3 kinase suggests that compounds inhibiting its activity might be useful in diverse conditions including the treatment of cancer (2, 3) or as anti-inflammatory/immune suppressive agents (4–6). Efforts to develop Wm derivatives as drugs involve the antiproliferative and anticancer activities (7, 8) of these compounds. However, using Wm to define the PI3 kinase dependence of biological processes, or as a scaffold for drug development, is complicated since both the fate of Wm in biological systems and mechanism of interaction with molecular targets are so unlike those of man-made kinase inhibitors. Wm has a short half-life in various culture media (9) and reacts with amino acids such as lysine or proline in PBS (10). These observations, as well as others, led us to propose that Wm’s instability in culture media was due to its reaction with amino acids at the C20 position but that the resulting WmC20 amino acid derivatives maintain biological activity because of their ability to regenerate Wm (11). The unusual properties of Wm suggest that understanding its fate in biological systems will be essential to understanding its mechanism of action. Our objectives were to demonstrate the antiproliferative bioactivity of a fluorescent wortmannin conjugate (NBD-Wm) and to describe the fate of NBD-Wm in cultured cells at the micromolar concentrations needed for the antiproliferative * To whom correspondence should be addressed. Tel: (617) 7266478. Fax: (617) 726-5708 . E-mail:
[email protected]. † Massachusetts General Hospital and Harvard Medical School. ‡ Beth Israel Deaconess Medical Center. § These authors contributed equally to this work.
Inhibition of PI3 Kinase (IC50 in nM) compound
Wm
NBD-Wm
IC50
17
24
Antiproliferative Activity of Wm and NBD-Wm (IC50 in µM) compound cell line A549 MCF-7 BT-20 HeLa HT29 SKOV3 a
Wm
NBD-Wm
p value
11.4 ( 0.5 n)4 5.3 ( 0.4 n)3 29.7 ( 1.0 n)4 32.4 ( 1.0 n)4 24.0 ( 0.6 n)4 14.1 ( 1.2 n)3
12.2 ( 0.8 n)5 4.1 ( 0.4 n)4 13.1 ( 0.4 n)4 15.8 ( 1.0 n)4 19.2 ( 0.8 n)4 10.5 ( 1.9 n)2
p > 0.05 p > 0.05 p < 0.001 p < 0.001 p < 0.001 p < 0.01
95% confidence limits.
effects of Wm or Wm derivatives (see below). By fate we mean both the chemical reactions Wm undergoes when added to a biological fluid and its disposition, which includes its time course, concentration, and intracellular location. Our studies employed 10 µM NBD-Wm, a concentration that is modest in relation to its antiproliferative effects but which is several orders of magnitude higher than the concentrations needed for many actions of Wm. The IC50 values for the inhibition of cell proliferation by Wm or Wm derivatives are in the micromolar range with all cell lines (see Table 1 or refs 7, 12, and 13. At concentrations below 100 nM, Wm antagonizes insulinstimulated glucose transport (14–16), histamine release (17, 18), or respiratory burst (19–21). Earlier studies examining Wmmodified proteins using fluorescent (22, 23) and radioactive (24) forms of Wm employed lower concentrations than that employed here. In addition, others have employed low concentrations of Wm to demonstrate its reaction with a lysine in the ATP site of PI3 kinase (25), with polo-like kinase (23) and DNA-PK (22, 23). The antiproliferative actions of Wm are typically measured by exposing cells to relatively high micromolar concentrations and for long
10.1021/bc7002204 CCC: $40.75 2008 American Chemical Society Published on Web 11/08/2007
Bioactive Fluorescent Wortmannin
