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Non-competitive effect of gambogic acid displaces fluorescence labelled ATP but requires ATP for binding to Hsp90/HtpG Qing Yue, Frank Stahl, Oliver Plettenburg, Andreas Kirschning, Athanasia Warnecke, and Carsten Zeilinger Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00155 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018
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Biochemistry
Non-competitive effect of gambogic acid displaces fluorescence labelled ATP but requires ATP for binding to Hsp90/HtpG Qing Yue1,4, Frank Stahl2, Oliver Plettenburg3, Andreas Kirschning3 Athanasia Warnecke4 and Carsten Zeilinger1,* [1] Institute of Biophysics and Center of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 38, 30167 Hannover (Germany) [2] Institute of Technical Chemistry and Center of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 1B, 30167 Hannover (Germany) [3] Institute of Organic Chemistry and Center of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 1B, 30167 Hannover (Germany) [4] Department of Otorhinolaryngology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover
KEYWORDS Hsp90, HtpG, heat shock proteins, reblastatin derivatives, protein microarray Supporting Information Placeholder ABSTRACT: The heat shock protein 90 (Hsp90) family
plays a critical role in maintaining the homeostasis of the intracellular environment for human and prokaryotic cells. Hsp90 orthologues were identified as important target proteins for cancer and plant disease therapies. It was shown that gambogic acid (GBA) has the potential to inhibit human Hsp90. However, it is unknown, whether it is also able to act on the bacterial high temperature protein (HtpG) analogue. In this work, we screened GBA and nine other novel potential Hsp90 inhibitors using a miniaturized high throughput protein microarray-based assay and found that GBA shows an inhibitory effect on different Hsp90s after dissimilarity analysis of the protein sequence alignment. The dissociation constant of GBA and HtpG Xanthomonas (XcHtpG) computed from micro scale thermophoresis is 682.2 ± 408 µM in the presence of ATP, which is indispensable for the GBA binding to XcHtpG. Our results demonstrate that GBA is a promising Hsp90/HtpG inhibitor. The work further demonstrates that our assay concept has great potential to find new potent Hsp / HtpG inhibitors. Introduction Gambogic acid is used in traditional medicine of southeast Asia.1,2 Recently, it was shown that the xanthonoid gambogic acid 1 (GBA, from the plant Garcinia hanburyi) a natural phenolic compound with a xanthone
backbone, shows antitumor activity.3-5 Newer data suggest anti-Hsp90 activity with specificity for Hsp90β 6 and modelling data indicate that unlike most Hsp inhibitors it binds outside the ATP pocket.7 Antibiotic compounds such as colistins and polymyxin B bind to hydrophobic sites of the N-terminal domain of HtpG without affecting its ATPase activity.8 In HeLa cells, gambogic acid binds to the N-terminal ATP-binding domain of Hsp90 acting as a non-competitive inhibitor of the Hsp90 ATPase activity. This leads to the degradation of Hsp90 client proteins in HeLa cells making this compound a promising anticancer agent.9
Since it may be possible that GBA targets plant pathogens, heat shock proteins found in typical plant pathogenic organisms such as Pseudomonas syringae and Xanthomonas campestris were tested in a target-oriented microarray based assay system. HtpG, the bacterial ortholog is not present in Archaebacteria and not all eubacteria seem to possess this gene. Those who lack it are Lactic acid bacteria, Chlamydia, Deinococcus and Thermus.10,11 In comparison to eukaryotic Hsp90, which is an essential stress marker, the HtpG functions differ within the eubacteria. HtpG from E. coli does not appear to be essential for the thermotolerance, but for Cyanobacteria it was shown that HtpG is essential for survival under thermal stress conditions. In addition, it is also involved in protection from oxidative stress and acclimation to cold.12-14 The plant pathogenic bacteria cause a number of symptoms in plants such as plant gall blight or rotting. In addition, phytotoxins are produced by the bacteria inside the host and enhance the effects of severe diseases.15
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These toxins frequently cause necrosis or chlorosis in the case of P. syringae. Besides the bacterially caused phenomena, Pseudomonas syringae also promotes ice nucleation e. g., for supercooled water by producing an ice nucleation protein (INP).15 As most plants do not tolerate intracellular ice crystals, a severe frost damage is the common cause of this process. The impact of P. syringae or X. campestris results in an immense economic damage and often requires the bulk use of copper compounds to control bacterial diseases. Because of the crosstalk tolerance of P. syringae pv. syringae there is a quest for finding new inhibitors and prevent major crop losses.15 Human Hsp90 and the bacterial homologue HtpG are targets for inhibitors with the potential to develop drug candidates.16-18 A famous inhibitor for the ATP-binding site of Hsp90 and antitumor agent is the benzoquinone ansamycin geldanamycin that was first isolated from Streptomyces lividans.19 Reblastatin is structurally related to geldanamycin except that it differs in the pquinone moiety that is exchanged by a phenolic group. It is also a potent cell cycle inhibitor, however, due to the absence of a Michael acceptor group, reblastatin does not show undesired hepatotoxic side effects as was reported for geldanamycin.20 Importantly, reblastatin shows a higher affinity for human Hsp90 compared to geldanamyin.21 Therefore, we22-26 and others27,28 developed strategies to access new reblastatin derivatives using mutasynthesis as a synthetic tool.29-31 Some of those are presented in Figure 1 (compounds 2-10).
