Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent

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Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent and Selective Inhibitors of the Transcription Factor STAT5b Nagarajan Elumalai,† Angela Berg,† Stefan Rubner, and Thorsten Berg* Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany S Supporting Information *

ABSTRACT: Design approaches for inhibitors of protein−protein interactions are rare, but highly sought after. Here, we report that O-phosphorylation of simple derivatives of the natural products dihydrocapsaicin and N-vanillylnonanamide leads to inhibitors of the SH2 domain of the transcription factor STAT5b. The most potent molecule is obtained from dihydrocapsaicin in only three synthetic steps. It has submicromolar affinity for the SH2 domain of STAT5b (Ki = 0.34 μM), while displaying 35fold selectivity over the highly homologous STAT5a (Ki = 13.0 μM). The corresponding pivaloyloxymethyl ester inhibits STAT5b with selectivity over STAT5a in human tumor cells. Importantly, it inhibits cell viability and induces apoptosis in human tumor cells in a STAT5-dependent manner. Our data validate O-phosphorylation of appropriately preselected natural products or natural product derivatives as a semirational design approach for small molecules that selectively inhibit phosphorylationdependent protein−protein interaction domains in cultured human tumor cells.

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respective peptide motifs only in their phosphorylated state. These interactions, collectively referred to as phosphorylationdependent protein−protein interactions, require a different strategy. By screening chemical libraries consisting of known bioactive compounds, we recently identified O-phosphorylated salicylic acid (Fosfosal, Figure 1A) and dexamethasone-21-phosphate as inhibitors of two phosphorylation dependent protein−protein interactions. 10 The protein target of Fosfosal is the phosphotyrosine-binding Src homology 2 (SH2) domain of STAT5b, a transcription factor which is constitutively activated in many human tumors. STAT5b has been validated as a target for cancer therapy.11,12 Fosfosal was subsequently modified to give Stafib-1 (Figure 1A), which displayed nanomolar affinity against the STAT5b SH2 domain and which displays high selectivity over STAT5a in biochemical and cell-based assays.13 Retrospective analysis of the composition of the screening library and the profile of compound activities displayed in the screen in which Fosfosal was identified10 revealed a high hit rate from a subset of compounds which can be described as Ophosphorylated natural product derivatives.10 Consequently, we proposed O-phosphorylation of preselected natural products or natural product derivatives as a straightforward approach for the design of nonpeptidic ligands of phosphorylation-depend-

rotein−protein interactions mediate the functions of most proteins.1−3 Selective targeting of protein interaction domains with cell-permeable organic molecules opens the door to functional modulation of the vast majority of proteins in living organisms. Unlike most current chemical biology approaches, inhibition of protein−protein interactions is not restricted to more established drug targets such as cell surface receptors and enzymes.4 This enables the functional analysis of the vast majority of proteins for basic research studies and allows the development of new therapeutic strategies to treat unmet medical needs. However, generally applicable strategies for the design of functional modulators of intracellular protein− protein interactions are rare.5 For interactions mediated by αhelices binding to hydrophobic grooves, numerous proteomimetic scaffolds have been developed,6,7 allowing rational design of competitive inhibitors of the respective protein−protein interaction. Another design approach targeting this class of protein−protein interactions involves conformational stabilization of the α-helical peptide motifs by introduction of hydrophobic cross-links.8,9 However, these approaches remain restricted to interactions mediated by α-helices, and new approaches need to be developed which address the specific requirements of other individual types of protein−protein interactions. Cellular signaling via protein kinases and phosphatases is mediated, to a large extent, by interactions between short peptide motifs containing phosphorylated serine, threonine, or tyrosine residues and protein domains which recognize the © XXXX American Chemical Society

Received: May 15, 2015 Accepted: October 15, 2015

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DOI: 10.1021/acschembio.5b00817 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Natural product based inhibitors of STAT5b. (A) Structures of known salicylic acid-derived inhibitors.10,13 (B) Synthesis of STAT5b inhibitors based on the natural products dihydrocapsaicin and N-vanillylnonanamide. (i) Dibenzyl phosphite, CCl4, DIEA, DMAP, CH3CN, 0 °C, 1 h. (ii) Pd/C, H2, EtOH, 1 h. (iii) 1 M BBr3 in DCM, 16 h.

ent protein−protein interaction domains.10 The rationale for this approach is that the combination of the intrinsic propensity of natural products to interact with proteins,14 together with the presence of a part of the molecule reminiscent of phosphorylated amino acid side chains, should result in a larger chance of binding between the small molecule and the phosphorylation-dependent protein−protein interaction domain.

