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Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics*

  • Jun-Young Kim
    Footnotes
    Affiliations
    From the Laboratory of Plant Systems Bio-Dynamics, Pohang University of Science and Technology, 790-784 Pohang, Korea,
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  • Sina Henrichs
    Footnotes
    Affiliations
    the Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zolliker Strasse 107, CH-8008 Zurich, Switzerland,
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  • Aurélien Bailly
    Affiliations
    the Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zolliker Strasse 107, CH-8008 Zurich, Switzerland,
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  • Vincent Vincenzetti
    Affiliations
    the Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zolliker Strasse 107, CH-8008 Zurich, Switzerland,
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  • Valpuri Sovero
    Affiliations
    the Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zolliker Strasse 107, CH-8008 Zurich, Switzerland,
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  • Stefano Mancuso
    Affiliations
    the Department of Horticulture, University of Firenze, I-50019 Sesto Fiorentino, Italy,
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  • Stephan Pollmann
    Affiliations
    the Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, D-44801 Bochum, Germany, and
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  • Daehwang Kim
    Affiliations
    the Biomaterials Research Center, Korea Research Institute of Chemical Technology, 305-600 Dae-jon, Korea
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  • Markus Geisler
    Correspondence
    To whom correspondence may be addressed. Tel.: 41-44-634-8277; Fax: 41-44-634-8204;
    Affiliations
    the Institute of Plant Biology, University of Zurich and Zurich-Basel Plant Science Center, Zolliker Strasse 107, CH-8008 Zurich, Switzerland,
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  • Hong-Gil Nam
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    From the Laboratory of Plant Systems Bio-Dynamics, Pohang University of Science and Technology, 790-784 Pohang, Korea,
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  • Author Footnotes
    * This work was supported by grants from the Forschungskredit of the University of Zurich (KN57150702, to A. B.), from the Novartis Foundation (to M. G.), from the Swiss National Funds (NF31-125001, to M. G.), from the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (no. 2009-0091504), and from the Crop Functional Genomics Frontier Research Program (CG3132) (to H.-G. N.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7.
    1 Both authors contributed equally to this work.
Open AccessPublished:May 14, 2010DOI:https://doi.org/10.1074/jbc.M110.105981
      Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC50 value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.

      Introduction

      In plants, the auxin indolyl-3-acetic acid (IAA)
      The abbreviations used are: IAA
      indole-3-acetic acid
      PAT
      polar auxin transport
      PGP
      phosphoglycoprotein
      TMD
      transmembrane domain
      NBD
      nucleotide-binding domain
      dag
      day(s) after germination
      ATI
      auxin transport inhibitor
      NPA
      1-N-naphtylphtalamic acid
      NBP
      NPA-binding protein
      gravacin
      3-(5-[3,4-dichlorophenyl]-2-furyl)acrylic acid
      GFP
      green fluorescent protein
      BA
      benzoic acid
      BRET
      bioluminescence resonance energy transfer.
      serves as a hormone-like signaling molecule that is a key factor in plant development and physiology (
      • Blakeslee J.J.
      • Peer W.A.
      • Murphy A.S.
      ,
      • Kepinski S.
      • Leyser O.
      ,
      • Vieten A.
      • Sauer M.
      • Brewer P.B.
      • Friml J.
      ,
      • Benjamins R.
      • Scheres B.
      ). Many of its functionalities are controlled by a unique, plant-specific process, the cell-to-cell or polar auxin transport (PAT) (
      • Vieten A.
      • Sauer M.
      • Brewer P.B.
      • Friml J.
      ). However, cellular efflux is the rate-limiting step of PAT, and in agreement with the chemiosmotic hypothesis, putative exporters of the PIN (pin-formed) and B subfamily of ABC transporter, ABCB/PGP/MDR (P-glycoprotein, multidrug resistance), families have been identified (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Geisler M.
      • Murphy A.S.
      ,
      • Petrásek J.
      • Mravec J.
