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Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine*

Open AccessPublished:November 21, 2016DOI:https://doi.org/10.1074/jbc.M116.750299
      Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

      Introduction

      Piperlongumine (PL),
      The abbreviations used are: PL
      piperlongumine
      hPL
      hydrolyzed PL
      ROS
      reactive oxygen species
      NBDHEX
      6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanoyl
      RMSD
      root mean square deviation
      CDNB
      1-chloro-2,4-dinitrobenzene
      G-site
      glutathione binding site
      H-site
      hydrophobic binding site.
      a natural product derived from the fruit of Piper longum, selectively causes apoptosis in numerous cancer cell lines as well as cancerous tumors in animal models, but does not demonstrate anti-proliferative behavior in non-transformed cells (
      • Raj L.
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      • Stern A.M.
      • Mandinova A.
      • Schreiber S.L.
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      Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
      ). PL has shown anti-tumor activity in prostate cancer (
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      • Ang W.
      • Ye W.
      • Wei Y.
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      • Luo Y.
      Biodegradable nanoassemblies of piperlongumine display enhanced anti-angiogenesis and anti-tumor activities.
      ,
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      • Kutikov A.
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      Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells.
      ), breast cancer (
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      Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.
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      Piperlongumine for enhancing Oral bioavailability and cytotoxicity of docetaxel in triple-negative breast cancer.
      ), lung cancer (
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      Piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of PI3K/Akt/mTOR pathway.
      ), primary brain tumors (
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      Piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent JNK and p38 activation.
      ), gastric cancer (
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      • Ying S.
      • Zhang J.
      • Zhang Z.
      • Liu Z.
      • Yang S.
      • Liang G.
      Auranofin induces apoptosis by ROS-mediated ER stress and mitochondrial dysfunction and displayed synergistic lethality with piperlongumine in gastric cancer.
      ), colon cancer (
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      • Reindl K.M.
      Activation of ERK signaling and induction of colon cancer cell death by piperlongumine.
      ), lymphoma (
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      • Son D.J.
      • Yun H.
      • Kamberos N.L.
      • Janz S.
      Piperlongumine inhibits proliferation and survival of Burkitt lymphoma in vitro.
      ), and leukemia (
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      • Callahan K.P.
      • Balys M.
      • Ashton J.M.
      • Neering S.J.
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      • et al.
      Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.
      ), making it an attractive molecule for further development to treat these cancers. Of note, PL is commonly used in traditional medicine on the Indian subcontinent to treat a range of maladies including cancer. The anti-cancer effects of PL are associated with an elevation of reactive oxygen species (ROS) and a decrease in cellular GSH (
      • Pei S.
      • Minhajuddin M.
      • Callahan K.P.
      • Balys M.
      • Ashton J.M.
      • Neering S.J.
      • Lagadinou E.D.
      • Corbett C.
      • Ye H.
      • Liesveld J.L.
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      • Li Z.
      • Shi L.
      • Greninger P.
      • Settleman J.
      • et al.
      Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.
      ). Consistent with those observations, a leading cellular target was identified as glutathione S-transferase-Pi 1 (GSTP1), based on stable isotope labeling (
      • Raj L.
      • Ide T.
      • Gurkar A.U.
      • Foley M.
      • Schenone M.
      • Li X.
      • Tolliday N.J.
      • Golub T.R.
      • Carr S.A.
      • Shamji A.F.
      • Stern A.M.
      • Mandinova A.
      • Schreiber S.L.
      • Lee S.W.
      Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
      ).
      PL is composed of a trimethoxyphenyl head and two reactive olefins, C2-C3 and C7-C8 (see Fig. 1A). Extensive structure-activity relationship analysis has shown the electrophilicity of PL to be essential for cellular activity, with the C2-C3 olefin playing a dominant role in causing cellular toxicity by elevation of cellular ROS and depletion of cellular GSH, and the C7-C8 olefin contributing to cellular toxicity. The aggregate of these observations led to a model wherein the C2-C3 olefin binds directly to GSH, while the C7-C8 olefin binds covalently to GSTP1 (
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      ). Further elaboration of PL showed the α-β unsaturated imide to be essential for supporting the reactivity of the C2-C3 olefin. Oligomerization was also found to enhance cellular potency, in a linker length-dependent manner (
      • Boskovic Z.V.
      • Hussain M.M.
      • Adams D.J.
      • Dai M.
      • Schreiber S.L.
      Synthesis of piperlogs and analysis of their effects on cells.
      ).
      Figure thumbnail gr1
      FIGURE 1Hydrolyzed piperlongumine inhibits GSTP1. A, structure of PL and hPL. B, architecture of GSTP1 dimer. Protomer A is colored brown, and protomer B is gray. The hPL-GSH complex is shown as green sticks, and the glutathione (G-site) and hydrophobic (H-site) binding sites are each labeled. C, 2FoFc map for hPL-GSH complex is shown in green and scaled to 1σ. D, binding of the hPL-GSH complex (green sticks) to GSTP1. Residues that make notable hydrogen-bonding or van der Waals interactions are shown as gray sticks.