Figure 1. Synthesis and ionic forms of NBD-Wm. (A) Synthesis of NBD-Wm. (B) Fluorescence of compound 1 as a function of pH. An apparent pKa of 7.5 was obtained. NBD-Wm exists as a neutral (pH >> 7.5) or positively charged species (pH 0.05 for 3 or 5 washes; see Figure 2E). We next examined the time course with which NBD-Wm modified cellular protein as shown in Figure 3A. Cells were incubated with 10 µM NBD-Wm for the indicated times and lysed, and the cell protein was analyzed by the Western blot method using anti-NBD. Six bands at 35, 50, 75 85, 95, and 130 kDa were seen. The time course of the six wortmannylated species was determined by quantitative analysis of band intensity as shown in Figure 3B, with the total amount of wortmannylated protein (sum of the intensities of six bands) shown in Figure 3C. Total wortmannylated protein reached a peak at 1.5 h and declined slowly thereafter. Proteins modified by reaction with NBD-Wm were next examined in greater detail as shown Figure 4. Wortmannylated proteins, visualized by reaction with anti-NBD, and total cell protein were compared as shown in Figure 4A. Wortmannylated proteins occurred with molecular weights and with band intensities that differed markedly from the pattern seen with the total protein, indicating that NBD-Wm-modified proteins were a distinct subset of total cell proteins. To demonstrate that the catalytic subunit of PI3 kinase, p110, was modified by NBD-Wm, we employed an immunoprecipitation (IP) with anti-p85, the regulatory subunit of the enzyme. As shown in Figure 4B, the clarified lysate (CL) from the anti-p85 IP did not appear to be missing a band in the vicinity of 110 kDa. However, when the IP pellet was then electrophoresed and stained with anti-NBD (Figure 4C), a wortmannylated band at 110 kDa was present in NBD-Wm-treated cells and not in control cells. However, the amount of total cellular p110 was sufficient to enable visualization by anti-p110 without IP purification (Figure 4D). We conclude that NBD-Wm reacts with the catalytic subunit of PI3
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Figure 3. Time course for the wortmanylation of proteins by NBD-Wm. (A) Cells were exposed NBD-Wm (10 µM) for the indicated time, lysed, and stained for modification by NBD-Wm with anti-NBD. Molecular weights of prominent wortmanylated proteins (asterisks) are given on the right. (B) Densitometric scan for the time course wortmannylated protein from A. The numbers refer to the molecular weights from A. RDU ) relative density units. (C) Time course for the total amount of wortmannylated protein. The values are the sum of the six wortmannylated proteins shown in B.
Figure 4. Comparison of total cell protein, wortmannylated protein, and wortmannylated p110 from cells. (A) Cells were exposed to NBDWm (4 h, 10 µM) and then stained for total protein. Wortmannylated proteins were visualized by Western blot (WB) with anti-NBD. Wortmannylated proteins were a distinct subset of total cell proteins. (B) Cells were treated with NBD-Wm as indicated and immunoprecipated (IP’ed) with anti-p85 to remove p110 from cell lysates. The IP supernatant (IP Sup, Anti-p85) was not missing a band at 110 kDa. WB Ab indicates the use of the Western blot method with the indicated antibody. (C) IP pellets from NBD-Wm were treated and control cells reacted with anti-NBD. A band at 110 kDa was visible. (D) WB with anti-p110 for total cell protein or the IP Pellet. p110 was visible in total cell protein and prominent in IP Pellet.
kinase (p110), but the amount of NBD-Wm-modified p110 is extremely small compared to the other proteins it modifies. The intracellular distribution of NBD-Wm was monitored by fluorescence microscopy as shown in Figure 5. Fluorescence at 1.5 h (Figure 5A) was distinctly higher than that at 24 h (Figure 5B), paralleling cell fluorescence data obtained from FACS analysis (Figure 3B). The fluorescence intensity from Figure 5B was increased through image processing (Figure 5D) to permit comparison of the intracellular fluorescence (Figure 5A and B). Fluorescence intensity by microscopy (compare Figure
Figure 5. Fluorescent microscopy with NBD-Wm. Cells were incubated with 10 µM NBD-Wm for 1.5 (A) or 24 h (B). Cells incubated for 24 h have far less fluorescence. (C) Cells stained for membranes, nuclei, or nuclei and NBD-Wm. (D) The intensity of the cells shown in (B) was increased to permit the comparison of intracellular distribution of cells shown in A and B.