Figure 1. Structures of Hsp90 inhibitors radicicol (2) and reblastatin derivatives 3-10 and of gambogic acid (1). Bacterial heat shock proteins also maintain the functional proteome under stressful environmental conditions.32 Therefore, small molecules that are able to inhibit bacterial heat shock proteins can potentially be used to combat bacterial infections, an emerging topic in the field of heat shock proteins. It is known that some bacteria produce secondary metabolites that target heat shock proteins (Hsp)32, but the physiological role of these compounds still need to be deciphered.
To identify new candidates for such targets, an effective screening system is essential. A recently developed miniaturized protein microarray test system from our laboratories requires only small quantities of both the heat shock protein and the test compound.27,28 For this purpose, the protein of interest is immobilized on nitrocellulose of the microarray thereby maintaining its active conformation and its ability to bind substrates. When ATP is the natural substrate, commonly the use of ATPanalogues labelled with a fluorescent dye is possible and allows to measure an optical binding signal. This is of relevance in competition experiments or displacement assays with other compounds such as radicicol or geldnamycin.27,28 Such binders not only displace the labelled ATP, but also prevent cell-physiologically relevant substrates/ proteins from binding to that position. Displacement assays of this kind can be used for the high throughput screening of synthetic compound libraries and the optimization of new Hsp inhibitors with pharmaceutical relevance.22-26 With this target-oriented screening system, active compounds can be identified whose potential for drug development is hardly recognizable in virtual screenings. Here, we report on the inhibitory properties of gambogic acid (1) in comparison to radicicol (2) and reblastatin derivatives 3-10 for human Hsp90, for the bacterial Hsp PsHtpG from Pseudomonas syringae and for XcHtpG from Xanthomonas campestris by binding to the ATP binding sites.22-26 We demonstrate that a non-competitive inhibitor can be assayed on functional protein microarrays and with microscale thermophoresis (MST). Comparison of Hsp90 orthologues by inhibitor susceptibility Comparison of the ATP binding site of human Hsp90 and bacterial HtpGs, namely PsHtpG and XcHtpG, reveal differences in the amino acid sequence, hydropathy and the interactome. Therefore, the ability of ATP as well as of small molecular inhibitors can differ with the consequence of specificity differences (see supplementary material, Figures S1a-c). However, further comparison of human with bacterial Hsp90s in the hydropathy and interactome plots showed that the differences are not present in key positions of the ATP-binding site. These are highly conserved (Figure S1a). Therefore, it we assumed that the susceptibility for the macrocycles present in radicicol and reblastatin derivatives shown in Figure 1 are conserved. To test the susceptibility of these inhibitors, the bacterial heat shock proteins PsHtpG and XcHtpG were cloned into the bacterial expression vector pETSUMO and synthesized in E. coli BL21DE3 cells. After purification by IMAC and removal of the fusion site, expressed PsHtpG or XcHtpG together with purified human Hsp90α and Hsp90β22 were spotted onto the nitrocellulose microarray chip for target-oriented assaying.
