Guttiferone A Aggregates Modulate Silent ... - ACS Publications

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Guttiferone A Aggregates Modulate Silent Information Regulator 1 (SIRT1) Activity Kévin Cottet,† Bin Xu,‡,⊥ Pascale Coric,§ Serge Bouaziz,§ Sylvie Michel,† Michel Vidal,‡,∥ Marie-Christine Lallemand,† and Sylvain Broussy*,‡ †

Laboratoire Pharmacognosie, Chimie des Substances Naturelles, Electrochimie UMR COMETE 8638 CNRS, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, 4 Avenue de l’Observatoire, Paris 75006 France ‡ Laboratoire Hétérocycles et Peptides: Approche Ciblée, Cancer et Angiogenèse UMR COMETE 8638 CNRS, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, 4 Avenue de l’Observatoire, Paris 75006 France § Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, 4 Avenue de l’Observatoire, Paris 75006 France ∥ UF Pharmacocinétique et Pharmacochimie, Hôpital Cochin, AP-HP, 27 Rue du Faubourg Saint Jacques, Paris 75014, France S Supporting Information *

ABSTRACT: Natural products guttiferone A, hyperforin, and aristoforin were able to inhibit or increase SIRT1 catalytic activity, depending on protein concentration and presence of detergent. On the basis of NMR data for guttiferone A, we demonstrated that the aggregation state of the natural product played a crucial role for its interaction with the enzyme. These results are useful to interpret future in vitro structure−activity relationship studies on these natural products in the quest of their biological target(s).

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INTRODUCTION In vitro enzymatic assays are usually performed under simple conditions, leading to a high false-positive rate of about 2% when compounds are screened at concentrations in the micromolar range.1 One common mechanism explaining such false-positive hits is the congregation of organic molecules into colloidal aggregates, which nonspecifically inhibit enzymes.2−7 Several methodologies have been developed to detect aggregates such as DLS1,2 and NMR spectroscopies.8,9 Typically, aggregate-induced inhibition is expected to be partially or completely reversed in the presence of detergent. Thus, the use of detergents is recommended to identify “promiscuous” inhibition of enzymes by aggregates.1,10−12 Natural products and especially phenolic compounds are known as aggregating molecules.13 With this in mind, we have investigated the effect on their reported enzyme targets of some polycyclic polyprenylated acylphloroglucinol (PPAPs). This class of natural products was mainly isolated from plants and trees of the Clusiaceae family (Guttiferae)14 and display an unusually broad range of biological activities.15 However, the molecular targets of PPAPs are often unknown or are still awaiting validation. In this respect, we were interested when the © 2016 American Chemical Society

PPAP guttiferone G (1, Chart 1) was described as an inhibitor of the NAD+-dependent deacetylase enzymes SIRT1 and SIRT2 (human sirtuins). Reported IC50 values were 9 μM for SIRT1 and 22 μM for SIRT2, employing a radioactivity-based deacetylation assay.16 An antiproliferative effect of the natural products on HUVE cells was also observed, and the authors proposed the direct involvement of SIRT1. In another recent study, a series of synthetic PPAPs were evaluated,17 and for the most potent compound (JQ-101, Chart 1), IC50 values of 30 μM for SIRT1 and 150 μM for SIRT2 were measured by a fluorescence-based assay. This PPAP compound was able to induce tumor cell apoptosis and senescence on several cancer cell lines and to suppress cancer cell invasion in a SIRT1dependent manner.17 SIRT1 constitutes a therapeutically relevant target for the treatment of several diseases, including some cancers.18,19 In addition to the above-mentioned PPAPs, several inhibitors have demonstrated cytotoxicity on cancer cell lines and antitumor activity on preclinical models.20 The development of potent and selective inhibitors of SIRT1 is Received: August 11, 2016 Published: September 27, 2016 9560