      • Bouchard R.
      • Blakeslee J.J.
      • Abas M.
      • Seifertová D.
      • Wisniewska J.
      • Tadele Z.
      • Kubes M.
      • Covanová M.
      • Dhonukshe P.
      • Skupa P.
      • Benková E.
      • Perry L.
      • Krecek P.
      • Lee O.R.
      • Fink G.R.
      • Geisler M.
      • Murphy A.S.
      • Luschnig C.
      • Zazímalová E.
      • Friml J.
      ).
      Most PIN efflux carriers show predominantly polar locations in PAT tissues and developmental, organogenetic loss-of-function phenotypes and are thought to be the determinants of a “reflux loop” in the root apex (
      • Vieten A.
      • Sauer M.
      • Brewer P.B.
      • Friml J.
      ,
      • Blilou I.
      • Xu J.
      • Wildwater M.
      • Willemsen V.
      • Paponov I.
      • Friml J.
      • Heidstra R.
      • Aida M.
      • Palme K.
      • Scheres B.
      ). ABCB isoforms have been identified as primary active (ATP-dependent) auxin pumps showing auxin-related, developmental (but not organogenetic) loss-of-function phenotypes (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Blakeslee J.J.
      • Bandyopadhyay A.
      • Lee O.R.
      • Mravec J.
      • Titapiwatanakun B.
      • Sauer M.
      • Makam S.N.
      • Cheng Y.
      • Bouchard R.
      • Adamec J.
      • Geisler M.
      • Nagashima A.
      • Sakai T.
      • Martinoia E.
      • Friml J.
      • Peer W.A.
      • Murphy A.S.
      ,
      • Mravec J.
      • Kubes M.
      • Bielach A.
      • Gaykova V.
      • Petrásek J.
      • Skůpa P.
      • Chand S.
      • Benková E.
      • Zazímalová E.
      • Friml J.
      ). Despite their mostly apolar locations, they have been demonstrated to contribute to PAT and long range auxin transport (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Bailly A.
      • Sovero V.
      • Vincenzetti V.
      • Santelia D.
      • Bartnik D.
      • Koenig B.W.
      • Mancuso S.
      • Martinoia E.
      • Geisler M.
      ,
      • Bouchard R.
      • Bailly A.
      • Blakeslee J.J.
      • Oehring S.C.
      • Vincenzetti V.
      • Lee O.R.
      • Paponov I.
      • Palme K.
      • Mancuso S.
      • Murphy A.S.
      • Schulz B.
      • Geisler M.
      ). Moreover, ABCB1/PGP1 and ABCB19/PGP19/MDR1 coordinately function in basipetal reflux of auxin maxima out of the root and shoot tip (
      • Blakeslee J.J.
      • Bandyopadhyay A.
      • Lee O.R.
      • Mravec J.
      • Titapiwatanakun B.
      • Sauer M.
      • Makam S.N.
      • Cheng Y.
      • Bouchard R.
      • Adamec J.
      • Geisler M.
      • Nagashima A.
      • Sakai T.
      • Martinoia E.
      • Friml J.
      • Peer W.A.
      • Murphy A.S.
      ). The immunophilin-like FKBP42, TWD1 (twisted DWARF1) protein, was characterized as a central regulator of ABCB-mediated auxin transport by means of protein-protein interaction (
      • Bailly A.
      • Sovero V.
      • Vincenzetti V.
      • Santelia D.
      • Bartnik D.
      • Koenig B.W.
      • Mancuso S.
      • Martinoia E.
      • Geisler M.
      ,
      • Bouchard R.
      • Bailly A.
      • Blakeslee J.J.
      • Oehring S.C.
      • Vincenzetti V.
      • Lee O.R.
      • Paponov I.
      • Palme K.
      • Mancuso S.
      • Murphy A.S.
      • Schulz B.
      • Geisler M.
