Yeast chemogenomic profiling reveals iron chelation to be the

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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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00692 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Journal of Medicinal Chemistry

Yeast chemogenomic profiling reveals iron chelation to be the principle cell inhibitory mode of action of gossypol Thomas A.K. Prescott1*, Tiphaine Jaeg2†, Dominic Hoepfner2* 1Royal

Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom Institutes for BioMedical Research, Developmental & Molecular Pathways, Novartis Pharma AG, Fabrikstrasse 22, CH-4056 Basel, Switzerland 2Novartis

KEYWORDS Gossypol; yeast; iron; chelation; mode-of-action; HIP HOP; antifungal

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.

INTRODUCTION Gossypol is a phenolic compound found in cotton plants, including the seeds and the seed oil1. 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 gossypol 13, 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 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 towards 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 exposed to an inhibitory but sub-lethal 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 re-testing 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. RESULTS

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Chemogenomic profiling reveals links to Fe2+ homeostasis To investigate the mechanism of action of gossypol in S. cerevisiae cells we subjected gossypol to unbiased, chemogenomic profiling using a fully automated system. The results are visualized by plotting the relative growth reduction of individual HIP/HOP strains by the compound (sensitivity) vs. a measure of significance (z-score). The substance was profiled at an IC30 concentration of 400 µM in two independent experiments (Fig. 1A, B). The HOP results obtained for gossypol were consistent across the different replicates (Fig. 1C). However, in the HIP assays only hypersensitive strains with a low z-score cold be identified and these were not conserved in the two experiments (Fig. 1A, B). The z-score is a statistical assessment of how specific the response of a strain to a given perturbation is. The value is computed across the HIP HOP profiles of ∼4000 diverse compounds and a zscore within a range of +/-5 indicates a strain that responds to many diverse chemotypes and is thus, not relevant for interpretation 18. The hypersensitive strains in the HOP assay with the best z-scores were strains with homozygous deletions in: FTR1, an iron permease 30, FET3 an iron oxidoreductase 31, CCC2 a copper transporting ATPase that is also involved in Fe2+ homeostasis 32 and GEF1 a voltage gated chloride channel involved in cation homeostasis 33. Two additional hits (YDR269c and YDR271c) are dubious ORFs and almost completely overlap the CCC2/YDR270w gene 34. Therefore, deletion affects Ccc2p function and supports the validity of the CCC2 HOP hit. The ATX1 HOP strain was mildly affected but present in all three profiles. Atx1p is annotated as a copper metallochaperone and reported to interact with Cccp2 and Fet3p 35. In both replicates the GRX6 deletion strain scored as slightly resistant to gossypol. Grx6 is annotated as a monothiol glutaredoxin and binds Fe-S clusters, linking this hit also to Fe2+ homeostasis and oxidative stress 36. In summary, profiling of gossypol did not yield relevant, reproducible hits in the HIP assay but did provide consistent hits in the HOP assay, and these all share functional annotations linking them to Fe2+ homeostasis. Individual validation of hypersensitive yeast strains identified in the HOP assay The HIP HOP assay makes use of a pooled format where sensitivity of individual yeast strains is monitored by the relative abundance of their unique molecular barcode. To separately confirm the hypersensitivity or resistance of the identified hits we tested individual HOP strains deleted for FTR1, FET3, CCC2, GEF1 and GRX6 respectively, in doseresponse experiments (Fig. 1D). We also tested one of the HIP hits (PIB2) but as expected, did not find any reproducible hypersensitivity (data not shown). The GRX6 deletion stain that scored as resistant showed some improved growth kinetics at all concentrations tested but the effect was marginal. In contrast, FTR1, FET3, CCC2 and GEF1 homozygous deletion strains were confirmed as markedly hypersensitive compared to the wild type HO control strain (Fig. 1D). This implicates Fe2+ related biology in the mode of action of gossypol.

The HOP profile of gossypol matches that of known iron chelators Next, we queried our in-house HIP HOP database comprising profiles of ∼4000 diverse chemical compounds and checked for closely related chemogenomic profiles. The HOP profile of gossypol clustered best with its own replicate (Pearson correlation value of 0.82) followed by the HOP profile of the cation chelator drug deferasirox which is approved for use in iron overload 37, 38 (0.77) and the natural iron chelator desferricoprogen 39 (0.71) (see figure 2). Performing a pairwise comparison between gossypol and deferasirox or desferricoprogen (Fig. 1E, F) not only revealed a conserved set of hypersensitive hits (FTR1, FET3, CCC2 (including the overlapping ORFs YDR269c and YDR271c) and GEF1 but also a highlyconserved sequence of hypersensitivity: FTR1 and FET3 were the most hypersensitive and GEF1 the least with the three CCC2 associated ORFs distributed in the middle. In summary, the HOP profile of gossypol corelated best with deferasirox and desferricoprogen, two compounds known to chelate Fe2+. Addition of Fe2+ rescues yeast from gossypol growth inhibition If the growth inhibitory effects of gossypol and deferasirox are due to lack of Fe2+ then addition of Fe2+ should rescue yeast from growth inhibition. To test this hypothesis, each compound was used to induce complete growth inhibition in the presence of increasing concentrations of Fe2+. Figures 1G and 1H, show that both gossypol and deferasirox-induced growth inhibition was indeed reversed by the addition of Fe2+. The rescue effect is greater with gossypol than with deferasirox, as toxicity of iron salts prevented higher Fe2+ concentrations being tested for the latter.


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Figure 1. Figure caption. 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 v3.2.1 (TIBCO Software Inc.). HIP, HOP, and microarray analysis was 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, Cat # YSC1056 and YSC1055) and testing log-phase cultures in 96-well microtiter plates in

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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 v3.2.1. Strain HO/YDL227C was used as the wildtype reference. Cation rescue experiment 1M 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 hours). 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 0.05. The final reaction volume per well was 50 µl. Cell growth at OD600 over 15 hours was measured as described previously 42. Supporting information LC-UV purity check of gossypol sample, UV spectrum of gossypol sample, high resolution MS of gossypol sample. Acknowledgements We would like to thank the Novartis Natural Product team for compound supply and Philipp Krastel for compound quality control, Thomas Aust, 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. The authors with affiliation Novartis Institutes for BioMedical research are employees of Novartis Pharma AG and may own stock in the company, all other authors state no conflict of interest. Corresponding Author *[email protected]; +44 (0) 208 3325393 *[email protected] +41 79 8634524 Present Addresses †Tiphaine Jaeg. Current address: Alms Therapeutics, 650 Boulevard Gonthier d'Andernach, 67400 Illkirch, France Author Contributions All authors have given approval to the final version of the manuscript. Abbreviations HIP HOP, profiling.


haploinsufficiency

profiling

homozygous

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