DOI: 10.1021/acs.jmedchem.6b01182 J. Med. Chem. 2016, 59, 9560−9566

Journal of Medicinal Chemistry

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SIRT1 batches and buffer compositions. Moreover, we did not detect any inhibition at 32 μM, suggesting a steep decrease in inhibitory activity as the inhibitor concentration decreases. Therefore, we decided to identify the reaction parameter(s) involved in this unexpected behavior. For simplicity, the following assay conditions were chosen: (i) a unique batch of SIRT1 enzyme (from Sigma) was employed in the entire study, (ii) the HPLC assay was preferred over the fluorescence-based assay because it is less prone to artifact, and (iii) a buffer free of any additive was used for initial experiments (TrisHCl/Tris, pH 8.0, 50 mM). We first measured the effect of the concentration of SIRT1 on its inhibition by 100 μM of 2, 3, and 4. We did not detect any inhibition with 250 nM of enzyme. However, some inhibition was obtained at 50 nM and a strong inhibition (about 90% for 2) at 25 nM (Figure 1A for 2 and 4, and

Chart 1. PPAPs and Control Compounds Studied for Their Modulation of SIRT1 Activity: Guttiferone G (1), Guttiferone A (2), Hyperforin (3), Aristoforin (4), JQ-101, Suramin, and Resveratrola

a

The absolute stereochemistry of 1 is tentative.

therefore a validated approach toward potential anticancer therapy. In the continuation of our program for the discovery of new biologically significant metabolites from medicinal plants, we initiated metabolomics studies of Symphonia globulifera L.f. (Clusiaceae). The comparative LC-MS based metabolite profiling study led to the identification of tissue-specific compounds.21 The results showed that renewable organs/ tissues could be used as starting material for the production of PPAPs, thereby reducing the impact on the biodiversity. We also succeeded in isolating the guttiferone A (2, Chart 1) from the latex in large quantity, permitting its use in biological research.22,23 Therefore, we decided to test the guttiferone A (2), structurally close to the guttiferone G, on SIRT1, and to carry out subsequent structure−activity relationship studies. Other PPAPs such as hyperforin (3) and its derivative aristoforin (4, Chart 1) were also included in the study to serve as positive control. Importantly, their reported IC50 values for SIRT1 and SIRT2 inhibition are in the same range as those of guttiferone G (1).16

Figure 1. Percent SIRT1 activity in the presence of indicated compound compared to activity under the same assay conditions in the absence of compound. TX−, absence of Triton X-100; TX+, presence of 0.01% Triton X-100 (v:v). Error bars are SD from the mean of 3−8 experiments. See the Experimental Section for details.

Supporting Information (SI), Figure S1 for 3). Such dependence of the inhibition on enzyme concentration can occur when the K d of the inhibitor is lower than the enzyme concentration.4,25 Here, because the inhibitor is present in much larger molar concentration than the enzyme, the most plausible hypothesis is the interaction of a large number of inhibitor molecules, likely as aggregates, with enzyme molecule(s).2,4 In any case, this first series of experiments ruled out a simple “one inhibitor/one enzyme” type interaction. Unexpectedly, the addition of a widely used detergent, Triton X-100 (TX-100, 0.01%, 170 μM), resulted in SIRT1 activation with diluted enzyme. Similar trends were obtained for 2, 3, and 4 (Figure 1A for 2 and 4, and SI, Figure S1 for 3). A nearly 2fold activation was achieved with 100 μM of 2, and the activating effect was still detectable at low micromolar concentration (SI, Figure S2). TX-100 alone (0.01%) had a weak inhibitory effect (SI, Figure S3). To evaluate the generality of this phenomenon, we assayed other commonly used detergents, either alone (SI, Figure S4) or in combination with 2 (SI, Figure S5). Interestingly, the effect of Tween 20

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RESULTS AND DISCUSSION Enzyme Inhibition Assays. SIRT1 activity was measured by two methods. The first one employed a commercially available fluorogenic substrate (Sigma).24 The second one was based on UV detection by HPLC with the substrate RHKK(Ac)W-NH2. For all tested compounds, including 3 and 4, nonreproducible inhibitions were observed at a concentration of 100 μM. The results were dependent on 9561