      ). Positive regulation of ABCB1- and ABCB19-mediated auxin transport accounts for overlapping phenotypes between twd1 and abcb1 abcb19 (
      • Bailly A.
      • Sovero V.
      • Vincenzetti V.
      • Santelia D.
      • Bartnik D.
      • Koenig B.W.
      • Mancuso S.
      • Martinoia E.
      • Geisler M.
      ,
      • Bouchard R.
      • Bailly A.
      • Blakeslee J.J.
      • Oehring S.C.
      • Vincenzetti V.
      • Lee O.R.
      • Paponov I.
      • Palme K.
      • Mancuso S.
      • Murphy A.S.
      • Schulz B.
      • Geisler M.
      ).
      ABCB- and PIN-mediated auxin efflux can function independently and play identical cellular but separate developmental roles (
      • Mravec J.
      • Kubes M.
      • Bielach A.
      • Gaykova V.
      • Petrásek J.
      • Skůpa P.
      • Chand S.
      • Benková E.
      • Zazímalová E.
      • Friml J.
      ). However, ABCBs and PINs are also able to interactively and coordinately transport auxin (
      • Blakeslee J.J.
      • Bandyopadhyay A.
      • Lee O.R.
      • Mravec J.
      • Titapiwatanakun B.
      • Sauer M.
      • Makam S.N.
      • Cheng Y.
      • Bouchard R.
      • Adamec J.
      • Geisler M.
      • Nagashima A.
      • Sakai T.
      • Martinoia E.
      • Friml J.
      • Peer W.A.
      • Murphy A.S.
      ). The current picture that emerges is that in interacting cells, multilaterally expressed ABCBs minimize apoplastic reflux, whereas polar ABCB-PIN interactions provide specific vectorial auxin stream (
      • Mravec J.
      • Kubes M.
      • Bielach A.
      • Gaykova V.
      • Petrásek J.
      • Skůpa P.
      • Chand S.
      • Benková E.
      • Zazímalová E.
      • Friml J.
      ). However, the individual roles of ABCB- and PIN-mediated auxin flows are far from being understood.
      The investigation of PAT streams was facilitated by using synthetic compounds that act as auxin transport inhibitors (ATIs), with the non-competitive IAA efflux inhibitor 1-N-naphtylphtalamic acid (NPA) being the most prominent. Until today, the identity, number, and affinity of putative NPA-binding proteins (NBPs) is still controversial (
      • Cox D.N.
      • Muday G.K.
      ,
      • Luschnig C.
      ,
      • Michalke W.
      • Katekar G.F.
      • Geissler A.E.
      ,
      • Petrásek J.
      • Cerná A.
      • Schwarzerová K.
      • Elckner M.
      • Morris D.A.
      • Zazímalová E.
      ,
      • Sussman M.R.
      • Gardner G.
      ). However, the current consensus is that the auxin efflux complex consists of at least two proteins: a membrane-integral transporter and an NBP-regulatory subunit (
      • Cox D.N.
      • Muday G.K.
      ,
      • Luschnig C.
      ,
      • Michalke W.
      • Katekar G.F.
      • Geissler A.E.
      ,
      • Bernasconi P.
      • Patel B.C.
      • Reagan J.D.
      • Subramanian M.V.
      ). Several lines of evidence suggest that PIN proteins do not themselves act as NBPs (
      • Lomax T.L.
      • Muday G.K.
      • Rubery P.H.
      ), although NPA application results in a pin-formed inflorescence, mimicking PIN1 loss of function (
      • Okada K.
      • Ueda J.
      • Komaki M.K.
      • Bell C.J.
      • Shimura Y.
      ). Therefore, it was suggested that NPA blocks PAT by interfering with the cycling of auxin transporters, like PIN1 (
      • Geldner N.
      • Friml J.
      • Stierhof Y.D.
      • Jürgens G.
      • Palme K.
      ). However, NPA itself does not directly affect PIN cycling, and concentrations necessary to perturb PIN cycling were much higher than was needed for efficiently blocking PAT (
      • Petrásek J.
