Yeast chemogenomic profiling reveals iron chelation to be the

ABSTRACT: Gossypol is an inhibitor of eukaryotic cells with an undetermined mode of action. Here we show that the chemogenomic profile of gossypol is ...
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Brief Article Cite This: J. Med. Chem. 2018, 61, 7381−7386

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Yeast Chemogenomic Profiling Reveals Iron Chelation To Be the Principle Cell Inhibitory Mode of Action of Gossypol Thomas A. K. Prescott,*,† Tiphaine Jaeg,‡,§ and Dominic Hoepfner*,‡ †

Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Fabrikstrasse 22, CH-4056 Basel, Switzerland

‡

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S Supporting Information *

ABSTRACT: Gossypol is an inhibitor of eukaryotic cells with an undetermined mode of action. Here we show that the chemogenomic profile of gossypol is strikingly similar to that of the iron chelators deferasirox and desferricoprogen. Iron import channels Fet1 and Fet3 are prominent in all three profiles. Furthermore, yeast inhibited by gossypol and deferasirox is rescued by the addition of Fe2+. We propose that Fe2+ chelation is in fact the principle mode of action of gossypol.

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INTRODUCTION Gossypol is a phenolic compound found in cotton plants, including the seeds and the seed oil.1 It has a diverse pharmacological profile, inhibiting fungi, trypanosomes, and amoebas.2−5 It also promotes apoptosis in cancer cell lines and has been entered into several cancer clinical trials.6−12 Separate to these cell inhibitory effects, gossypol has been shown to promote anemia and reduce male fertility. In the United States, anemia in livestock fed with cotton-seed-containing feed was found to be caused by gossypol13,14 and iron chelation was suggested as a possible mechanism.6 In China, medical observations linked consumption of cotton seed oil to reduced male fertility. Gossypol was identified as the active compound and tested as a male contraceptive in large scale clinical trials in China, but these were stopped due to hypokalemia and irreversible effects on spermatogenesis.15 The apparent diversity of all these separate activities raises the possibility that they are linked by a simple conserved mechanism. Surprisingly little is known about the molecular targets of gossypol. Various studies have presented oxidoreductases, transferases, hydrolases, lyases, and kinases as potential targets (reviewed by ref 6). In the cancer field the current hypothesis is that gossypol acts as a BH3 mimetic and binds to the BHC3 domain of the Bcl-2 and Bcl-xL proteins, thereby promoting apoptosis in cells resistant to chemotherapeutic agents.16,17 A potentially useful feature of gossypol is its inhibitory activity toward yeast. The modes of action of compounds targeting conserved areas of cell biology have been successfully elucidated using yeast chemogenomic HIP HOP profiling.18−21 HIP HOP profiling uses a genome wide collection of more than 6000 yeast deletion strains. The yeast strains are © 2018 American Chemical Society

exposed to an inhibitory but sublethal dose of the test compound, and the pattern of sensitivity across the collection is characteristic of the mode of action of the test compound. The yeast can have both copies of a gene deleted (homozygous deletion) or for essential genes just one copy (heterozygous deletion). Haploinsufficiency profiling (HIP) takes advantage of a pool of heterozygous deletion strains and identifies proteins or pathways directly affected by the compound. Homozygous profiling (HOP) is based on a pool of homozygous deletion strains and can reveal synthetic lethal effects and delineate compensating pathways.22 The usefulness of chemogenomic yeast HIP HOP assays to decipher the molecular mechanism of bioactive molecules, including those that lack a protein drug target, has been demonstrated in several studies, including those performed in our laboratory.22−29 To investigate the effect of gossypol on a eukaryotic cell, we have subjected gossypol to HIP HOP profiling in the model organism S. cerevisiae with a view to understanding its antifungal mode of action and by extension its mode of action in other eukaryotic systems that share fundamental conserved biochemistry. The calculated profiles have been validated by retesting individual deletion strains, and the gained mechanism of action hypothesis was tested by follow up experiments. In summary we present evidence that Fe2+ chelation is the principle antifungal mechanism of action of gossypol. Received: May 1, 2018 Published: July 17, 2018 7381