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resulted in a strong increase in scattering intensity (166 ± 2 kcps), along with well-formed autocorrelation curves, indicating the formation of large aggregates (Supporting Information). In that case, the size calculated by the software from autocorrelation curves was 120 nm. Apart from the last experiment, we believe that the results from DLS measurement are difficult to interpret because of the high polydispersity and low overall scattering intensities. Therefore, to confirm these observations by another spectroscopic method, we decided to use a recently developed NMR assay, detecting the formation of aggregates using dilution experiments.8,9 The NMR assay consists in recording a series of 1H NMR spectra over a suitable concentration range, typically about 10− 200 μM. The presence of both insoluble and soluble aggregates (from compound “self-aggregation”) can be detected by unusual spectral features, for example, broadening of the signals, changes in chemical shifts, or unexpected changes in signal intensities. When assayed alone in solution in deuterated phosphate buffer (50 mM, pH 8.0, 1% DMSO-d6), 2, and suramin were devoid of any unusual spectral feature at concentrations up to 100 μM (SI, Figure S16). The effect of TX-100 was then tested (Figure 2A). In the presence of TX100 (0.01%, 170 μM), a large broadening of the signals of a 200 μM solution of 2 occurred, confirming the formation of aggregates with the detergent (Figure 2A, top). To determine the dependence of aggregate formation as a function of TX-100 concentration, we carried out an additional series of experiments with 100 μM 2 and 0.001% or 0.01% TX-100. At 0.001% TX-100 (17 μM, corresponding to about a 6-fold deficit compared to the concentration of 2), we observed a decrease in signal intensities, which we attributed to an intermediate exchange mode between 2 and the detergent combined with a slight aggregation. At the highest detergent concentration (0.01%), the large broadening observed demonstrated that 2 and TX-100 coaggregated (SI, Figure S17, and accompanying discussion). These coaggregates are apparently able to interact with and stabilize SIRT1. In control experiments with suramin, the addition of TX-100 (0.01%, 170 μM) did not induce any significant effect on the NMR spectra (Figure 2A, bottom). To complete the study, we evaluated by NMR the interaction between 2 and the detergents Tween 20 and CHAPS. The addition of 0.01% Tween 20 to a 100 μM solution of 2 had an aggregating effect similar to that of TX-100 (SI, Figure S19). On the contrary, the addition of 0.01% CHAPS had no effect (SI, Figure S20). Tween 20 interacted with 2, forming aggregates that activated SIRT1, whereas CHAPS did not form aggregates with 2 and did not prevent SIRT1 inhibition by 2 (see above and SI, Figure S5). In the absence of detergent, we hypothesized that the natural product could form aggregates upon interaction with the protein. When large aggregates interact with their protein target, micromolar concentrations of compound bind to nanomolar concentrations of protein. Therefore, we thought that the NMR assay could be conveniently extended to in situ aggregation by addition of nanomolar concentrations of protein. Thus, a series of spectra was recorded while adding SIRT1 to a 100 μM solution of 2 directly in the NMR tube (Figure 2B, top). Interestingly, the intensity of all 1H resonances of 2 decreased when only 25 nM enzyme were added. Importantly, this effect was already visible when the natural product concentration was 4000 times higher than the enzyme concentration. The maximum enzyme concentration added was 100 nM because the addition of glycerol present in