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STAT5a and STAT5b in competitive binding assays based on fluorescence polarization13,18 revealed their inhibitory activity against the SH2 domains of both STAT5 proteins (Table 1). The highest activity was observed for 3b, with an inhibitory constant (Ki) of 49 ± 4 μM against STAT5a and a Ki of 96 ± 4 μM against STAT5b. In contrast, the nonphosphorylated natural products 1a and 1b were completely inactive against either STAT5 protein (Table 1). Thus, although the activities of 3a/b against STAT5a/b are relatively weak, O-phosphorylation of natural products was indeed an effective measure by which to create starting points for inhibitor development. Since the catechol bisphosphate structure had been essential for high activity against STAT5b in the previous study,13 we also aimed to convert the natural products to the 1,2dihydroxyphenyl (catechol) derivatives, followed by phosphorylation of both hydroxyl groups. To this end, the catechol moieties of dihydrocapsaicin (1a) and N-vanillylnonanamide (1b) were liberated by demethylation using boron tribromide (Figure 1B). Subsequently, the hydroxyl groups of diols 4a and 4b were converted to dibenzylphosphates 5a and 5b by reaction with dibenzylphosphite. Finally, Pd/C-catalyzed hydrogenation resulted in the bisphosphates 6a and 6b. Biochemical analysis of the bisphosphates 6a and 6b revealed both compounds to be submicromolar inhibitors of the

RESULTS AND DISCUSSION

In order to validate our design concept for inhibitors of phosphorylation-dependent protein−protein interactions, we turned our attention to the natural product class of the capsaicinoids. Most peppers contain capsaicinoids such as dihydrocapsaicin (1a) and N-vanillylnonanamide (1b),15−17 which are responsible for the fruits’ pungency (Figure 1B). Both capsaicinoids possess a masked catechol moiety bearing an N-acylated aminomethyl group, reminiscent of structural features contained in Stafib-1, and are therefore promising starting points for the development of STAT5 inhibitors. Conversion of the two natural products 1a/b to the benzyl phosphates 2a/b and their subsequent hydrogenation afforded O-phosphorylated dihydrocapsaicin (3a) and N-vanillylnonanamide (3b) in good yields. Analysis of their activities against B


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Table 1. Activity of Natural Products and Phosphorylated Derivatives in a Fluorescence Polarization Assay against the SH2 Domain of STAT5b and STAT5aa

a

N/A = not applicable. Conversion of IC50 data to Ki values was carried out using the published equation.19

STAT5b domain (Table 1).18 6a (Ki = 0.34 ± 0.01 μM) is slightly more potent than 6b (Ki = 0.39 ± 0.03 μM). Thus, 6a is almost 3 times as potent as catechol bisphosphate (Ki = 0.93 ± 0.07 μM).13 Both 6a and 6b displayed high selectivity over the SH2 domain of the close homologue STAT5a, which is 93% identical on the amino acid level20 (6a, Ki (STAT5a) = 13.0 ± 0.7 μM; 6b, Ki (STAT5a) = 12.3 ± 0.7 μM). In-depth analysis of the selectivity profile of the best compound 6a against the SH2 domains of other STAT proteins and of the tyrosine kinase Lck21 confirmed the high degree of selectivity for STAT5b (Figure 2A and Table S1). 6a displayed more than 35-fold selectivity for STAT5b over STAT5a, more than 20-fold selectivity over STAT6, and more than 45-fold selectivity over STAT4. The SH2 domains of STAT1, STAT3, and Lck were inhibited to an even lesser extent. These data join our previous results13 in challenging the dogma that SH2 domains cannot be targeted selectively with small organic molecules. Molecular docking with AutoDock Vina22 using a homology model of the STAT5b SH2 domain10 (based on the crystal structure of STAT5a)23 placed the catechol bisphosphate

moiety of compound 6a into the phosphotyrosine pocket of STAT5b (Figure 2B).13 Binding of the catechol bisphosphate moiety to the phosphotyrosine binding pocket of the STAT5b SH2 domain had previously been experimentally confirmed using point mutants in a direct binding assay.13 The amide bond is assumed to be involved in hydrogen bonds with the backbone of Asn642 (Figure S1), and the hydrophobic tail of the molecule is placed along the indole ring of Trp641, reaching toward Tyr665. In order to analyze the ability of 6a to selectively inhibit the STAT5b SH2 domain in cultured cells, 6a was converted to the pivaloyloxymethylester 7 (Figure 3A). This class of prodrugs has been thoroughly demonstrated to mediate cell permeability of phosphonates24−26 and phosphates13,27 and liberates the active agent after intracellular cleavage of the pivaloyloxymethyl groups by esterases. Since the function of the SH2 domain is required for activation of STAT5 proteins by phosphorylation of the conserved tyrosine residue (STAT5b, Tyr699; STAT5a, Tyr694) by tyrosine kinases such as Bcr-Abl,28 inhibition of the SH2 domain leads to reduced tyrosine phosphorylation (Figure 3B). This can be monitored by Western blot analysis using C