      • Cerná A.
      • Schwarzerová K.
      • Elckner M.
      • Morris D.A.
      • Zazímalová E.
      ,
      • Geldner N.
      • Friml J.
      • Stierhof Y.D.
      • Jürgens G.
      • Palme K.
      ). Independently, ABCB1 and ABCB19 have been identified as targets of NPA (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Nagashima A.
      • Uehara Y.
      • Sakai T.
      ,
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ) and high affinity NBPs (
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ,
      • Murphy A.S.
      • Hoogner K.R.
      • Peer W.A.
      • Taiz L.
      ,
      • Noh B.
      • Murphy A.S.
      • Spalding E.P.
      ). Surprisingly, NPA was additionally shown to bind to TWD1, and NPA binding disrupted TWD1-ABCB1 interaction (
      • Bailly A.
      • Sovero V.
      • Vincenzetti V.
      • Santelia D.
      • Bartnik D.
      • Koenig B.W.
      • Mancuso S.
      • Martinoia E.
      • Geisler M.
      ). In planta, this leads to disruption of ABCB1 activity, suggesting that TWD1 and ABCB1 represent high and low affinity components of the NPA-sensitive efflux complex (
      • Bailly A.
      • Sovero V.
      • Vincenzetti V.
      • Santelia D.
      • Bartnik D.
      • Koenig B.W.
      • Mancuso S.
      • Martinoia E.
      • Geisler M.
      ).
      In the last few years, chemical genomic screens have allowed for the identification of several synthetic compounds and, in some cases, respective molecular targets that interfere with auxin signaling (
      • Armstrong J.I.
      • Yuan S.
      • Dale J.M.
      • Tanner V.N.
      • Theologis A.
      ,
      • Zhao Y.
      • Dai X.
      • Blackwell H.E.
      • Schreiber S.L.
      • Chory J.
      ), membrane trafficking (
      • Norambuena L.
      • Hicks G.R.
      • Raikhel N.V.
      ,
      • Robert S.
      • Chary S.N.
      • Drakakaki G.
      • Li S.
      • Yang Z.
      • Raikhel N.V.
      • Hicks G.R.
      ,
      • Surpin M.
      • Rojas-Pierce M.
      • Carter C.
      • Hicks G.R.
      • Vasquez J.
      • Raikhel N.V.
      ), and auxin transport (
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ,
      • Savaldi-Goldstein S.
      • Baiga T.J.
      • Pojer F.
      • Dabi T.
      • Butterfield C.
      • Parry G.
      • Santner A.
      • Dharmasiri N.
      • Tao Y.
      • Estelle M.
      • Noel J.P.
      • Chory J.
      ). 3-(5-[3,4-dichlorophenyl]-2-furyl)acrylic acid (gravacin) was recently identified as a strong inhibitor of root and shoot gravitropism, auxin responsiveness, and protein trafficking to the tonoplast in Arabidopsis (
      • Surpin M.
      • Rojas-Pierce M.
      • Carter C.
      • Hicks G.R.
      • Vasquez J.
      • Raikhel N.V.
      ). In a follow-up screen, inhibition of gravitropism and protein trafficking was shown to employ independent mechanics (
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ). Mutations in ABCB19 confer resistance to the effect of gravacin on hypocotyl gravitropism and result in reduced binding of gravacin to microsomal fractions, implicating ABCB19 as the major target of gravacin (
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ). Consequently, gravacin was found to be a strong inhibitor of ABCB19-mediated auxin transport in Arabidopsis and HeLa cells.
      In this study, we screened chemical libraries of small organic compounds for plant physiological and developmental regulators and identified a novel, highly potent ATI by means of chemical genomics. A direct comparison of compound 10824 (BUM) and NPA effects on auxin-controlled plant physiology, auxin transport, and drug binding reveals that BUM shares many features with NPA, like induction of pin-formed inflorescences and ABCB binding and blocking of transport. Unlike NPA and gravacin, BUM lacks growth activation at lower concentrations and does not interfere with membrane trafficking, respectively. BUM might therefore act as a powerful tool in dissecting ABCB- and PIN-mediated auxin streams in plant physiology.