(0.01%), another nonionic detergent, was similar to the effect of TX-100, whereas CHAPS (0.01%), an ionic detergent, had no effect. Glycerol contained in commercial sources of SIRT1 might have biased the results due to changes in viscosity of the reaction mixtures. However, we did not detect any effect of 0.4% glycerol, the highest percentage used throughout the present study, on SIRT1 activity modulation by 2 (SI, Figure S6). As additional control experiments, we assayed suramin, a known inhibitor, and resveratrol, a known activator (structures are in Chart 1).26,27 As expected, SIRT1 inhibition by suramin was almost unaffected by reaction conditions (Figure 1B). Resveratrol appeared as a weak inhibitor with diluted SIRT1. Under such experimental conditions, resveratrol could not activate SIRT1 because the substrate concentration (100 μM) was well above its KM value (3 μM).28 We verified that the same batch of resveratrol did activate SIRT1 under favorable conditions with the fluorogenic substrate assay.24 Therefore, it could be concluded that the activation observed with PPAPs in the presence of TX-100 did not involve a lowering of the KM value for substrate. We hypothesized that the presence of PPAPs and TX-100 caused enzyme stabilization, similar to the well-known effect of addition of proteins like bovine serum albumin (BSA) or human serum albumin (HSA).29,30 This effect was particularly pronounced under diluted conditions. Supporting this hypothesis, the addition of BSA (1 mg/mL, 15 μM) in the assay mixtures resulted in a loss of both the activation and inhibition potencies of 2 (SI, Figure S7), whereas it did not affect suramin inhibition. Therefore, the PPAPs binding ability for SIRT1 was not specific for the enzyme and could be reversed by BSA. Finally, we performed control experiments regarding the enzyme source and nature. First we verified that using SIRT1 enzyme purchased from another provider (Enzo Life Science) gave qualitatively the same results with 100 μM of 2: a very strong inhibition in the absence of detergent (1% remaining activity) and an activation in the presence of 0.01% TX-100 (158%, SI, Figure S8). Second, we chose a completely unrelated enzyme target. Interestingly, 2 was reported to inhibit the enzyme trypsin with an IC50 value of 9 μM.31 Therefore, we developed an HPLC-based assay to measure trypsin activity on the peptide substrate c[YKDEGLEE]-NH2. Trypsin activity inhibition by 100 μM of 2 was highly dependent on the enzyme concentration. Moreover, most of the inhibition observed at low enzyme concentration was suppressed by addition of 0.01% TX-100 (SI, Figure S9). Mechanistic Studies Using Dynamic Light Scattering (DLS) and NMR Spectroscopies. In the subsequent mechanistic investigations, we decided to focus our study on guttiferone A (2). We realized DLS measurements in order to explore the mechanism by which this compound inhibits and activates SIRT1. Buffer alone (TrisHCl/Tris, pH 8.0, 50 mM) gave a weak scattering intensity, lower than 10 kcps (kilocounts per second). When incubated in buffer, 100 μM of 2 induced a slight increase to 18 ± 2 kcps, which could indicate the marginal formation of colloidal aggregates. The proteins BSA (1 mg/mL, 15 μM) and SIRT1 (250 nM) alone gave 15 ± 4 and 9 ± 1 kcps, respectively. Interestingly, the combination of 2 with either BSA or SIRT1 gave higher scattering intensities: 32 ± 3 and 29 ± 2 kcps, respectively. These values should however be taken with caution, owing to the comparatively high intensities measured for 2 and for the proteins alone. Finally, addition of TX-100 (0.01% v:v) to a 100 μM solution of 2 9562



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SIRT1 was slightly stronger than with BSA. As a control, no spectral changes were observed for a 100 μM solution of suramin with the increase of BSA concentrations up to 1.5 μM (Figure 2C, bottom). Moreover, the addition of 100 nM SIRT1 and 1.5 μM BSA to a 0.01% solution of TX-100 had no effect on its NMR spectra. Therefore, no aggregation occurred between these proteins and the detergent in the absence of 2 (SI, Figure S24). We performed 2D diffusion-ordered spectroscopy (DOSY) NMR experiments32,33 to evaluate and compare the relative size of coaggregates of 2 formed in the presence of TX-100 and BSA. These species were characterized according to their diffusion coefficients, measured on residual resonances of 2. The diffusion coefficient measured for 2 alone at 100 μM (2.706 × 10−10 m2/s) served as a reference because at this concentration, the natural product did not significantly aggregate (SI, Table S2 and Figure S25). When the concentration of 2 was increased to 1 mM, the diffusion coefficient value decreased (2.246 × 10−10 m2/s), proving that at high concentration the natural product had a propensity to aggregate. When 0.001% TX-100 (17 μM) was added to a 100 μM solution of 2, the diffusion coefficient decreased to 2.298 × 10−10 m2/s. This value was comparable to the one measured for a solution of 2 alone at 1 mM. Therefore, even at low concentration, TX-100 was able to partially aggregate 2. Furthermore, the diffusion coefficient decreased to 1.808 × 10−10 m2/s for 2 at 100 μM in the presence of TX-100 at 0.01% (170 μM). This confirmed that the phenomenon of aggregation was intensified by increasing the concentration of the detergent. Similarly, the binding of 2 to BSA was experimentally verified with 100 μM 2 in the presence of 1.5 μM BSA. The low value of the diffusion coefficients (1.610 × 10−10 m2/s) showed unambiguously that 2 coaggregated with BSA. The aggregation of 2 seemed to be more important in the presence of 1.5 μM BSA than 170 μM TX-100 (SI, Table S2 and Figure S26).