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phosphates in the cellular milieu, we conducted a time-course experiment using 10 μM of 7 on STAT5b-GFP-transfected K562 cells. The compound was found to exert its strongest effect between 1 and 4 h (Figure S2), with inhibitory activity partially reduced after 8 h of exposure. Although leukemia cells transformed by the Bcr-Abl oncoprotein display constitutive activation of both STAT5a and STAT5b,29 there is strong indication that the activation of STAT5b is of larger functional relevance than the activation of STAT5a. Inhibition of Bcr-Abl mediated signaling by a dominant negative STAT5b mutant, which lacks the ability to dimerize via reciprocal phosphotyrosine-SH2 domain interactions, strongly inhibited cell growth, and viability of Bcr-Abl expressing cells.30 Proliferation of K562 cells is inhibited by shRNA-mediated depletion of STAT5b to a larger extent than by depletion of STAT5a.31 In addition, short hairpin RNA (shRNA)-mediated depletion of STAT5b, but not shRNAmediated depletion of STAT5a, sensitizes K562 cells to the Bcr-Abl inhibitor Imatinib.31 Sh-mediated depletion of STAT5b caused a strong increase in the apoptotic rate in Bcr-Abl expressing leukemia cells, while depletion of STAT5a was ineffective.32 Consequently, selective inhibition of STAT5b has been proposed as a therapeutic strategy to combat tumorigenesis driven by Bcr-Abl.31 Cell viability assays conducted on K562 cells indicated a dose-dependent effect of 7 (Figure 4A). To exclude the possibility that the reduced cell viability was caused by an effect of 7 on targets other than STAT5 proteins, we also tested the effect of 7 on MDA-MB-231 cells, which do not harbor constitutively active STAT5a/b (Figure S3) and thus do not proliferate via STAT5 signaling. We observed virtually no inhibition of the cell viability in MDA-MB-231 cells in the presence of 7 (Figure 4B), indicating that the reduction of cell viability in K562 cells was caused by a STAT5-dependent effect. In order to analyze whether the reduced cell viability of K562 cells in the presence of 7 was caused by an increased rate of apoptosis, we analyzed K562 cells treated with 7 for exposure of phosphatidylserine on the outside of the cell membrane by annexin V staining, followed by flow cytometry. K562 cells treated with 7 showed a dose-dependent increase in apoptotic cells (Figure 4C and Figure S4). In contrast, there was no significant increase in apoptotic cells in the STAT5independent control cell line MDA-MB-231 (Figure 4D and Figure S5). These data strongly indicate that the induction of apoptosis in K562 cells is caused by a STAT5-dependent effect. Given the high selectivity of 7 against STAT5b over STAT5a (Figure 3C−F) and the literature reports31,32 describing the crucial relevance of STAT5b, but not STAT5a, for proliferation and survival of Bcr-Abl expressing tumor cells, our data strongly suggest that the effects of 7 on K562 cells are mediated via the inhibition of STAT5b. Activity and specificity of 7 against the individual STAT5 proteins at the time point at which cell viability and apoptosis were analyzed (48 h) cannot be reliably tested using STAT5a/b-transfected cells because of the transient nature of the plasmid transfection, which would lead to a loss of the STAT5a/b expression plasmid in the course of the experiment. We attribute the higher concentrations required to achieve the effects in cell viability assays and in apoptosis assays to the longer time of exposure in these assays, which presents a larger challenge for the stability of the active agent against phosphatases, as indicated by the reduced activity of 7 already after 8 h of exposure (Figure S2).

Figure 2. (A) Selectivity profile of 6a in fluorescence polarization assays. (B) Binding mode of 6a to the STAT5b SH2 domain as proposed by AutoDock Vina.

phospho-specific antibodies that recognize STAT5b only when phosphorylated at Tyr699. However, these antibodies also recognize STAT5a when phosphorylated at Tyr694, since they are raised against a short peptide motif which is identical in both STAT5 proteins. To enable us to differentiate between the phosphorylated forms of the proteins, we designed fusion constructs between STAT5a or STAT5b and GFP.13 K562 cells, in which STAT5 proteins are constitutively activated by phosphorylation of STAT5b Tyr699 and STAT5a Tyr694 by Bcr-Abl, were transfected with plasmids encoding either STAT5a-GFP or STAT5b-GFP. The added molecular weight of the GFP allows for differentiation between endogenous STAT5, consisting of both STAT5a and STAT5b, and the respective transfected GFP-STAT5 protein. Treatment of STAT5b-GFP-transfected K562 cells with 7 led to a strong, dose-dependent decrease of STAT5b Tyr699 phosphorylation (Figure 3C, D). In contrast, phosphorylation of the STAT5aGFP fusion protein at STAT5a Tyr694 was reduced only to a minor extent (Figure 3E,F). This demonstrates that 6a is able to inhibit the phosphorylation of STAT5b in cells, and that specificity for STAT5b over STAT5a is maintained in the cellular environment to a major extent. Tyrosine phosphorylation of endogenous STAT5 was inhibited to a lesser extent than STAT5b-GFP and a higher extent than STAT5a-GFP (Figure 3G,H), reflecting the presence of both STAT5 proteins in K562 cells. Phosphates are prone to intracellular enzymatic cleavage by phosphatases. To investigate the stability of 7 against D