      GSTP1 belongs to a family of detoxification proteins referred to as glutathione S-transferases or GSTs that catalyze the conjugation of GSH to electrophiles, and is frequently overexpressed in cancerous tumors (
      • Mannervik B.
      • Castro V.M.
      • Danielson U.H.
      • Tahir M.K.
      • Hansson J.
      • Ringborg U.
      Expression of class Pi glutathione transferase in human malignant melanoma cells.
      ,
      • Hayes J.D.
      • Pulford D.J.
      The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.
      • O'Brien M.L.
      • Tew K.D.
      Glutathione and related enzymes in multidrug resistance.
      ). GST is involved in metabolism of alkylating chemotherapeutic drugs such as cisplatin, adriamycin, etoposide, thiotepa, chlorambucil, and ethacrynic acid. Indeed, GST overexpression has also been associated with multi-drug resistance (
      • O'Brien M.L.
      • Tew K.D.
      Glutathione and related enzymes in multidrug resistance.
      ,
      • Kodera Y.
      • Isobe K.
      • Yamauchi M.
      • Kondo K.
      • Akiyama S.
      • Ito K.
      • Nakashima I.
      • Takagi H.
      Expression of glutathione-S-transferases alpha and pi in gastric cancer: a correlation with cisplatin resistance.
      • Chen J.
      • Solomides C.
      • Simpkins H.
      Sensitization of mesothelioma cells to platinum-based chemotherapy by GSTpi knockdown.
      ). Additionally, this effect may also be mediated by direct interactions with signaling proteins such as c-Jun N-terminal kinase leading to pro-survival (anti-apoptotic) responses (
      • Elsby R.
      • Kitteringham N.R.
      • Goldring C.E.
      • Lovatt C.A.
      • Chamberlain M.
      • Henderson C.J.
      • Wolf C.R.
      • Park B.K.
      Increased constitutive c-Jun N-terminal kinase signaling in mice lacking glutathione S-transferase Pi.
      ,
      • Adler V.
      • Yin Z.
      • Fuchs S.Y.
      • Benezra M.
      • Rosario L.
      • Tew K.D.
      • Pincus M.R.
      • Sardana M.
      • Henderson C.J.
      • Wolf C.R.
      • Davis R.J.
      • Ronai Z.
      Regulation of JNK signaling by GSTp.
      ).
      GST proteins have been well characterized, in large part motivated by their potential as drug targets. Numerous GST inhibitors have been reported including: a GSH analogue, TLK117 (γ-glutamyl-S-(benzyl)cysteinyl-R(-)-phenylglycine) (
      • Morgan A.S.
      • Ciaccio P.J.
      • Tew K.D.
      • Kauvar L.M.
      Isozyme-specific glutathione S-transferase inhibitors potentiate drug sensitivity in cultured human tumor cell lines.
      ); a GSH-doxorubicin conjugate (GSH-DXR) (
      • Asakura T.
      • Sasagawa A.
      • Takeuchi H.
      • Shibata S.
      • Marushima H.
      • Mamori S.
      • Ohkawa K.
      Conformational change in the active center region of GST P1–1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis.
      ); small molecule inhibitors ethacrynic acid (
      • Ploemen J.H.
      • van Ommen B.
      • Bogaards J.J.
      • van Bladeren P.J.
      Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.
      ) and 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) (
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      ); the natural products benastatins A and B; and finally, an irreversible inhibitor, chloroacetamide-containing KT53 (
      • Tsuboi K.
      • Bachovchin D.A.
      • Speers A.E.
      • Spicer T.P.
      • Fernandez-Vega V.
      • Hodder P.
      • Rosen H.
      • Cravatt B.F.
      Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.
      ). Structural information is available for many of these bound to their respective GST targets, and in each case, GSH resides in the glutathione binding site, or G-site, while the inhibitor occupies the hydrophobic region of the active site adjacent to GSH, or the H-site. Although several of these inhibitors have been shown to exhibit anti-tumor effects, general toxicity has prevented them from moving forward in human trials (
      • Raj L.
      • Ide T.
      • Gurkar A.U.
      • Foley M.
      • Schenone M.
      • Li X.
      • Tolliday N.J.
      • Golub T.R.
      • Carr S.A.
      • Shamji A.F.
      • Stern A.M.
      • Mandinova A.
      • Schreiber S.L.
      • Lee S.W.
      Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
      ,
      • Wang W.
      • Liu G.
      • Zheng J.
      Human renal UOK130 tumor cells: a drug resistant cell line with highly selective over-expression of glutathione S-transferase-pi isozyme.
      ).
      To understand the molecular basis for the effect of PL on GSTP1 and explain the prior structure-activity relationship analysis, we determined the high resolution X-ray crystal structure of GSTP1 in complex with PL and GSH. Surprisingly, PL becomes hydrolyzed to a trimethoxycinnamic acid, which we term hydrolyzed PL (hPL), eliminating the C2-C3 olefin. Additionally, a covalent complex is formed between the C7-C8 olefin of PL and the thiol from GSH, which is in contrast to the predicted model where GSH undergoes a Michael addition at the C2-C3 olefin. Mass spectrometric analysis of the reactions between PL and a cysteine-containing peptide confirmed the hydrolysis of PL through nucleophilic attack of PL at the C2-C3 olefin. Further, we demonstrate that in the presence of physiologically relevant concentrations of PL, PL does not covalently modify GSTP1, suggesting that the biological mechanism for GSTP1 inhibition is not through covalent modification by PL. Instead, our X-ray data show that the hPL-GSH conjugate binds non-covalently in the GSTP1 active site, through the formation of multiple van der Waals and hydrogen-bonding interactions. Coupled with in vitro kinetic data, which show a concentration-dependent inhibition of GSTP1 by hPL, our data suggest that PL is a prodrug, which becomes activated by hydrolysis to hPL, resulting in the formation of an hPL-GSH conjugate and inhibition of GSTP1.