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Biochemistry
Figure 2. Experimental microarray design: After target proteins (HSP) are spotted on the microarray and incubated in the presence of the small molecule and fluorescent labelled Cy5-ATP, the fluorescence signal is measured. A low intensity of the fluorescence signal is detected in the case of effective competition of the small molecule for the binding site compared to Cy5-ATP. Larger signal intensities mean that only small amounts of Cy5ATP are displaced by the small molecule. After capping unspecific binding sites on the nitrocellulose surface, the spotted proteins were incubated in the presence of fluorescence labelled ATP-Cy5 to monitor displacement of bound ATP by Hsp90 inhibitors (Figure 2). The data reveal that several reblastatin derivatives effectively displaced labelled ATP-Cy5 from the heat shock proteins (Figure 3a). HsHsp90α
a)
HsHsp90β
PsHtpG
XcHtpG
0 1 2 3 4 5 6 7 8 9 10
b)
Figure 3. (a) Heat map compounds 1-10. The normalized signals are ranked from 0 to 100 displaying different colors. Low signal values (grey) indicate stronger competition against bound Cy5-ATP (position 0, dark blue) and higher affinity for Hsps. Compounds were tested at a concentration of 500 nM. (b) Dose response curves (EC50) for gambogic acid (1) with different HSPs based on calculated microarray data. The EC50–values for Hsp90α and Hsp90β were determined to be 19.83 mM~27.28 mM (12.47 µg/ml ~17.16 µg/ml) and 46.41 mM (29.19 µg/ml) for PsHtpG and finally 14.82 mM (9.32 µg/ml) for XcHtpG. Fitting curve was based on the dose-response model. Radicicol (2), and reblastatin derivatives 6-8 and 10 showed high activity on human Hsp90α,β. By contrast, radicicol (2) was not active on PsHtpG (Figure 3a). This result is in accordance with studies on the bacterial heat shock protein HtpG from H. pylori.22 Additionally, all candidates were tested for displacement activity by 17allylamino geldanamycin (17AAG, Tanespimycin), a
semisynthetic derivative of geldanamycin that had reached phase 2 clinical trials with EC50 values for XcHtpG (670 nM) PsHtpG (180 nM) HsHsp90α (80 nM) HsHsp90β (150 nM). Bacterial HtpGs showed a much lower susceptibility tofor 17AAG. Interestingly, reblastatin derivatives 5, 7 and 9 bearing a phenolic group at position 17 exerted best activity for bacterial HtpG (Figures 1, 3a). As was demonstrated before23, Asp54 in HsHsp90 is important for binding to a phenolic group at C17. This site is also conserved in the bacterial HtpGs.22 When testing gambogic acid (1) in a concentration dependent manner, the obtained dose-response curve revealed that the effective concentrations (EC50) for Hsp90α and Hsp90β is at ~8 mM, while for PsHtpG 30 mM and for XcHtpG 40 mM were found (Figure 3b). Confirmation of GBA by MST We recently reported that the position of the fluorescence label attached to ATP can influence the binding to HSPs.25. With respect to sterically congested gambogic acid (1) one may consider uncompetitive effects instead. Therefore, we included a modified orthogonal assay into our studies. First, the binding was analyzed in the presence as well as in the absence of unlabelled ATP. Clearly, for binding of gambogic acid (1) to XcHtpG ATP is essential (Figure 3b). In the presence of ATP (1 mM), the dose responsive determination of binding of 1 on XcHtpG gave 682.2 ± 408 µM (429 ± 256.6 µg; Fig. 4 and Fig. S2). The EC50 values determined for GBA are higher than those reported by Davenport et al., 2011 and Zhang et al. 2010, respectively.7,9 Similar affinities have been observed by the direct competitive assay format using the ATP binding site as the site of target. Here, we demonstrate for the first time that our competition assay concept can also be employed for small molecules that do not compete for the ATP binding site. However, the lower of EC50 values reported by other groups may be rationalized if one assumes different accessibilities for the formation of the GBA-Hsp90 complex as we utilzed the full-length dimeric protein and labeled ATP and conducted the MST technique. Since it is known that dimeric Hsp90 has high and low affinity sites for ATP binding it is reasonable to assume that the high affinity site is saturated at 100 nM ATP which is the concentration of the label.33 In contrast higher ATP concentrations are necessary to saturate the low affinity site, which may not be required for GBA binding. This explains why we found enhanced labelled ATP background in the microarray experiment and that may be responsible for an elevated EC50 value.
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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Qing Yue was financed by Elite Young Doctors study abroad plan of Shaanxi province people hospital Notes The authors declare no competing financial interest. ABBREVIATIONS Hsp heat shock protein; HtpG the homologue of Hsp90; GBA gambogic acid Figure 4. The affinity binding curve of gambogic acid (1) and XcHtpG. 1 was titrated from 874.5 µM down to 53 nM at room temperature. XcHtpG concentration was kept constant at 50 nM. The ∆F data were normalized and were fitted in the Kd model using three runs. No binding was detected in the reaction buffer without ATP (red symbol). Conclusions A microarray-assisted target displacement assay with different Hsp90 proteins was established for the simultaneous evaluation of the binding properties for the ATP binding sites using radicicol, reblastatin derivatives and gambogic acid as potential Hsp inhibitors. The studies revealed that different Hsps have different susceptibilities for the test compounds.22,25 Surprisingly, ATP displacement was also induced by gambogic acid (1), that supposedly binds outside the ATP binding pocket (Figure S3). In addition, orthogonal MST experiments revealed that ATP is required for the binding of 1. These new findings show that the gambogic acid binding site displays a unique position for developing non- or uncompetitive binders against Hsp90/HtpG to either regulate Hsp90/HtpG activities in human or in bacterial cells. ASSOCIATED CONTENT Supporting Information. S1 Material and Methods, S2 Figures, Figure S1 a) Sequence alignment HtpG related proteins. b) Hydropathy plot of heat shock proteins. c) Interactome of Hsp90 and HtpG proteins computed by the STRING database. Figure S2 MST-traces. Figure S3 Sequence and structural alignment of putative GBA binding sites.
AUTHOR INFORMATION Corresponding Author *Email:
[email protected] Center of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 38, 30167 Hannover, Germany, Phone: (+49) 511-762-16351; Fax: (+49) 511-762-5436 Author Contributions
prokaryotic
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