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Figure 2. Representative aromatic and aliphatic regions of NMR spectra of 2 and suramin. (A) At 200 μM compound concentration, before (black) and after (blue) addition of TX-100 (0.01% v:v). (B) At 100 μM compound concentration, without SIRT1 (black) and with 25 nM (blue), 50 nM (green), and 100 nM (red) SIRT1. (C) At 100 μM compound, without BSA (black) and with 150 nM (blue) and 1.5 μM (green) BSA. *signal from TX-100. See the Experimental Section for details.

CONCLUSIONS Several conclusions can be drawn from this study regarding both the NMR assay method and the natural product guttiferone A (2) biochemical behavior. First, 2 alone in aqueous solution did not show unusual NMR spectral features at concentrations up to 100 μM. It indicated an absence of “self-aggregation” according to the previously described NMR assay.8,9 However, it was detected as “co-aggregator” in the presence of protein by the herein described in situ NMR assay, using either SIRT1 or BSA. Conveniently, this assay only required the addition of nanomolar concentrations of the protein of interest or BSA. It may be applied to other cases where an aggregation mechanism is suspected but not easily evidenced with the inhibitor alone. Consequently, it may constitute a complementary assay to identify non-self-aggregating compounds displaying a high off-target rate.9 Second, the activating effect of a combination of TX-100 and 2−4 on diluted SIRT1 was intriguing. Rare cases of promiscuous enzyme activation have been reported, mainly with detergents and detergent-like molecules.12,34−36 Therefore, the use of detergents has been recommended for both inhibition and activation enzyme screens.12,34,36 The case described herein is particular because the combination of two inhibitors (2, a strong inhibitor under diluted enzyme conditions, and TX-100, a weak inhibitor) resulted in enzyme activation through aggregate formation. It will be interesting to

the commercial SIRT1 solution prevented accurate NMR measurement beyond this limit. Under these conditions, a dose response effect was observed, demonstrating the interaction of many molecules of 2 with each molecule of enzyme (Figure 2B, top). As a control experiment, the addition of 0.2% glycerol (the maximum amount added in NMR experiments in the presence of SIRT1) had no effect on the spectra of a 100 μM solution of 2 (SI, Figure S21). In control experiments with suramin, the addition of SIRT1 did not induce any significant effect on the NMR spectra (Figure 2B, bottom). This is what was expected for the simple “one inhibitor/one enzyme” type interaction between these two partners. To study the interaction between 2 and BSA, the addition of BSA to a 100 μM solution of 2 was monitored by standard 1H NMR (Figure 2C). A small decrease in signal intensity was observed for 2 with 150 nM BSA and a large decrease with 1.5 μM (Figure 2C, top). Therefore, 2 was able to interact with BSA in a similar way to SIRT1. Qualitatively, from the NMR signal intensity, we could estimate that its interaction with 9563