      Results

      Structure Determination of PL-GSTP1 Complex

      We performed X-ray crystallography to understand the molecular interactions between GSTP1 and PL. GSTP1 was expressed and purified from Escherichia coli, and protein crystals were obtained using methods described previously (
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      ). The crystals were grown in the presence of 1 mm final concentration of GSH and PL and appeared within 6 days, growing to 200 μm in size. After cryoprotection in 25% glycerol, diffraction data extended to a resolution of 1.1 Å. Crystals were found to have C2 symmetry with two molecules in the asymmetric unit, forming a functional dimer, as reported previously (
      • Oakley A.J.
      • Lo Bello M.
      • Battistoni A.
      • Ricci G.
      • Rossjohn J.
      • Villar H.O.
      • Parker M.W.
      The structures of human glutathione transferase P1–1 in complex with glutathione and various inhibitors at high resolution.
      ). Phases were obtained by molecular replacement using a previously solved structure of GSTP1 as a search model, Protein Data Bank (PDB) ID 3GUS, followed by positional refinement and 2-fold noncrystallographic symmetry averaging. Refinement resulted in final Rwork and Rfree values of 16.3 and 18.6, respectively. Final crystallographic and refinement statistics are given in Table 1.
      TABLE 1Crystallography statistics
      Data collection
          X-ray sourceAPS 19–1D
          Wavelength (Å)0.97924
          Space groupC2
          Unit cell
              a, b, c (Å)78.5, 89.5, 69.0
              α, β, γ (°)90.0, 98.2, 90.0
          Resolution (Å)50.00–1.18
          Unique reflections152,714
          Redundancy3.6 (2.5)
      Statistics for the last shell are given in parentheses.
          Completeness (%)99.3 (87.6)
          Wilson B-factor9.2
          Rmerge (%)4.6 (33.6)
          〈I/σ 〉24.16 (2.65)
      Refinement
          Resolution50–1.18
          Reflections Used145,077
          Rfree reflections7,637
          Rwork/Rfree (%)16.3/18.6
          Non-hydrogen atoms4117
              Protein3,291
              Water728
              Ligand98
          RMSD
              Bond lengths (Å)0.009
              Bond angles (°)1.1
          Average B-factor (Å2)14.0
          Ramachandran plot (%)
              Favored97.3
              Allowed1.2
              Disallowed1.5
      a Statistics for the last shell are given in parentheses.

      Overall Architecture and Piperlongumine/Glutathione Binding

      The topology and overall organization of GSTP1 have been reported previously as a biological dimer between chains A and B, with each chain/protomer consisting of eight α-helices and two sets of anti-parallel β-sheets (
      • Oakley A.J.
      • Lo Bello M.
      • Battistoni A.
      • Ricci G.
      • Rossjohn J.
      • Villar H.O.
      • Parker M.W.
      The structures of human glutathione transferase P1–1 in complex with glutathione and various inhibitors at high resolution.
      ). Consistent with prior reports, each protomer is subdivided into N and C-terminal domains. The G-site is formed by the N-terminal domain, whereas the H-site is formed with contributions from the C terminus (Fig. 1B). Structural alignment with a previously solved X-ray structure of GSTP1 bound with the GSH-ethacrynic acid conjugate (PDB ID 3GSS) (
      • Oakley A.J.
      • Lo Bello M.
      • Battistoni A.
      • Ricci G.
      • Rossjohn J.
      • Villar H.O.
      • Parker M.W.
      The structures of human glutathione transferase P1–1 in complex with glutathione and various inhibitors at high resolution.
      ) showed an almost identical fold (0.22 Å RMSD for Cα atoms).
      Inspection of the difference electron density map clearly demonstrated GSH bound to each protomer. The interactions between GSH and GSTP1 have been described in detail previously (
      • Oakley A.J.
      • Lo Bello M.
      • Battistoni A.
      • Ricci G.
      • Rossjohn J.
      • Villar H.O.
      • Parker M.W.
      The structures of human glutathione transferase P1–1 in complex with glutathione and various inhibitors at high resolution.
      ) and are consistent with our findings, as described below for binding in chain A. The carbonyl oxygen from the cysteine portion of GSH makes a 3.0 Å hydrogen bond with the backbone amino nitrogen of Leu-52, whereas the nitrogen from the glycine portion of GSH forms a 3.0 Å hydrogen bond with HOH209, and the C-terminal oxygens are engaged in hydrogen-bonding interactions with HOH617 and with the side chain nitrogen atoms of Lys-44 and Trp-38, respectively. The glutamate portion of GSH can be summarized by the following interactions: the backbone nitrogen makes a 2.8 Å hydrogen bond with the carbonyl oxygen of Leu-52; the carbonyl oxygen makes a 2.7 Å hydrogen bond with HOH53; the α-amino group makes a 2.8 Å hydrogen bond with the side chain Asp-98 O δ2 from chain B; and oxygen δ1 of the α-carboxylic acid makes hydrogen-bonding interactions of 2.9 Å to HOH3 and 2.9 Å to the backbone amino nitrogen of Ser-65, whereas oxygen δ2 makes a 2.9 Å hydrogen bond with HOH99 and a 2.7 Å hydrogen bond with the side chain oxygen of Ser-65.
      Electron density for PL was also found in each subunit, occupying the H-site, where other hydrophobic electrophiles such as chlorambucil and ethacrynic acid have been reported to bind. Continuous electron density between the thiol portion of GSH and the C7-C8 olefin of PL indicated that a covalent bond had been formed between the two (Fig. 1C). This is in contrast to the proposed adduct formation that would occur between the C2-C3 olefin of PL and the GSH thiol (
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      ). Density for the PL portion of the complex extends from the thiol portion of GSH into a hydrophobic pocket of GSTP1, formed by the side chains of residues Tyr-108, Ile-104, Phe-8, and Val-35 (Fig. 1D). The trimethoxyphenyl portion of PL fit the electron density very well, and was found to form a parallel-displaced pi-stacking interaction with the side chain of Tyr-108.
      Surprisingly, there was not sufficient density to model the dihydropyridine portion of PL. Rather, hPL, which replaces the dihydropyridine with a carboxyl group, fit the density well. The hydroxyl oxygen of the carboxyl group makes hydrogen-bonding interactions of 2.6 Å with HOH53 and of 3.0 Å with the ɛ nitrogen of Arg-13. The carbonyl oxygen makes hydrogen-bonding interactions of 2.8 Å with HOH36 and HOH48, respectively. No additional density corresponding to a dihydropyridine conjugate was observed near the binding pocket.