DOI: 10.1021/acs.jmedchem.8b00692 J. Med. Chem. 2018, 61, 7381−7386

Journal of Medicinal Chemistry

Brief Article

Figure 1. Gossypol target identification by yeast chemogenomic profiling. (A) HIP and HOP profiles using a sublethal dose of gossypol (400 μM). Relevant hypersensitive strains (95% according to HPLC analysis. The compound was stored as powder at 4 °C until use and then dissolved in DMSO to make a stock concentration of 10 mM. Solutions were kept at 4 °C for up to 6 months. Chemogenomic Profiling (HIP/HOP). The growth-inhibitory potency of test substances was determined using wild-type S. cerevisiae BY4743. OD600 values of exponentially growing cultures in rich medium were recorded with a robotic system. Twelve-point serial dilutions were assayed in 96-well plates with a reaction volume of 150 μL, start OD600 was 0.05 with DMSO normalized to 2%. IC30 values were calculated using logistic regression curve fits generated by TIBCO Spotfire version 3.2.1 (TIBCO Software Inc.). HIP, HOP, and microarray analyses were performed as described previously (Hoepfner et al., 2014). Sensitivity was computed as the median absolute deviation logarithmic (MADL) score for each compound/concentration combination. z-Scores are based on a robust parametric estimation of gene variability from >4000 different profiles and were computed as described in detail in Hoepfner et al.18 Growth Curves. HIP/HOP profiles were validated by picking the individual strains from the HIP and HOP collections (OpenBiosystems, catalog nos. YSC1056 and YSC1055) and testing logphase cultures in 96-well microtiter plates in yeast peptone dextrose medium with serial dilutions of the compound. The assay volume was 150 μL/well, start OD600 was 0.01, DMSO was normalized to 2%. Curves were calculated by taking the 11 h OD600 measurements and applying a logistic regression curve fit in TIBCO Spotfire version 3.2.1. Strain HO/YDL227C was used as the wild-type reference. Cation Rescue Experiment. 1 M FeCl3 and FeSO4 aqueous stock solutions were prepared from powdered FeCl3·6H2O and FeSO4·7H2O and adjusted to pH 2 with HCl. Serial dilutions of these solutions combined with SC culture medium were used to check that precipitation of iron salts did not occur within the time frame of the experiment (15 h). Yeast strain BY4743 was then tested with varying concentrations of gossypol and deferasirox to determine the minimal inhibitory concentration of each. The compounds were then tested at the minimal inhibitory concentration in the presence of increasing concentrations of FeCl3 and FeSO4 to look for restoration of growth. The experiment was performed in a transparent 384-well microplate using gossypol and deferasirox in SC medium along with serial dilutions of iron solutions and log phase BY4743 cells diluted to OD600 of 0.05. The final reaction volume per well was 50 μL. Cell growth at OD600 over 15 h was measured as described previously.42

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Notes

The authors declare the following competing financial interest(s): The authors with affiliation Novartis Institutes for Bio-Medical Research are employees of Novartis Pharma AG and may own stock in the company; all other authors state no conflict of interest.

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ACKNOWLEDGMENTS We thank the Novartis Natural Product team for compound supply and Philipp Krastel for compound quality control, Thomas Aust and Ralph Riedl for execution of the HIP HOP assay, and Nicole Hartmann and Juerg Eichenberger for processing the HIP HOP microarrays. This work was funded by the Novartis Institutes for BioMedical Research.

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ABBREVIATIONS USED HIP HOP, haploinsufficiency profiling and homozygous profiling