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gave approximately 250 nM concentration of SIRT1. Dilutions of the SIRT1 stock solution (in TrisHCl/Tris, pH 8.0, 50 mM) were prepared directly before use. The reaction mixture was then incubated at 37 °C. The incubation time was chosen so that the conversion was strictly kept between 1% and 10%: 5−10 min for 250 nM SIRT1, 30 min for 50 nM SIRT1, and 1 h for 25 nM SIRT1. This allowed for accurate measurement by HPLC and ensured initial velocity conditions. Typically, the conversion of control experiments was adjusted at 2−4% for activation reactions and 4−6% for inhibition reactions. Reactions were stopped by addition of 50 μL of 4% TFA in water, and the resulting mixtures were analyzed by analytical HPLC. Analytical HPLC (Shimadzu Prominence) was carried out using a Phenomenex Luna C18 column (5 μm, 4.6 mm × 250 mm). Solvent A was water with 0.1% TFA, and solvent B was 70% acetonitrile aqueous solution with 0.09% TFA. The products were detected at 220 nm. The peptide substrate RHKK(Ac)W-NH2 and deacetylated product RHKKW-NH2 were separated with a linear gradient from 10 to 50% B in 20 min at a flow rate of 1 mL/min. Peak areas were integrated to calculate the percentage of conversion. Trypsin. Trypsin from bovine pancreas was purchased from Sigma (T8802), as an essentially salt free powder. Stock solutions were prepared by dissolving the powder (molecular weight of 24000 g/mol) in aqueous HCl 1 mM. Reactions were prepared and analyzed with the same procedure as for SIRT1, except that no NAD+ was added and the substrate c[YKDEGLEE]-NH2 (250 μM final concentration) was used. Dynamic Light Scattering Measurements. DLS data were recorded on a DSC125 instrument (Malvern) with the software Zetasizer. Stock solutions of 2 were prepared in DMSO at a concentration of 10 mM. Then 100 μM solutions of 2 were obtained by addition of 5 μL of stock in a final volume of 500 μL with TrisHCl/ Tris, pH 8.0, 50 mM, giving a final concentration of 1% DMSO. When required, TX-100, BSA, and SIRT1 were added from stock solutions at 1% in water, 100 mg/mL and 6 μM, respectively. Kilocounts per second (kcps) values are given as means of at least triplicate measurements, accompanied by standard deviations. NMR Assay. The NMR assay of 2 and suramin followed the previously reported procedure, with compound concentrations of 200, 100, 50, and 25 μM and a final concentration of 1% DMSO.8 Deuterated phosphate buffer (50 mM, pH 8.0) was used for all experiments. TX-100 was added from a freshly prepared solution at 1% in D2O. BSA was added from stock solutions at 50 and 5 mg/mL in D2O. SIRT1 was added from a commercial stock solution at 6 μM (see above). All spectra were recorded at 298 K on a Bruker 400 MHz spectrometer (1H frequency) or at 293 K on a Bruker 600 MHz spectrometer (1H frequency) equipped with a cryoprobe. Spectra were calibrated relative to residual solvent signals or according to tetramethylsilane (TMS) chemical shift that was set to zero ppm. Solvent peak suppression was achieved using excitation sculpting with gradients.43 At 400 MHz, spectra were recorded with 1000−2000 scans, a relaxation delay of 1 s and an acquisition time of 0.4 or 1 s. At 600 MHz, spectra were recorded with a spectral width of 6009 Hz, 128 scans, a relaxation delay of 1.0 s, and an acquisition time of 1.36 s over 16 k points. All parameters, including the receiver gain and number of scans, were kept constant within each set of experiments. Diffusion-ordered spectroscopy (DOSY) experiments were recorded on a 600 MHz spectrometer using a pulse sequence program for diffusion measurement using stimulated echo, bipolar gradients pulses for diffusion and spoil gradients.44 For each DOSY experiment, 32 experiments have been performed in F1 with 32 scans, a relaxation delay of 1 s, an acquisition time of 0.68 s, a diffusion time of 50 ms (Δ), and a gradient pulse of 2.2 ms (δ) over 8192 points and a spectral width of 6009 Hz. NMR spectra were processed with the software Topspin 3.5.

characterize further these aggregates. They should be different from inhibitory aggregates of 2 formed in the presence of SIRT1 alone. Finally, this study raises questions concerning the PPAPs selectivity for their reported protein targets and the interpretation of results provided by structure−activity relationship studies. Some tools are available to identify false positive hits. Early on in the validation process, any hits obtained should be evaluated with computational filters such as Aggregator Advisor37 and PAINS.38 With the first filter, 2 was not identified as a known aggregator, but a very high Log P value of 8.3 was calculated and the catechol group was flagged as potentially troublesome in PAINS filter. 1, 3, and 4 showed a low selectivity for SIRT1 over SIRT2.16 2 was reported to inhibit four different proteases, including trypsin, with comparable potency (IC50 values from 2 to 9 μM).31 In these biochemical assays on 1−4, no BSA was used. We demonstrate here with SIRT1, trypsin, and BSA that the total protein content in the test tube is crucial rather than enzyme units. Promiscuous inhibition will likely occur with other enzymes, if assayed at low concentrations. Therefore, we recommend the use of excess BSA (for example 1 mg/mL, 15 μM, for 100 μM compound) in biochemical assays involving 2−4 and possibly other PPAPs. If the biologically relevant protein target(s) have a high affinity for the natural product, they should displace bound BSA. Only the identification of such affine protein targets will lead to meaningful structure−activity relationship studies.