      Comparison with Other GSTP1 Inhibitor Structures

      GSTP1 has been shown to conjugate several chemotherapeutics including chlorambucil (PDB 4HJ2) and ethacrynic acid (PDB 3GSS). It has also been shown to be inhibited by NBDHEX (PDB 3GUS), although no conjugation occurs in the reported GSTP1 structure. Structural alignment of our PL-GSH crystal structure with each shows nearly identical overall folds for GSTP1 (RMSD Cα ≤0.25 Å), and the binding of GSH to the G site is identical, with the exception of the thiol, which can rotate ∼45° relative to the inhibitor in the H site. Despite the conservation in the overall fold of GSTP1 and of GSH binding, the H site binders show subtle yet significant differences in positioning, hydrogen bonding, and side chain orientation, supporting the assignment of GSTP1 as a significant cellular target of PL.
      Most notably, hPL extends deeper into the H pocket by 2.8 Å, which allows it to form a direct hydrogen bond with Arg-13 described above, making four hydrogen-bonding interactions altogether (Fig. 2, A–D). The positioning also causes the side chain of Ile-104 to flip away from the H site relative to the other three structures, and then make van der Waals interactions with the aliphatic backbone of hPL. If Ile-104 did not flip, then presumably there would be a steric clash with the carboxyl group of hPL. The entropy lost due to the rotation of Ile-104 may be offset by the enthalpy gained through the multiple hydrogen-bonding interactions of the carboxylate oxygens. Of note, I104V is a common polymorphism found in malignant tumors (
      • Lo H.W.
      • Ali-Osman F.
      Genetic polymorphism and function of glutathione S-transferases in tumor drug resistance.
      ). NBDHEX shows ∼4-fold greater affinity for the I104V mutant GSTP1 (Kd = 0.21 ± 0.06 μmol/min) (
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      ), in part because of a shift in position resulting in a direct hydrogen bond from the carbonyl oxygen to the ɛ nitrogen of Arg-13, which in the wild type only hydrogen-bonds through a bridging water molecule. This suggests the importance of this particular direct hydrogen bond toward the binding affinity, which is also found in the hPL-GSH structure even without an I104V mutation.
      Figure thumbnail gr2
      FIGURE 2Surface representation of GSTP1 bound by inhibitors: hPL-GSH (A); chlorambucil-GSH (B); NBDHEX-GSH (C); and ethacrynic acid-GSH (D). Inhibitor structures are shown as green sticks. The red arrow indicates the portion of the hPL-GSH structure that extends deeper into the binding cleft when compared with the other inhibitor-bound structures shown.

      No Covalent Labeling of PL to GSTP1

      Previous hypotheses about PL binding to GSTP1 suggested a model whereby a covalent adduct forms between the C7-C8 olefin and a side chain cysteine of the protein (
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      ). Despite no indication in our crystal structure of a covalent adduct between PL and GSTP1 at either olefin, we sought to determine whether PL could efficiently covalently modify GSTP1 in solution. Recombinant GSTP1 at a concentration of 10 μm was incubated with 10 μm PL and 1 mm l-glutathione. These conditions were chosen to imitate those in which the cellular effects of PL are seen. Electrospray ionization mass spectrometry revealed that none of the GSTP1 protein became labeled with PL at these concentrations (Fig. 3, A and B). Notably, the conjugate of GSH and PL was also detected (M+H+ at m/z 625; Fig. 3B).
      Figure thumbnail gr3
      FIGURE 3PL does not label GSTP1. A and B, purified GSTP1 was incubated at 10 μm without (A) or with (B) PL and GSH. Deconvoluted electrospray mass spectra (A and B, upper panels) detected GSTP1 (23,349 Da; A and B; green circles correspond to full-length protein, while yellow circles indicate loss of N-terminal methionine) but not GSTP1+PL (expected molecular weight 23,666 Da). C–E, PL reacts with cysteine thiols and readily forms hydrolysis products. A model peptide, FGLCSGPADTGR, was incubated with PL and GSH, and the reaction mixture was analyzed by mass spectrometry. Prominent peaks corresponding to [M+H]+ of FPEP-PL hydrolysis product (peak 1), FPEP-PL (peak 2), FPEP-PL-GSH (peak 3), GSH-PL (peak 4), GSH-PL hydrolysis product (peak 5), PL (peak 6), and GSH (peak 7) were observed (C and D). MALDI-MS/MS analysis confirmed labeling of the FPEP cysteine residue (E). Ions of type y are marked by red circles, water loss is denoted by *, and C(*) corresponds to the cysteine-PL adduct. An ion at m/z 1277, colored green, corresponds to the indicated PL hydrolysis product (indicated by (A) in panel F). F, proposed PL, GSH, and FPEP reaction scheme. MW, molecular weight.