      DISCUSSION

      Recent analyses of PIN and ABCB transport mechanisms suggest independent (or sometimes even opposite) and at certain domains additive and synergistic actions (
      • Blakeslee J.J.
      • Bandyopadhyay A.
      • Lee O.R.
      • Mravec J.
      • Titapiwatanakun B.
      • Sauer M.
      • Makam S.N.
      • Cheng Y.
      • Bouchard R.
      • Adamec J.
      • Geisler M.
      • Nagashima A.
      • Sakai T.
      • Martinoia E.
      • Friml J.
      • Peer W.A.
      • Murphy A.S.
      ,
      • Mravec J.
      • Kubes M.
      • Bielach A.
      • Gaykova V.
      • Petrásek J.
      • Skůpa P.
      • Chand S.
      • Benková E.
      • Zazímalová E.
      • Friml J.
      ). In this study, we have identified by means of chemical genomics a novel ATI that efficiently blocks auxin-related plant physiology and development. Quantification of physiological parameters, drug binding, and transport data indicates that ABCBs, primarily ABCB1, are direct BUM/NPA targets, whereas PINs are apparently less affected, which is in agreement with previous findings on NPA (
      • Luschnig C.
      ,
      • Nagashima A.
      • Uehara Y.
      • Sakai T.
      ,
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ). This makes BUM a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. On the other hand, our work also suggests that pin-formed inflorescences, which are caused by BUM and NPA and that phenocopy PIN1 loss-of-function mutations are primarily caused by ABCB transport inhibition of the functional ABCB-PIN efflux complex.
      Our findings also demonstrate that BUM inhibition, like NPA inhibition, is light-dependent. This leads to the suggestion that ABCBs, obviously the cellular targets of BUM- and NPA-induced inhibition in Arabidopsis, are part of a light-controlled developmental pathway, which is in agreement with the concept that auxin has a more important role in elongation and bending responses in the light (
      • Jensen P.J.
      • Hangarter R.P.
      • Estelle M.
      ,
      • Nagashima A.
      • Suzuki G.
      • Uehara Y.
      • Saji K.
      • Furukawa T.
      • Koshiba T.
      • Sekimoto M.
      • Fujioka S.
      • Kuroha T.
      • Kojima M.
      • Sakakibara H.
      • Fujisawa N.
      • Okada K.
      • Sakai T.
      ). This concept was recently genetically supported by demonstrating that the photoreceptors, phytochromes and cryptochromes, regulate differential growth of Arabidopsis hypocotyls in an ABCB-dependent manner (
      • Nagashima A.
      • Suzuki G.
      • Uehara Y.
      • Saji K.
      • Furukawa T.
      • Koshiba T.
      • Sekimoto M.
      • Fujioka S.
      • Kuroha T.
      • Kojima M.
      • Sakakibara H.
      • Fujisawa N.
      • Okada K.
      • Sakai T.
      ).
      In many physiological respects and partially also structurally, BUM resembles NPA functionality. Based on our transport and drug binding data, both have a stronger effect on ABCB1 than on ABCB19 (FIGURE 5, FIGURE 6). However, BUM has the advantage of not showing activation of plant growth at lower concentrations and acting roughly a factor of 30 stronger than NPA, which seems to be mainly caused by apically shifted root influx maxima. This again might be the result of altered binding preferences or affinities to the ABCBs. An alternative, simpler explanation that we cannot rule out at the moment is that BUM, being less hydrophobic compared with NPA, has a higher solubility. BUM shares this higher apparent solubility with the phytotropin l-(2′-carboxyphenyl)-3-phenylpropane-1,3-dione, which competes for the same binding site and does affect the same processes of auxin transport and geotropic curvature as NPA (
      • Katekar G.F.