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EXPERIMENTAL SECTION

Natural Products Extraction, Synthesis, And Purification. Guttiferone A (2) was extracted from Symphonia globulifera extract following a reported procedure.22 Hyperforin (3) was extracted from a petroleum ether extract of Hypericum perforatum following a reported procedure.39 Aristoforin (4) was prepared from 3 following a reported procedure.40 All three compounds gave NMR, αD, and mass spectroscopy data matching literature data.39−41 Compound purity was determined by HPLC-UV (at 220 and 254 nm). All compounds were ≥95% pure. Substrates Synthesis. The SIRT1 substrate RHKK(Ac)W-NH2 was synthesized by standard Fmoc solid phase synthesis (Liberty 1 CEM automatic peptide synthesizer) and purified by reversed phase C18 semipreparative HPLC (Shimadzu Prominence system). The identity of the product was confirmed by MALDI mass spectrometry: expected m/z 795.46 (mono), observed 795.86 [M + H]+. The trypsin substrate c[YKDEGLEE]-NH 2 was synthesized as previously described42 and characterized by MALDI mass spectrometry: expected m/z 963.44 (mono), observed 963.69 [M + H]+. Enzyme Activity Assays. SIRT1. SIRT1 was purchased from Sigma (S8446 batch 092M4102). The stock solution nominal concentration was 480 μg protein/mL. With a molecular weight of 81681 g/mol, the concentration was approximately 6 μM. The buffer contained 10% glycerol and no BSA. Suramin sodium salt, TX-100, and BSA (fraction V) were purchased from Sigma. Stock solutions of TX-100 (0.1% v:v in water) were prepared daily. Reactions mixtures were prepared in 0.5 mL Eppendorf vials (protein low binding quality) with a final volume of 50 μL. The reaction mixtures contained buffer (TrisHCl/Tris, pH 8.0, 50 mM), NAD+ (200 μM), peptide substrate RHKK(Ac)W-NH2 (100 μM), and additive and compound if required (added from stock solutions 10 times more concentrated). The compounds were prepared as stock solutions with 10% DMSO in water so that the final DMSO concentration was 1% in the reaction mixture. In control experiments without compound, the final DMSO concentration was kept at 1% by addition of a solution of 10% DMSO in water. The reaction was started by addition of SIRT1, without preincubation. Addition of 2 μL of stock SIRT1 in 50 μL final volume 9564