      Mass Spectrometry Analysis of PL Hydrolysis

      To address the possibility that PL may react non-specifically in the cell with free cysteines in solution, we conducted experiments using the model peptide FGLCSGPADTGR (FPEP; containing a single cysteine residue) with PL and GSH using the same buffer conditions employed for the crystallography experiment. Mass spectrometry analysis of the resulting reaction products identified FPEP-PL, FPEP-PL-GSH, and PL-GSH conjugates (Fig. 3, C and D). MALDI MS/MS analysis of FPEP-PL (Fig. 3E) confirmed PL labeling of the peptide cysteine residue. Interestingly, peaks at m/z 405 (Fig. 3D, peak 5) and 1277.8 (Fig. 3C, peak 1) did not correspond to starting materials or expected products. Consideration of the possible products of the reaction led us to the realization that PL likely reacts with a thiol at the C2-C3 olefin, but ultimately becomes unstable, leading to amide hydrolysis and the formation of hPL. The proposed reaction scheme is shown in Fig. 3F. Of note, we did not detect the hPL adduct of GSH, suggesting that GSTP1 is necessary for the reaction at the C7-C8 olefin to occur, which is also consistent with the kinetic observations reported for chlorambucil (
      • Ciaccio P.J.
      • Tew K.D.
      • LaCreta F.P.
      The spontaneous and glutathione S-transferase-mediated reaction of chlorambucil with glutathione.
      ).

      hPL Does Not Inhibit Cell Proliferation

      With a predicted pKa of 3.7, we considered that in physiologic pH conditions, hPL likely exists in a deprotonated form and therefore does not efficiently pass through cell membranes because of its negative charge. To confirm this, we studied cancer cell lines that had previously shown sensitivity to PL (Raj et al. (
      • Raj L.
      • Ide T.
      • Gurkar A.U.
      • Foley M.
      • Schenone M.
      • Li X.
      • Tolliday N.J.
      • Golub T.R.
      • Carr S.A.
      • Shamji A.F.
      • Stern A.M.
      • Mandinova A.
      • Schreiber S.L.
      • Lee S.W.
      Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
      ) and Adams et al. (
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      )). We assayed for cell viability using cellular ATP content as a surrogate following a 72-h treatment with PL or hPL. IC50 values for PL were 5.8, 7.9, and 17 μm for HeLa, SW620, and PANC-1 cells, respectively, consistent with prior observations. However, under identical conditions, hPL had little effect on cell viability (Fig. 4A), suggesting that hydrolysis of PL occurs only after passage into cells.
      Figure thumbnail gr4
      FIGURE 4PL requires cell entry prior to being hydrolyzed to active form. A, cell proliferation in the presence of PL versus hPL. Exposure of cancer cell lines previously shown to be sensitive to PL (PANC1, HELA, SW620) shows impaired growth in the micromolar range when exposed to PL (blue hue curves) consistent with prior studies (
      • Raj L.
      • Ide T.
      • Gurkar A.U.
      • Foley M.
      • Schenone M.
      • Li X.
      • Tolliday N.J.
      • Golub T.R.
      • Carr S.A.
      • Shamji A.F.
      • Stern A.M.
      • Mandinova A.
      • Schreiber S.L.
      • Lee S.W.
      Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
      ,
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      ). Cells are unaffected at similar concentrations by hPL (red hue curves). Error bars indicate ± S.E. B, dose-response curve shows the inhibition of GSTP1 activity by hPL. C, schematic of the entry of PL, activation by free thiols, and inhibition of GSTP1, leading to reduced cellular glutathione, increased ROS, and cell death. TMCA, trimethoxycinnamic acid.

      hPL Inhibits GSTP1 in a Concentration-dependent Manner in Vitro

      To confirm inhibition of GSTP1 by hPL, we developed an enzymatic in vitro assay that utilizes purified GSTP1 in solution. Catalysis of the conjugation of glutathione and 1-chloro-2,4-binitrobenzene by GSTP1 was found to be inhibited by hPL in a concentration-dependent manner (IC50 = 384 μm) (Fig. 4B). It should be noted that in this case, the IC50 is not interchangeable with a binding constant because hPL is competitive with the substrate needed for the assay (400 μm 1-chloro-2,4-dinitrobenzene, see “Experimental Procedures”). Using the Cheng-Prusoff equation, the Ki for hPL was calculated to be 199 μm.