(1) Stipanovic, R. D.; Puckhaber, L. S.; Bell, A. A.; Percival, A. E.; Jacobs, J. Occurrence of (+)- and (−)-gossypol in wild species of cotton and in Gossypium hirsutum Var. marie-galante (Watt) Hutchinson. J. Agric. Food Chem. 2005, 53, 6266−6271. (2) Mellon, J. E.; Dowd, M. K.; Beltz, S. B.; Moore, G. G. Growth inhibitory effects of gossypol and related compounds on fungal cotton root pathogens. Lett. Appl. Microbiol. 2014, 59, 161−168. (3) Puckhaber, L. S.; Dowd, M. K.; Stipanovic, R. D.; Howell, C. R. Toxicity of (+)- and (−)-gossypol to the plant pathogen, Rhizoctonia solani. J. Agric. Food Chem. 2002, 50, 7017−7021. (4) Gonzalez-Garza, M. T.; Matlin, S. A.; Mata-Cardenas, B. D.; Said-Fernandez, S. Differential effects of the (+)- and (−)-gossypol enantiomers upon Entamoeba histolytica axenic cultures. J. Pharm. Pharmacol. 1993, 45, 144−145. (5) Montamat, E. E.; Burgos, C.; Gerez de Burgos, N. M.; Rovai, L. E.; Blanco, A.; Segura, E. L. Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi. Science 1982, 218, 288−289. (6) Dodou, K.; Anderson, R. J.; Small, D. A.; Groundwater, P. W. Investigations on gossypol: past and present developments. Expert Opin. Invest. Drugs 2005, 14, 1419−1434. (7) Fulda, S. Modulation of apoptosis by natural products for cancer therapy. Planta Med. 2010, 76, 1075−1079. (8) Lu, M. D.; Li, L. Y.; Li, P. H.; You, T.; Wang, F. H.; Sun, W. J.; Zheng, Z. Q. Gossypol induces cell death by activating apoptosis and autophagy in HT-29 cells. Mol. Med. Rep. 2017, 16, 2128−2132. (9) Wang, X.; Howell, C. P.; Chen, F.; Yin, J.; Jiang, Y. Gossypol−a polyphenolic compound from cotton plant. Adv. Food Nutr. Res. 2009, 58, 215−263. (10) Ready, N.; Karaseva, N. A.; Orlov, S. V.; Luft, A. V.; Popovych, O.; Holmlund, J. T.; Wood, B. A.; Leopold, L. Double-blind, placebocontrolled, randomized phase 2 study of the proapoptotic agent AT101 plus docetaxel, in second-line non-small cell lung cancer. J. Thorac. Oncol. 2011, 6, 781−785. (11) Sonpavde, G.; Matveev, V.; Burke, J. M.; Caton, J. R.; Fleming, M. T.; Hutson, T. E.; Galsky, M. D.; Berry, W. R.; Karlov, P.; Holmlund, J. T.; Wood, B. A.; Brookes, M.; Leopold, L. Randomized phase II trial of docetaxel plus prednisone in combination with placebo or AT-101, an oral small molecule Bcl-2 family antagonist, as first-line therapy for metastatic castration-resistant prostate cancer. Ann. Oncol 2012, 23, 1803−1808. (12) Swiecicki, P. L.; Bellile, E.; Sacco, A. G.; Pearson, A. T.; Taylor, J. M.; Jackson, T. L.; Chepeha, D. B.; Spector, M. E.; Shuman, A.; Malloy, K.; Moyer, J.; McKean, E.; McLean, S.; Sukari, A.; Wolf, G.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00692.

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REFERENCES

LC−UV purity check of gossypol sample, UV spectrum of gossypol sample, high resolution MS of gossypol sample (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

*T.A.K.P.: e-mail, [email protected]; phone, +44 (0) 208 3325393. *D.H.: e-mail, [email protected]; phone, +41 79 8634524. ORCID

Thomas A. K. Prescott: 0000-0002-3039-7067 Dominic Hoepfner: 0000-0001-8450-7543 7384