5A at 1.5 h to Figure 5B at 24 h) closely paralleled the time course of fluorescence seen with FACS (Figure 2B). To shed light on the subcellular localization of NBD fluorescence, cells were stained for nuclei and membranes and NBD fluorescence viewed against those stains (Figure 5C). At both 1.5 h (Figure 5A) and 24 h (Figure 5D), cells treated with NBD-Wm showed a distinct pattern of perinuclear fluorescence that colocalized with a membrane stain (red stain, Figure 5C). The colocalization of NBD fluorescence with membrane structures, high cellular concentrations of NBD-Wm (Figure 2D), and the hydrophobic nature of NBD-Wm suggest that a large pool of NBD-Wm is solubilized in membranes. Our results on the fate of NBD-Wm in A549 cells are summarized in Figure 6. The model describes the major
Bioactive Fluorescent Wortmannin
Figure 6. Pools of NBD-Wm in cells. NBD-Wm (or NBD-WmC20 amino acid derivatives) enters cells. NBD-Wm is found in two pools, an intramembrane pool and as modified proteins. Once in cells, NBDWm is converted to a second chemical entity termed NBD-Wm extrudable, whose efflux from cells causes the drop in cell fluorescence (1.5–24 h; see Figure 2B).
intracellular pools of NBD, the form that enters cells, and the formation of a fluorescent form that is extruded by cells. Form of NBD-Wm Entering Cells. The model proposes that either NBD-Wm or NBD-WmC20 amino acid derivatives enter the cells. The possibility that NBD-WmC20 derivatives may enter cells is based on (i) the fact that C20 of Wm reacts rapidly with amino acids (half-life of 0.99 min in culture media (11)) and (ii) the fact that with antiproliferation assays many Wm amino derivatives, which bear negative, positive, or zwitter ion charges, are more active than Wm (7, 11, 35). Since NBD-Wm reacts with amino acids under physiological conditions, it is difficult to determine which species enters the cells. Major Intracellular Pools of NBD-Wm. Two major pools of NBD-Wm proposed by the model are a membrane-localized pool, based on the colocalization of NBD fluorescence and a stain for membranes shown in Figure 5, and as covalently reacted with protein, based on the NBDylated protein (see in Figure 3). Other minor pools or forms of NBD-Wm may exist, and the model does not propose that any specific form gives rise to biological or pharmacological effects of Wm. For example, a minor cytoplasmic form of NBD-Wm might not appear on fluorescence microscopy and yet react with kinases and cause important physiological effects. Using an HPLC method (11), the ester bond in NBD-Wm was found to be highly stable in PBS, with a half-life longer than 700 h at 37 °C in PBS (data not shown). Nonenzymatic hydrolyzis of the ester bond of NBD-Wm is therefore unlikely. As mentioned with respect to Figure 2D, NBD-Wm was added to cell culture media, lysis buffer, or lysis buffer with cell protein, and each time yielded a time independent, stable fluorescence. For these reasons, the fluorescence from NBD is assumed to be from NBD-Wm in Figure 6, a simplifying assumption. Formation of an Extrudable Form of NBD. The characteristic peak accumulation of cell fluorescence at 1.5 h and the decrease thereafter was seen when A549 cells were incubated with NBD-Wm with multiple techniques. These included (i) FACS (Figure 2B), (ii) lysate fluorescence (Figure 2D), and (iii) fluorescence microscopy (compare Figure 5A at 1.5 h vs Figure 5B at 24 h)). This transient peak of cell-associated NBD was inconsistent with a model where a single fluorescent entity (or single class of fluorescent molecules) equilibrated between two compartments (media and cells), a mechanism through which the cell-associated NBD-Wm would asymptotically approach a maximum value. Since cells extrude fluorescence into the culture media (Figure 2C), we propose that NBD-Wm must be converted to a second form, which undergoes rapid cell extrusion.