      Discussion

      The structure of GSTP1 in complex with a hydrolysis product of PL gives novel structural insights into how PL interacts with GSTP1 and demonstrates that PL is able to sequester GSH through GSTP1-assisted conjugation at the C7-C8 olefin, with no covalent bond formation between PL and GSTP1. The extensive van der Waals and hydrogen-bonding contacts between GSTP1 and the hPL-GSH conjugate provide a possible mechanism for the anti-cancer activity of PL, through inhibition of GSTP1. Additionally, the hydrolyzed form of PL may hold biological significance.
      Identification of hPL as the inhibitory form of PL is consistent with prior studies concerning the structure-activity relationship of PL. In the absence of C2-C3 olefin, PL derivatives exerted no effect on H1703 or HeLa breast cancer cell lines (
      • Adams D.J.
      • Dai M.
      • Pellegrino G.
      • Wagner B.K.
      • Stern A.M.
      • Shamji A.F.
      • Schreiber S.L.
      Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
      ). Similarly, an analog lacking the more reactive C7-C8 olefin produced no increase in ROS or reduction of cell viability in all cancer cells tested. In conjunction with our results, this suggests a model wherein PL requires activation to its hydrolyzed form to effectively interact with GSH in the GSTP1 binding site (Fig. 4C). Indeed, hydrolysis of the C2-C3 olefin is predicted to enhance the reactivity of the C7-C8 olefin due to the electron-withdrawing effects of the carboxyl group, as well as a decrease in steric hindrance.
      We note that the high Ki of hPL relative to the active concentration of PL in cells suggests that additional mechanisms must contribute to the activity of hPL. A leading hypothesis is that entrapment of hPL within cells leads to elevated intracellular concentrations of hPL that are required for inhibition of GSTP1. Of note, this mechanism of entrapment where compound exit from the cell is impeded has been reported for other compounds including chemotherapeutics such as vinblastine (
      • Singer W.D.
      • Himes R.H.
      Cellular uptake and tubulin binding properties of four Vinca alkaloids.
      ).
      It should be noted that despite initial reports that the primary target of PL is GSTP1, additional potential targets for the molecular mechanism of the action of PL have been put forward, including: Keap1 (
      • Lee H.N.
      • Jin H.O.
      • Park J.A.
      • Kim J.H.
      • Kim J.Y.
      • Kim B.
      • Kim W.
      • Hong S.E.
      • Lee Y.H.
      • Chang Y.H.
      • Hong S.I.
      • Hong Y.J.
      • Park I.C.
      • Surh Y.J.
      • Lee J.K.
      Heme oxygenase-1 determines the differential response of breast cancer and normal cells to piperlongumine.
      ), PI3K/AKT/mTOR (mechanistic target of rapamycin) (
      • Wang F.
      • Mao Y.
      • You Q.
      • Hua D.
      • Cai D.
      Piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of PI3K/Akt/mTOR pathway.
      ), the nuclear transporter CRM (chromosome maintenance region) (
      • Niu M.
      • Xu X.
      • Shen Y.
      • Yao Y.
      • Qiao J.
      • Zhu F.
      • Zeng L.
      • Liu X.
      • Xu K.
      Piperlongumine is a novel nuclear export inhibitor with potent anticancer activity.
      ), NF-κB pathway (
      • Ginzburg S.
      • Golovine K.V.
      • Makhov P.B.
      • Uzzo R.G.
      • Kutikov A.
      • Kolenko V.M.
      Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells.
      ,
      • Niu M.
      • Shen Y.
      • Xu X.
      • Yao Y.
      • Fu C.
      • Yan Z.
      • Wu Q.
      • Cao J.
      • Sang W.
      • Zeng L.
      • Li Z.
      • Liu X.
      • Xu K.
      Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import.
      ,
      • Han J.G.
      • Gupta S.C.
      • Prasad S.
      • Aggarwal B.B.
      Piperlongumine chemosensitizes tumor cells through interaction with cysteine 179 of IκBα kinase, leading to suppression of NF-κB-regulated gene products.
      ), peroxiredoxin 4 (PRDX4) (
      • Kim T.H.
      • Song J.
      • Kim S.H.
      • Parikh A.K.
      • Mo X.
      • Palanichamy K.
      • Kaur B.
      • Yu J.
      • Yoon S.O.
      • Nakano I.
      • Kwon C.H.
      Piperlongumine treatment inactivates peroxiredoxin 4, exacerbates endoplasmic reticulum stress, and preferentially kills high-grade glioma cells.
      ), C/EBP homologous protein (CHOP) activation (
      • Jin H.O.
      • Lee Y.H.
      • Park J.A.
      • Lee H.N.
      • Kim J.H.
      • Kim J.Y.
      • Kim B.
      • Hong S.E.
      • Kim H.A.
      • Kim E.K.
      • Noh W.C.
      • Kim J.I.
      • Chang Y.H.
      • Hong S.I.
      • Hong Y.J.
      • et al.
      Piperlongumine induces cell death through ROS-mediated CHOP activation and potentiates TRAIL-induced cell death in breast cancer cells.
      ), signal transducer and activator of transcription (STAT) 3 (
      • Bharadwaj U.
      • Eckols T.K.
      • Kolosov M.
      • Kasembeli M.M.
      • Adam A.
      • Torres D.
      • Zhang X.
      • Dobrolecki L.E.
      • Wei W.
      • Lewis M.T.
      • Dave B.
      • Chang J.C.
      • Landis M.D.
      • Creighton C.J.
      • Mancini M.A.
      • Tweardy D.J.
      Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.
      ), p38 (
      • Liu J.M.
      • Pan F.
      • Li L.
      • Liu Q.R.
      • Chen Y.
      • Xiong X.X.
      • Cheng K.
      • Yu S.B.
      • Shi Z.
      • Yu A.C.
      • Chen X.Q.
      Piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent JNK and p38 activation.
      ,
      • Wang Y.
      • Wang J.W.
      • Xiao X.
      • Shan Y.
      • Xue B.
      • Jiang G.
      • He Q.
      • Chen J.
      • Xu H.G.
      • Zhao R.X.
      • Werle K.D.
      • Cui R.
      • Liang J.
      • Li Y.L.
      • Xu Z.X.
      Piperlongumine induces autophagy by targeting p38 signaling.
      ), and the ubiquitin-proteasome system (UPS) (
      • Jarvius M.
      • Fryknäs M.
      • D'Arcy P.
      • Sun C.
      • Rickardson L.
      • Gullbo J.
      • Haglund C.
      • Nygren P.
      • Linder S.
      • Larsson R.
      Piperlongumine induces inhibition of the ubiquitin-proteasome system in cancer cells.
      ). This long list of targets, combined with the fact that relatively high concentrations of PL are required for anti-cancer effects, raises the possibility that PL also acts on these targets through relatively nonspecific means, perhaps through direct interactions with biologically important small molecules such as GSH. The observation that treatment of leukemia cells with PL depletes reduced glutathione stores in a dose-dependent fashion (
      • Pei S.
      • Minhajuddin M.
      • Callahan K.P.
      • Balys M.
      • Ashton J.M.
      • Neering S.J.
      • Lagadinou E.D.
      • Corbett C.
      • Ye H.
      • Liesveld J.L.
      • O'Dwyer K.M.
      • Li Z.
      • Shi L.
      • Greninger P.
      • Settleman J.
      • et al.
      Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.
      ) is consistent with this model. Although our data provide supporting evidence for this idea, it does not exclude other possibilities such as direct inhibition of GSTP1, nor does it rule out the possibility of effects from a different byproduct of PL hydrolysis or conjugation. Additional work will be required to clarify this question.
      In conclusion, we have provided the first structural model for interactions between PL, GSH, and GSTP1 as summarized in Fig. 4C. In this model, PL is a prodrug that enters the cell and conjugates to GSH through Michael addition at the C7-C8 olefin, but only after PL is activated by hydrolysis of the C2-C3 olefin. GSTP1 is inhibited by the hPL-GSH conjugate, and rotation of Ile-104 allows the formation of several important hydrogen-bonding interactions, most notably with Arg-13, which resides deeper in the H-site and does not form a direct hydrogen bond with any other known inhibitors. This suggests that PL has thermodynamic advantages that enable the cellular effects seen with PL, but not with other compounds previously demonstrated to interact with GSTP1 (
      • Asakura T.
      • Sasagawa A.
      • Takeuchi H.
      • Shibata S.
      • Marushima H.
      • Mamori S.
      • Ohkawa K.
      Conformational change in the active center region of GST P1–1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis.
      • Ploemen J.H.
      • van Ommen B.
      • Bogaards J.J.
      • van Bladeren P.J.
      Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.
      ,
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      • Tsuboi K.
      • Bachovchin D.A.
      • Speers A.E.
      • Spicer T.P.
      • Fernandez-Vega V.
      • Hodder P.
      • Rosen H.
      • Cravatt B.F.
      Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.
      ). Finally, our results give an atomic level understanding of the mechanism of action for PL, and form the basis for a structure-guided design approach for novel PL or PL-GSH conjugates, which may be used as chemotherapeutic agents.