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aggregation-based inhibition in a large compound library. J. Med. Chem. 2007, 50, 2385−2390. (13) Pohjala, L.; Tammela, P. Aggregating behavior of phenolic compounds  a source of false bioassay results? Molecules 2012, 17, 10774−10790. (14) From the species Symphonia globulifera, see, for example: Fromentin, Y.; Cottet, K.; Kritsanida, M.; Michel, S.; Gaboriaud-Kolar, N.; Lallemand, M.-C. Symphonia globulifera, a widespread source of complex metabolites with potent biological activities. Planta Med. 2015, 81, 95−107. (15) Ciochina, R.; Grossman, R. B. Polycyclic polyprenylated acylphloroglucinols. Chem. Rev. 2006, 106, 3963−3986. (16) Gey, C.; Kyrylenko, S.; Hennig, L.; Nguyen, L.-H. D.; Büttner, A.; Pham, H. D.; Giannis, A. Phloroglucinol derivatives guttiferone G, aristoforin, and hyperforin: inhibitors of human sirtuins SIRT1 and SIRT2. Angew. Chem., Int. Ed. 2007, 46, 5219−5222. (17) Zhu, L.; Qi, J.; Chiao, C. Y.-C.; Zhang, Q.; Porco, J. A., Jr.; Faller, D. V.; Dai, Y. Identification of a novel polyprenylated acylphloroglucinol-derived SIRT1 inhibitor with cancer-specific antiproliferative and invasion-suppressing activities. Int. J. Oncol. 2014, 45, 2128−2136. (18) Sebastián, C.; Satterstrom, F. K.; Haigis, M. C.; Mostoslavsky, R. From sirtuin biology to human diseases: An update. J. Biol. Chem. 2012, 287, 42444−42452. (19) Jing, H.; Lin, H. Sirtuins in epigenetic regulation. Chem. Rev. 2015, 115, 2350−2375. (20) Blum, C. A.; Ellis, J. L.; Loh, C.; Ng, P. Y.; Perni, R. B.; Stein, R. L. SIRT1 modulation as a novel approach to the treatment of diseases of aging. J. Med. Chem. 2011, 54, 417−432. (21) Cottet, K.; Genta-Jouve, G.; Fromentin, Y.; Odonne, G.; Duplais, C.; Laprévote, O.; Michel, S.; Lallemand, M.-C. Comparative LC−MS-based metabolite profiling of the ancient tropical rainforest tree Symphonia globulifera. Phytochemistry 2014, 108, 102−108. (22) Cottet, K.; Fromentin, Y.; Kritsanida, M.; Grougnet, R.; Odonne, G.; Duplais, C.; Michel, S.; Lallemand, M.-C. Isolation of guttiferones from renewable parts of Symphonia globulifera by centrifugal partition chromatography. Planta Med. 2015, 81, 1604− 1608. (23) Fromentin, Y.; Gaboriaud-Kolar, N.; Lenta, B. N.; Wansi, J. D.; Buisson, D.; Mouray, E.; Grellier, P.; Loiseau, P. M.; Lallemand, M.-C.; Michel, S. Synthesis of novel guttiferone A derivatives: In-vitro evaluation toward Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani. Eur. J. Med. Chem. 2013, 65, 284−294. (24) Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem. Biol. 2003, 10, 61−68. (25) Straus, O. H.; Goldstein, A. Zone behavior of enzymes: illustrated by the effect of dissociation constant and dilution on the system cholinesterase-physostigmine. J. Gen. Physiol. 1943, 26, 559− 585. (26) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L.-L.; Scherer, B.; Sinclair, D. A. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425, 191−196. (27) Trapp, J.; Meier, R.; Hongwiset, D.; Kassack, M. U.; Sippl, W.; Jung, M. Structure−activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins). ChemMedChem 2007, 2, 1419−1431. (28) Hubbard, B. P.; Gomes, A. P.; Dai, H.; Li, J.; Case, A. W.; Considine, T.; Riera, T. V.; Lee, J. E.; Yen, E. S.; Lamming, D. W.; Pentelute, B. L.; Schuman, E. R.; Stevens, L. A.; Ling, A. J. Y.; Armour, S. M.; Michan, S.; Zhao, H.; Jiang, Y.; Sweitzer, S. M.; Blum, C. A.; Disch, J. S.; Ng, P. Y.; Howitz, K. T.; Rolo, A. P.; Hamuro, Y.; Moss, J.; Perni, R. B.; Ellis, J. L.; Vlasuk, G. P.; Sinclair, D. A. Evidence for a common mechanism of SIRT1 regulation by allosteric activators. Science 2013, 339, 1216−1219. (29) Ohtake, S.; Kita, Y.; Arakawa, T. Interactions of formulation excipients with proteins in solution and in the dried state. Adv. Drug Delivery Rev. 2011, 63, 1053−1073.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01182. Additional SIRT1 activity assay results, trypsin activity assay results, additional NMR data, DLS data, and representative HPLC chromatograms (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (33) 153731564. E-mail: sylvain.broussy@ parisdescartes.fr. Present Address ⊥

School of Pharmacy, Shanghai University of TCM, 1200 Cailun Road, Shanghai 201203, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. C. Charrueau is acknowledged for a gift of resveratrol. Drs. C. Richard and D. Scherman are acknowledged for the use of the DLS instrument. Financial support from the Université Paris Descartes, Sorbonne Paris Cité (grant to K.C.) and the French CNRS (“Chaire de Partenariat CNRS” to S.Br.) is acknowledged.

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REFERENCES

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Guttiferone A Aggregates Modulate Silent ... - ACS Publications

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