DISCUSSION Here, we describe the bioactivity of a fluorescent Wm probe, NBD-Wm, and its fate in a cell-based system. Our experiments
Bioconjugate Chem., Vol. 19, No. 1, 2008 135
differ from those of others who have used Bodipy or tetramethylrhodamine-based Wm probes to label cell protein (22, 23). First, we have demonstrated that NBD-Wm is similar to or better than Wm in a quantitative bioassay using intact cells, the antiproliferation assay. The physical basis of the similar bioactivity of NBD-Wm and Wm is further discussed below. Second, we employed the fluorescence of NBD-Wm to determine its uptake and visualize its intracellular disposition in cells by FACS and fluorescence microscopy. Third, we employed NBD-Wm to determine the average cellular concentrations of NBD, demonstrating the ability of cells to concentrate this compound above the concentrations found in media. Using a linker different from our own linker, Giner et al. conjugated NBD to Wm and showed that the conjugate was an effective inhibitor of PI3 kinase (34). The similar or superior bioactivity of NBD-Wm relative to Wm may reflect the relatively small increase in size that results from NBD attachment. The nominal molecular weights of Wm and NBD-Wm were 428 and 677 Da, respectively. Support for the view that the size of the fluorochrome attached at the C11 of Wm can be an important determinant of the bioactivity of the resulting conjugate can be obtained from our earlier studies. Attachment of fluorescein to Wm, a fluorochrome far larger than NBD, resulted in a conjugate that was a poor inhibitor of PI3 kinase. (See compound 7c of Table 1 from ref 30.) The charge of NBD-Wm may also contribute to its bioactivity since it exists with a single positively charged species below pH 7.5 and as a neutral species above pH 7.5 (Figure 1B). Although our biotinylated-Wm inhibited PI3 kinase (compound 7a of Table 1 from 30), its antiproliferative IC50 (A549 cells) was 63.0 ( 2.2 µM, compared to the IC50 of 11.4 ( 0.5 µM for Wm and 12.2 ( 0.8 µM for NBD-Wm (see Table 1). Similar poor results with the biotinylated-Wm were obtained in other cell lines examined (HeLa, MCF-7, and HT-29; data not shown). For these reasons, NBD-Wm was superior to the fluorescent and biotinylated Wm probes we have synthesized earlier. At a concentration of 10 µM, NBD-Wm initially labeled a 50 kDa protein (10 min exposure) followed by a labeling of four additional proteins, a distinct subset of the total cell protein. The p110 catalytic subunit of PI3 kinase was a minor band; however, when p110 was concentrated by immunoprecipitation, labeling of this band was apparent. The time course for the formation of wortmannylated protein exhibited a broad plateau between 1 and 24 h, with substantial levels present 24 h after NBD-Wm was added to cells (Figure 3). Using 20 nM tritiated Wm, Thelen et al. showed that a single protein (110 kDa) was labeled in human neutrophils (24). However, when Bodipy-Wm was used to label YZ5 cells, multiple labeled bands were obtained (see Figure 6 of ref 22), as was the case with a tetramethyrhodamine-Wm (50 nM) and Jurkat T cell lysates (see Figure 2 of ref 23). In both studies, the reaction of fluorescent Wm probes was specific for proteins of certain molecular weights (Wm displaceable) and nonspecific for others (nonWm displaceable). Given the very high levels of NBD-Wm achieved in cells (Figure 2D) and the ability of Wm to react with the epsilon amino groups of lysine in PBS (11), NBDWm may react with the epsilon amino groups of a select subset of cellular proteins (Figure 4A), whose function remains unknown, in addition to labeling the lysines in the ATP sites of kinases. Though we considered it surprising that A549 cells accumulated NBD-Wm at far higher concentrations than those of the culture media of 10 µM (see Figure 2D), a variety of factors indicate that this observation is correct. First, our method of determining the concentration of cell-associated NBD-Wm employed a high detergent lysis buffer, one containing 1% w/v of the nonionic detergent NP-40, which disrupts cell structure