      Experimental Procedures

      Expression and Purification of GSTP1

      A construct encoding human GSTP1 cDNA was synthesized and cloned into the pJExpress 411 vector (DNA2.0) and then transformed into the BL21 (DE3) bacterial strain. Cells were grown in Luria Broth to an A600 of 0.8 and induced with 250 μm isopropyl β-d-1-thiogalactopyranoside. Cells were grown at 16 °C for 18 h, and then harvested by centrifugation for 15 min at 6000 × g and resuspended in lysis buffer (100 mm sodium phosphate (pH 8.0), 750 mm NaCl, 10 mm imidazole, 10 mm EDTA (pH 8.0), 25% (v/v) glycerol, 1 mm PMSF, 1 mm benzamidine, and 1 mg/ml lysozyme). This preparation was flash-frozen and stored at −80 °C. Thawed cells were pelleted by centrifugation to remove debris, and soluble protein was loaded onto a glutathione-agarose column (Bio-Rad) and eluted with glutathione-containing buffer (40 mm glutathione, 200 mm Tris, pH 7.0, and 10 mm EDTA (pH 8.0)). The purified protein was then buffer-exchanged to 10 mm HEPES, pH 7.0, 50 mm ammonium sulfate, and 5 mm glutathione using Amicon 10-kDa cutoff filters (EMD Millipore) and concentrated to 10 mg/ml for crystallization experiments.

      Crystallization

      The crystallization was carried out as described previously; however, 2 mm final concentration of PL and reduced l-glutathione were added to the protein prior to crystallization (
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      ). Hanging drop vapor diffusion plates were set up at a ratio of 1:1 (protein to well solution containing 1.6 m ammonium sulfate and 100 mm MES, pH 6.5). Crystals were grown at 4 °C and appeared after 5 days. Crystals were cryoprotected in a mother liquor solution supplemented with 25% glycerol and flash-frozen in liquid nitrogen prior to data collection.

      X-ray Diffraction, Structure Determination, and Refinement

      X-ray diffraction data were collected at the Advanced Photon Source beamline 19-ID. Images were processed, integrated, and scaled with HKL-2000/3000 packages (HKL Research Inc.) (
      • Minor W.
      • Cymborowski M.
      • Otwinowski Z.
      • Chruszcz M.
      HKL-3000: the integration of data reduction and structure solution—from diffraction images to an initial model in minutes.
      ). Molecular replacement and model refinement were performed using Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.-W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ) and CCP4 software (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ) with PDB ID 3GUS as the initial search model (
      • Federici L.
      • Lo Sterzo C.
      • Pezzola S.
      • Di Matteo A.
      • Scaloni F.
      • Federici G.
      • Caccuri A.M.
      Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione S-transferases.
      ). The final structure was submitted to the Protein Data Bank under ID code 5J41. Images were prepared using PyMOL, version 1.5.0.4 (Schrödinger, LLC).