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Figure 3. Western blot analysis of compound 7 on K562 cells. (A) Synthesis of compound 7. (B) In K562 leukemia cells, STAT5a/b are constitutively phosphorylated on STAT5a Tyr694 and STAT5b Tyr699 by Bcr-Abl, leading to signal transduction via STAT5a/b. An inhibitor of the SH2 domain (symbolized by the triangle) prevents STAT5 tyrosine phosphorylation and subsequent signal transduction. (C) 7 inhibits phosphorylation of STAT5b-GFP in K562 cells in a dose-dependent manner. (D) Quantification of the pSTAT5b-GFP bands from C, normalized against total STAT5b-GFP. Error bars represent the standard deviations from two independent experiments. (E) 7 has only a minor effect on tyrosine phosphorylation of STAT5a-GFP in K562 cells. (F) Quantification of the pSTAT5a-GFP bands, normalized against total STAT5a-GFP. Error bars represent the standard deviations from two independent experiments. (G) Effect of 7 on endogenous STAT5a/b in K562 cells. (H) Quantification of the endogenous pSTAT5 bands, normalized against total endogenous STAT5. Error bars represent the standard deviations from four independent experiments.

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CONCLUSIONS In summary, we have demonstrated that O-phosphorylation of the widely abundant natural products dihydrocapsaicin and Nvanillylnonanamide, or simple derivatives thereof, creates chemical entities with activity against STAT5a/b. The bisphosphates 6a/b based on the demethylated compounds 4a/b are nanomolar inhibitors of the STAT5b SH2 domain and display a high degree of selectivity over the highly homologous SH2 domain of STAT5a. Conversion of the most potent compound 6a, dubbed Capstafin (for “capsaicinoid-based STAT f ive b inhibitor”), to its pivaloyloxymethylester resulted in compound 7, which caused selective inhibition of STAT5b phosphorylation, STAT5-dependent reduction in cell viability, and a STAT5-dependent increased rate of apoptosis in human

tumor cells. Our data validate O-phosphorylation of appropriately preselected natural products or derivatives thereof as a semirational design approach for small molecules that selectively inhibit phosphorylation-dependent protein−protein interaction domains in cultured human tumor cells. On a wider note, since the discussed chemical transformations involved in the conversion of the capsaicinoids (demethylation and phosphorylation) could also be carried out by plant enzymes, our study might stimulate natural product chemists to attempt the isolation of phosphorylated and thus water-soluble natural products, which may well be overlooked using current natural product isolation protocols.33 E


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Figure 4. Compound 7 inhibits cell viability and increases the rate of apoptosis in tumor cells in a STAT5-dependent manner. (A) Effect of 7 on cell viability on STAT5-dependent K562 cells and (B) on STAT5-independent MDA-MB-231 cells. Cells were treated for 48 h. (C) Effect of 7 on the rate of apoptosis in STAT5-dependent K562 cells and (D) in STAT5-independent MDA-MB-231 cells. Cells were treated for 48 h. Error bars depict the standard deviations from three independent experiments.

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METHODS

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ASSOCIATED CONTENT

service group (L. Hennig) and the mass spectrometry group (C. Birkemeyer) of the Faculty of Chemistry and Mineralogy at Leipzig University. This work was supported by the Core Unit Fluorescence-Technologies of the Interdisciplinary Centre for Clinical Research (IZKF) Leipzig, at the Faculty of Medicine of the University of Leipzig (K. Jäger and A. Lösche).

Details on chemical synthesis, spectroscopic characterization, plasmid construction, and assay design are summarized in the Supporting Information. S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00817. Table S1, Figures S1−S5, plasmid construction and protein expression, fluorescence polarization assays, cell culture, Western blots, cell viability assay, apoptosis assay, synthesis and characterization of compounds, supporting references, NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was generously supported by the Deutsche Forschungsgemeinschaft (BE 4572/4-1 and INST 268/281-1 FUGG). We extend our thanks to K. Natarajan for experimental support. We extend out thanks to the NMR F


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Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent

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