      • Geissler A.E.
      ). However, based on our binding, the primary BUM target seems to be ABCB1. This makes BUM complementary to NPA that has been shown to affect besides ABCB19 also other ABCBs (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Nagashima A.
      • Uehara Y.
      • Sakai T.
      ,
      • Terasaka K.
      • Blakeslee J.J.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bandyopadhyay A.
      • Makam S.N.
      • Lee O.R.
      • Richards E.L.
      • Murphy A.S.
      • Sato F.
      • Yazaki K.
      ). Most interestingly, BUM reveals a more drastic effect (even at lower concentrations) on root transport (Fig. 5) and root elongation (Fig. 2) than found in the hypocotyl in comparison with NPA, which shows an inverse behavior. The molecular reasons are unknown and under current investigation. However, this finding is in agreement with higher ABCB19 expression in the hypocotyl (
      • Blakeslee J.J.
      • Bandyopadhyay A.
      • Lee O.R.
      • Mravec J.
      • Titapiwatanakun B.
      • Sauer M.
      • Makam S.N.
      • Cheng Y.
      • Bouchard R.
      • Adamec J.
      • Geisler M.
      • Nagashima A.
      • Sakai T.
      • Martinoia E.
      • Friml J.
      • Peer W.A.
      • Murphy A.S.
      ) and a lower inhibition ABCB19 by BUM compared with ABCB1.
      BUM apparently also acts differently compared with the recently identified ATI, gravacin, which primarily inhibits ABCB19 (
      • Rojas-Pierce M.
      • Titapiwatanakun B.
      • Sohn E.J.
      • Fang F.
      • Larive C.K.
      • Blakeslee J.
      • Cheng Y.
      • Cutler S.R.
      • Peer W.A.
      • Murphy A.S.
      • Raikhel N.V.
      ). However, as shown by BRET analysis, BUM, unlike gravacin, also alters TWD1 function, suggesting that BUM might indirectly also regulate ABCB19 activity by disrupting TWD1-ABCB19 interaction. Another advantage is that BUM, unlike gravacin, apparently does not interfere with ABCB trafficking.
      Besides its academic usage as an ATI and its obvious potential as powerful weed herbicide, BUM might have a direct clinical impact because multidrug resistance toward many anti-cancer drugs is largely caused by human ABCB1, leading often to chemotherapy ineffectiveness. Interestingly, human and plant ABCBs share broad inhibitor sensitivities, which was demonstrated for the flavonol quercetin, which acts both as modulator of auxin transport and as inhibitor of mammalian and plant ABCBs, and for clinically relevant ABCB inhibitors, like cyclosporine A and verapamil (
      • Geisler M.
      • Blakeslee J.J.
      • Bouchard R.
      • Lee O.R.
      • Vincenzetti V.
      • Bandyopadhyay A.
      • Titapiwatanakun B.
      • Peer W.A.
      • Bailly A.
      • Richards E.L.
      • Ejendal K.F.
      • Smith A.P.
      • Baroux C.
      • Grossniklaus U.
      • Müller A.
      • Hrycyna C.A.
      • Dudler R.
      • Murphy A.S.
      • Martinoia E.
      ,
      • Bouchard R.
      • Bailly A.
      • Blakeslee J.J.
      • Oehring S.C.
      • Vincenzetti V.
      • Lee O.R.
      • Paponov I.
      • Palme K.
      • Mancuso S.
      • Murphy A.S.
      • Schulz B.
      • Geisler M.
      ). Based on our findings, plant ABCB inhibitors, such as BUM and NPA, are therefore bona fide human ABCB inhibitors that might suppress multidrug resistance when co-administered with anti-cancer drugs.

      Acknowledgments

      We thank A. Murphy for gravacin, S. pombe strains, and expression plasmids, E. Martinoia for discussion and support, and the Korean Chemical Bank for the chemical library donation.

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