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and completely solubilizes cell membranes and organelles. To obtain cell-associated NBD concentrations, fluorescent standards were employed; these standards were obtained by diluting known amounts of NBD-Wm into lysate buffer containing protein from lysed cells. Our method of determining cellassociated NBD concentrations destroyed cell structure and read the fluorescence of standards and unknowns in precisely the same media so that possible environmental effects on NBD fluorescence would not yield incorrect values. Second, the fluorescence of NBD-Wm when added to culture media, lysis buffer, or lysis buffer plus cell protein was stable for at least 24 h, indicating a lack of enzymatic or nonenzymatic mechanisms for altering fluorescence (data not shown). Third, the use of our standard three wash protocol reduced cell-associated fluorescence to a value that was independent of the number of washes employed. At both 1.5 and 24 h of incubation, cellassociated fluorescence reached a plateau after three washes that was not affected by additional washes (Figure 2E). Fourth, we considered that the high cellular concentrations of NBD-Wm that we obtained might be secondary to toxic effects of the NBDWm. However, cells exposed to the nonfluorescent Wm (10 µM, 23 h) then internalized 10 µM NBD-Wm in a manner identical to that of non-Wm exposed cells. Finally, key observations made with A549 cells (Figure 2B, time course of cell fluorescence, and Figure 2D, average cellular concentration) were also made with HeLa cells as well, indicating that our observations were not cell line specific. On the basis of these experiments, A549 cells apparently accumulate NBD-Wm to far higher levels than that present in the media, reaching average cell-associated concentrations as high as 300 µM when incubated with 10 µM NBD-Wm (1.5 h exposure). An interesting issue is the implication of these findings for the molecular targets of Wm and Wm derivatives. Literature values for the IC50 values of Wm for kinases are 1–4 nM (PI3 kinase 1, 29), 24 nM (polo-like kinase (23)), 200 nM (mTOR (36)), and 200 nM (DNA-dependent protein kinase (37)). The micromolar concentrations of NBD-Wm needed to inhibit cell proliferation, coupled to the observation that cells concentrate NBD-Wm to values far above that in culture media (Figure 2D), means that the average cellular concentrations of NBD-Wm far exceeds the IC50 values of all known kinases for Wm. For example, if a selective PI3 kinase inhibition were the basis of NBD-Wm’s antiproliferative effects, on the basis of known IC50 values, a cytoplasmic concentration on the order of 10 nM NBDWm would be required. This concentration is some 30,000fold lower than the 300 µM concentration of cell-associated NBD-Wm we obtained. While it is technically difficult to determine the concentration of NBD-Wm in the environment of an enzyme, we regard such a massive reduction in concentration unlikely and instead propose that the antiproliferative effects of NBD-Wm result from the inhibition of a variety of targets, one of which is PI3 kinase. Consistent with the proposed lack of selectivity of NBD-Wm as a specific PI3 kinase inhibitor in vivo was its ability to modify many proteins, of which the p110 catalytic subunit of PI3 kinase was an extremely minor band. Others have determined Wm-modified proteins using radioactive (24) or fluorescent Wm probes at lower concentrations than those employed here (22, 23). A different pattern of Wmmodified proteins maybe obtained at these low Wm concentrations, which are adequate to produce many important effects on cells. Our goal, however, was to describe the fate of NBDWm in cells at concentrations associated with its antiproliferative effects. Our objectives were to demonstrate that the bioactivity of NBD-Wm was similar to (or better than) Wm and to describe the fate of NBD-Wm in cultured cells associated with its antiproliferative effects using a variety of techniques (micros-
Barnes et al.
copy, FACS, and Western blot). Immunochemical methods of detecting of NBD can in principle be extended from Wm to other NBD-based probes or drugs that covalently modify proteins in cell-based systems. Antibody-based methods are not subject to interference from background fluorescence and amenable to enzyme amplification to increase sensitivity. On the basis of the variety of fluorescence-based and immunochemical methods for detecting NBD, we envision NBD-Wm as a powerful tool for future investigations concerning the mechanism of action of viridins.
ACKNOWLEDGMENT This work was supported in part by NIH Grants R01EB00662 (to L.J.), R01 EB004472(to L.J.), and 2P50CA86355 (to R.W.). Supporting Information Available: Proton NMR spectra of NBD-Wm and 13C NMR spectra of NBD-Wm. This material is available free of charge via the Internet at http://pubs.acs.org.
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