      Mass Spectrometry of Intact GSTP1

      Labeling and liquid chromatography-electrospray ionization-MS analysis of GSTP1 by PL were performed similarly to the previously described procedure (
      • Lim S.M.
      • Westover K.D.
      • Ficarro S.B.
      • Harrison R.A.
      • Choi H.G.
      • Pacold M.E.
      • Carrasco M.
      • Hunter J.
      • Kim N.D.
      • Xie T.
      • Sim T.
      • Jänne P.A.
      • Meyerson M.
      • Marto J.A.
      • Engen J.R.
      • Gray N.S.
      Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor.
      ). Briefly, a solution of 10 μm GSTP1 was mixed with 10 μm PL and glutathione and incubated at 27 °C for 2 h. Mass spectra were deconvoluted using MagTran software (version 1.03b2 (
      • Zhang Z.
      • Marshall A.G.
      A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra.
      )). Proteolytic digestion, nano-LC/MS, and data analysis were performed essentially as described (
      • Parikh J.R.
      • Askenazi M.
      • Ficarro S.B.
      • Cashorali T.
      • Webber J.T.
      • Blank N.C.
      • Zhang Y.
      • Marto J.A.
      multiplierz: an extensible API based desktop environment for proteomics data analysis.
      ,
      • Ficarro S.B.
      • Zhang Y.
      • Lu Y.
      • Moghimi A.R.
      • Askenazi M.
      • Hyatt E.
      • Smith E.D.
      • Boyer L.
      • Schlaeger T.M.
      • Luckey C.J.
      • Marto J.A.
      Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells.
      ).

      Mass Spectrometry of PL and Peptide FGLCSGPADTGR

      The peptide FGLCSGPADTGR was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by reversed phase HPLC. The peptide was reconstituted with 10 mm HEPES, pH 7.0, 50 mm ammonium sulfate, incubated with a 10-fold molar excess of PL and a 5-fold molar excess of GSH, and then incubated for 6 days at 4 °C. An aliquot of the reaction was acidified with 10% TFA and further diluted with 0.1% TFA, and peptides were extracted using μC18 zip tips (Millipore, Billerica, MA). After desalting, peptides were eluted to a MALDI plate (384-well Opti-TOF), mixed with matrix (1 μl of 10 mg/ml α-cyano-4-hydroxycinnamic acid in 80% acetonitrile, 0.1% TFA), allowed to air dry, and analyzed by MALDI-MS and -MS/MS as described (
      • Ficarro S.B.
      • Adelmant G.
      • Tomar M.N.
      • Zhang Y.
      • Cheng V.J.
      • Marto J.A.
      Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.
      ).

      Cell Culture

      Cells were maintained in a 37 °C incubator (5% CO2). HeLa (cervical cancer) cells were obtained from the ATCC and cultured in Eagle's minimum essential medium containing 10% FBS and 1% penicillin/streptomycin/amphotericin B (antibiotics). PANC-1 (pancreatic carcinoma) cells were a gift of Paul Chiao, MD Anderson Cancer Center, Houston, TX and were cultured in DMEM medium containing 10% FBS and antibiotics. SW620 (colorectal adenocarcinoma) cells were a gift of Nathanael Gray, Dana Farber Cancer Institute, Boston and were cultured in L-15 medium containing 10% FBS and antibiotics.

      Cell Viability Assay

      Cells were plated at 1000 viable cells/well in 50 μl of medium in white 96-well plates and allowed to attach for 4 h. At that time, another 50 μl of medium containing PL or hPL was added to create nine sets of triplicate wells of serial 3-fold dilutions spanning a final concentration range of 0.015–100 μm compound and 1% DMSO. A triplicate set of wells with medium containing 1% DMSO alone served as untreated controls. After the addition of compounds, plates were incubated for 72 h. At that time, 100 μl of a 1:1 solution of PBS containing 1% Triton X-100:CellTiter-Glo (Promega) was added to each well. Following 10 min of gentle shaking, luminescence was read using a BioTek Synergy NEO plate reader. Cell viability (% of DMSO alone) was calculated from the data and plotted using GraphPad Prism 6. IC50 values were extrapolated as best-fit values of log(inhibitor) versus response (three parameters) curves.

      GSTP1 Kinetic Assay

      Inhibition of GSTP1 activity by hPL was analyzed using the Glutathione S-Transferase Assay Kit (Sigma-Aldrich), which measures the conjugation of l-glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). Reactions in Dulbecco's phosphate-buffered saline containing 10 nm GSTP1, 2000 mm glutathione, and various concentrations of CDNB and hPL were performed at 25 °C in 96-well UV Flat Bottom Microtiter Plates (Fisher Scientific). The resultant time-dependent change of A340 was determined with a Biotech Synergy NEO plate reader. Data were graphed and processed using GraphPad Prism 7 software. A Km value was determined by nonlinear regression of initial rates of GSTP1-dependent catalysis of various concentrations of CDNB. Nonlinear regression of first-order rate constants from assays containing fixed CDNB and various hPL concentrations was used to obtain an IC50 value for the latter compound.

      Author Contributions

      K. D. W. conceived and coordinated the study. W. H., S. G., and K. D. W. wrote the paper with contributions from all authors. S. G. and D. U. purified the protein. J. H. and D. G. collected the X-ray data. W. H. and J. H. solved the structure and performed refinement. S. B. F., J. A. M., and S. G. conducted mass spec experiments. W. H., J. H., and K. D. W. analyzed and interpreted structural data. W. D. S. and Y. L. performed cell-based assays. W. D. S. and L. L. performed kinetic assays. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      Results shown in this report are derived from work performed at the Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U. S. Department of Energy, Office of Biological and Environmental Research under Contract DEAC02-06CH11357.

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