DOI: 10.1021/acs.jmedchem.8b00692 J. Med. Chem. 2018, 61, 7381−7386

Journal of Medicinal Chemistry

Brief Article

T.; Eisbruch, A.; Prince, M.; Bradford, C.; Carey, T. E.; Wang, S.; Nor, J. E.; Worden, F. P. A phase II trial of the BCL-2 homolog domain 3 mimetic AT-101 in combination with docetaxel for recurrent, locally advanced, or metastatic head and neck cancer. Invest. New Drugs 2016, 34, 481−489. (13) Gadelha, I. C.; Fonseca, N. B.; Oloris, S. C.; Melo, M. M.; SotoBlanco, B. Gossypol toxicity from cottonseed products. Sci. World J. 2014, 2014, 231635. (14) Rigdon, R. H.; Crass, G.; Ferguson, T. M.; Couch, J. R. Effects of gossypol in young chickens with the production of a ceroid-like pigment. AMA Arch. Pathol. 1958, 65, 228−235. (15) Wu, D. An overview of the clinical pharmacology and therapeutic potential of gossypol as a male contraceptive agent and in gynaecological disease. Drugs 1989, 38, 333−341. (16) Bodur, C.; Basaga, H. Bcl-2 inhibitors: emerging drugs in cancer therapy. Curr. Med. Chem. 2012, 19, 1804−1820. (17) Kitada, S.; Leone, M.; Sareth, S.; Zhai, D.; Reed, J. C.; Pellecchia, M. Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. J. Med. Chem. 2003, 46, 4259− 4264. (18) Hoepfner, D.; Helliwell, S. B.; Sadlish, H.; Schuierer, S.; Filipuzzi, I.; Brachat, S.; Bhullar, B.; Plikat, U.; Abraham, Y.; Altorfer, M.; Aust, T.; Baeriswyl, L.; Cerino, R.; Chang, L.; Estoppey, D.; Eichenberger, J.; Frederiksen, M.; Hartmann, N.; Hohendahl, A.; Knapp, B.; Krastel, P.; Melin, N.; Nigsch, F.; Oakeley, E. J.; Petitjean, V.; Petersen, F.; Riedl, R.; Schmitt, E. K.; Staedtler, F.; Studer, C.; Tallarico, J. A.; Wetzel, S.; Fishman, M. C.; Porter, J. A.; Movva, N. R. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions. Microbiol. Res. 2014, 169, 107− 120. (19) Hughes, T.; Andrews, B.; Boone, C. Old drugs, new tricks: using genetically sensitized yeast to reveal drug targets. Cell 2004, 116, 5−7. (20) Hoon, S.; Smith, A. M.; Wallace, I. M.; Suresh, S.; Miranda, M.; Fung, E.; Proctor, M.; Shokat, K. M.; Zhang, C.; Davis, R. W.; Giaever, G.; St Onge, R. P.; Nislow, C. An integrated platform of genomic assays reveals small-molecule bioactivities. Nat. Chem. Biol. 2008, 4, 498−506. (21) Parsons, A. B.; Lopez, A.; Givoni, I. E.; Williams, D. E.; Gray, C. A.; Porter, J.; Chua, G.; Sopko, R.; Brost, R. L.; Ho, C. H.; Wang, J.; Ketela, T.; Brenner, C.; Brill, J. A.; Fernandez, G. E.; Lorenz, T. C.; Payne, G. S.; Ishihara, S.; Ohya, Y.; Andrews, B.; Hughes, T. R.; Frey, B. J.; Graham, T. R.; Andersen, R. J.; Boone, C. Exploring the modeof-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 2006, 126, 611−625. (22) Giaever, G.; Shoemaker, D. D.; Jones, T. W.; Liang, H.; Winzeler, E. A.; Astromoff, A.; Davis, R. W. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat. Genet. 1999, 21, 278−283. (23) Hoepfner, D.; Karkare, S.; Helliwell, S. B.; Pfeifer, M.; Trunzer, M.; De Bonnechose, S.; Zimmerlin, A.; Tao, J.; Richie, D.; Hofmann, A.; Reinker, S.; Frederiksen, M.; Movva, N. R.; Porter, J. A.; Ryder, N. S.; Parker, C. N. An integrated approach for identification and target validation of antifungal compounds active against Erg11p. Antimicrob. Agents Chemother. 2012, 56, 4233−4240. (24) Hoepfner, D.; McNamara, C. W.; Lim, C. S.; Studer, C.; Riedl, R.; Aust, T.; McCormack, S. L.; Plouffe, D. M.; Meister, S.; Schuierer, S.; Plikat, U.; Hartmann, N.; Staedtler, F.; Cotesta, S.; Schmitt, E. K.; Petersen, F.; Supek, F.; Glynne, R. J.; Tallarico, J. A.; Porter, J. A.; Fishman, M. C.; Bodenreider, C.; Diagana, T. T.; Movva, N. R.; Winzeler, E. A. Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell Host Microbe 2012, 11, 654−663. (25) Lum, P. Y.; Armour, C. D.; Stepaniants, S. B.; Cavet, G.; Wolf, M. K.; Butler, J. S.; Hinshaw, J. C.; Garnier, P.; Prestwich, G. D.; Leonardson, A.; Garrett-Engele, P.; Rush, C. M.; Bard, M.; Schimmack, G.; Phillips, J. W.; Roberts, C. J.; Shoemaker, D. D.

Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 2004, 116, 121−137. (26) Nyfeler, B.; Hoepfner, D.; Palestrant, D.; Kirby, C. A.; Whitehead, L.; Yu, R.; Deng, G.; Caughlan, R. E.; Woods, A. L.; Jones, A. K.; Barnes, S. W.; Walker, J. R.; Gaulis, S.; Hauy, E.; Brachmann, S. M.; Krastel, P.; Studer, C.; Riedl, R.; Estoppey, D.; Aust, T.; Movva, N. R.; Wang, Z.; Salcius, M.; Michaud, G. A.; McAllister, G.; Murphy, L. O.; Tallarico, J. A.; Wilson, C. J.; Dean, C. R. Identification of elongation factor G as the conserved cellular target of argyrin B. PLoS One 2012, 7, e42657. (27) Richie, D. L.; Thompson, K. V.; Studer, C.; Prindle, V. C.; Aust, T.; Riedl, R.; Estoppey, D.; Tao, J.; Sexton, J. A.; Zabawa, T.; Drumm, J.; Cotesta, S.; Eichenberger, J.; Schuierer, S.; Hartmann, N.; Movva, N. R.; Tallarico, J. A.; Ryder, N. S.; Hoepfner, D. Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents. Antimicrob. Agents Chemother. 2013, 57, 2272−2280. (28) Roemer, T.; Xu, D.; Singh, S. B.; Parish, C. A.; Harris, G.; Wang, H.; Davies, J. E.; Bills, G. F. Confronting the challenges of natural product-based antifungal discovery. Chem. Biol. 2011, 18, 148−164. (29) Sadlish, H.; Galicia-Vazquez, G.; Paris, C. G.; Aust, T.; Bhullar, B.; Chang, L.; Helliwell, S. B.; Hoepfner, D.; Knapp, B.; Riedl, R.; Roggo, S.; Schuierer, S.; Studer, C.; Porco, J. A., Jr.; Pelletier, J.; Movva, N. R. Evidence for a functionally relevant rocaglamide binding site on the eIF4A-RNA complex. ACS Chem. Biol. 2013, 8, 1519− 1527. (30) Stearman, R.; Yuan, D. S.; Yamaguchi-Iwai, Y.; Klausner, R. D.; Dancis, A. A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 1996, 271, 1552−1557. (31) De Silva, D. M.; Askwith, C. C.; Eide, D.; Kaplan, J. The FET3 gene product required for high affinity iron transport in yeast is a cell surface ferroxidase. J. Biol. Chem. 1995, 270, 1098−1101. (32) Fu, D.; Beeler, T. J.; Dunn, T. M. Sequence, mapping and disruption of CCC2, a gene that cross-complements the Ca(2+)sensitive phenotype of csg1 mutants and encodes a P-type ATPase belonging to the Cu(2+)-ATPase subfamily. Yeast 1995, 11, 283− 292. (33) Gaxiola, R. A.; Yuan, D. S.; Klausner, R. D.; Fink, G. R. The yeast CLC chloride channel functions in cation homeostasis. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4046−4050. (34) Fisk, D. G.; Ball, C. A.; Dolinski, K.; Engel, S. R.; Hong, E. L.; Issel-Tarver, L.; Schwartz, K.; Sethuraman, A.; Botstein, D.; Cherry, J. M. Saccharomyces cerevisiae S288C genome annotation: a working hypothesis. Yeast 2006, 23, 857−865. (35) Lin, S. J.; Pufahl, R. A.; Dancis, A.; O’Halloran, T. V.; Culotta, V. C. A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport. J. Biol. Chem. 1997, 272, 9215−9220. (36) Mesecke, N.; Mittler, S.; Eckers, E.; Herrmann, J. M.; Deponte, M. Two novel monothiol glutaredoxins from Saccharomyces cerevisiae provide further insight into iron-sulfur cluster binding, oligomerization, and enzymatic activity of glutaredoxins. Biochemistry 2008, 47, 1452−1463. (37) Nick, H.; Acklin, P.; Lattmann, R.; Buehlmayer, P.; Hauffe, S.; Schupp, J.; Alberti, D. Development of tridentate iron chelators: from desferrithiocin to ICL670. Curr. Med. Chem. 2003, 10, 1065−1076. (38) Bollig, C.; Schell, L. K.; Rucker, G.; Allert, R.; Motschall, E.; Niemeyer, C. M.; Bassler, D.; Meerpohl, J. J. Deferasirox for managing iron overload in people with thalassaemia. Cochrane Database Syst. Rev. 2017, 8, CD007476. (39) Enyedy, E. A.; Pocsi, I.; Farkas, E. Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores. J. Inorg. Biochem. 2004, 98, 1957−1966. (40) Przybylski, P.; Wojciechowski, G.; Brzezinski, B.; Kozubek, H.; Marciniak, B.; Paszyc, S. Spectroscopic and semiempirical studies of gossypol complexes with Fe 2+ and Fe 3+ cations. J. Mol. Struct. 2001, 569, 147−155. 7385

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Journal of Medicinal Chemistry

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(41) Tvrda, E.; Peer, R.; Sikka, S. C.; Agarwal, A. Iron and copper in male reproduction: a double-edged sword. J. Assist Reprod Genet 2015, 32, 3−16. (42) Prescott, T. A. K.; Panaretou, B. A mini HIP HOP assay uncovers a central role for copper and zinc in the antifungal mode of action of allicin. J. Agric. Food Chem. 2017, 65, 3659−3664.

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DOI: 10.1021/acs.jmedchem.8b00692 J. Med. Chem. 2018, 61, 7381−7386