Noncovalent Fluorescent Probes of Human Immuno- and Constitutive

Brief Article pubs.acs.org/jmc

Noncovalent Fluorescent Probes of Human Immuno- and Constitutive Proteasomes Audrey Desvergne,†,‡ Yan Cheng,†,‡ Sophie Grosay-Gaudrel,§,∥ Xavier Maréchal,†,‡ Michèle Reboud-Ravaux,*,†,‡ Emilie Genin,*,§,∥ and Joel̈ le Vidal*,§,∥ †

Sorbonne Universités, UPMC Univ Paris 06, UMR 8256, Biological Adaptation and Ageing (B2A), Integrated Cellular Ageing and Inflammation, 7 Quai St Bernard, 75005 Paris, France ‡ CNRS, UMR 8256, Biological Adaptation and Ageing (B2A), 75005 Paris, France § Université de Rennes 1, CPM, Bâtiment 10A, Campus de Beaulieu, 35042 Rennes Cedex, France ∥ CNRS, UMR 6510, Chimie et Photonique Moléculaires, 35042 Rennes, France S Supporting Information *

ABSTRACT: We report here the synthesis and biological evaluation of fluorescent probes functioning as inhibitors that noncovalently block human immuno- and constitutive proteasomes. These cell-penetrating linear analogues of the natural cyclopeptide TMC-95A were efficient on cells at the nanomolar level and assessed by confocal microscopy and flow cytometry. They may constitute an alternative to previously reported fluorescent probes that all bind covalently to proteasomes.

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INTRODUCTION

with bortezomib and carfilzomib are needed because clinical studies revealed severe drawbacks, resistance, and failure in solid tumors.11 Recently, it was shown that the α-ketoamide motif is a peptide C-terminus electrophilic motif, leading to reversible inhibitors conversely to most of covalent inhibitors.12 Neglected during a long period, noncovalent inhibitors introduce today a plausible alternative mechanism of inhibition to that of covalent inhibitors.8,13 Inhibitor noncovalency favors deep solid tissue penetration, and noncovalent inhibitors can be more specific, stable, and less reactive than the covalent ones. Among the first reported noncovalent inhibitors are found the natural cyclopeptide 1 (TMC-95A)14 and its synthetic cyclic15−17 and simpler linear mimics18,19 such as 2a,b and 3 (Figure 1). The latter molecules have been dimerized by linking two inhibitory heads by PEG spacers20 then by polyaminohexanoic (Ahx) ones21 to produce highly efficient and selective noncovalent inhibitors 4 that simultaneously inhibited the chymotrypsin-like (ChT-L) and trypsin-like (T-L) activities of human constitutive 20S proteasome (cPR) (Figure 1). Other peptide and pseudopeptide noncovalent inhibitors were identified by in vitro screening of collections of peptides22−25 as well as of that of organic inhibitors like hydroxyureas.26 In silico screening of a large collection of organic compounds led to structurally distinct noncovalent inhibitors such as

The ubiquitin−proteasome system plays a pivotal role in protein homeostasis by degrading key regulatory proteins as well as misfolded and damaged ones.1 Extensive studies on constitutive proteasome revealed its implication in many cellular processes such as cell-cycle control, differentiation, transcription, apoptosis, and immune response. The inducible immunoproteasome generates optimal peptidic antigens but has also other cellular roles that remain to be better understood.2−4 Activity-based probes have been developed to facilitate functional studies of active proteasomes in complex biological samples.5−7 All probes are composed of an inhibitory molecule linked to a fluorescent dye (DANSYL, rhodamine, or BODIPY) and covalently bind, selectively or not, to the proteasome active site(s). Such probes are essential to establish procedures for in vivo proteasome activity profiling and imaging, but their covalent and irreversible mechanism of action may be a disadvantage to assess the cross reactivity of novel noncovalent inhibitors7 because covalent probes may eventually displace all noncovalent inhibitors. Today, the catalytic particle 20S proteasome is a central drug target in pharmaceutical research.2,8−10 The peptide boronate bortezomib and the peptide α′,β′-epoxyketone carfilzomib are approved cancer drugs that react covalently with proteolytic sites of the 20S proteasome core particle as most of all the second-generation proteasome inhibitors in current clinical studies.8−10 Alternative proteasome inhibitors in comparison © XXXX American Chemical Society

Received: July 27, 2014

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dx.doi.org/10.1021/jm5011429 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structures of the studied molecules: compound 1; reference linear mimics of 1 (2a, 2b, 3); bivalent inhibitor (4). Fluorescent molecules: inhibitory head linked to fluorescein (5), directly to Tamra (6), or by one (6b) or three (6c) Ahx; Tamra linked to one Ahx (7).

sulfonamides27 and 1,2,4-oxadiazoles with an unusual mechanism of inhibition for the latter.28 The potentiality of homodimerized linear mimics 4 as potent and selective inhibitors of cPR due to the simultaneous inhibition of two types of activities at the nanomolar level prompted us to study the cellular penetration of the tripeptidic inhibitory head 2b. In that aim, we designed fluorescent probes formed by the inhibitory head linked to fluorescein (5) or tetramethylrhodamine (Tamra) (6a). On the basis of crystallographic data obtained with 2a,b and 3 (Supporting Information (SI), Figure 1S),19,21 compounds 6b and 6c in which Tamra was linked to the inhibitory head by one or three Ahx were also designed and synthesized in order to mimic the conjugated monomer 3 and dimeric inhibitors 4 we previously described.21 This report describes the synthesis of the fluorescent probes 5 and 6a−c, their in vitro and in cellulo evaluation on the three activities of cPR and inducible human 20S immunoproteasome (iPR), and their cellular penetration by flow cytometry analysis (FACS) and confocal microscopy in experimental conditions where no cellular toxicity was observed.

spacers in compounds 6b,c. The syntheses of 5, 6a−c, and reference compound 7 (Tamra linked to Ahx) started from 5carboxyfluorescein 831 or 5-carboxy Tamra 9,32 both efficiently prepared as pure isomers according to literature (Scheme 1). Scheme 1. Synthesis of Compounds 5, 6a−c, and 7a

a Reagents and conditions: (a) 2b, EDC, HOBt, Et3N, DMF, RT, (18− 32% yield); (b) 10b, EDC, HOBt, Et3N, DMF, RT, (65% yield); (c) LiOH, H2O/THF, 0 °C to rt, (57−58% yield); (d) 10c, EDC, HOBt, Et3N, DMF, rt, (36% yield).

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RESULTS AND DISCUSSION The design of the fluorescent probes was based on both kinetics and crystallographic data of the noncovalent inhibitors 2a,b and 3 in complex with yeast cPR (SI, Figure 1S).19,21 As a bulky and lipophilic R group was shown to be favorable for the inhibitory power, we chose to conjugate the fluorescent dye to the N-terminal amino group of 2b. Among the diversity of fluorescent probes used to label biomolecules,29 the popular fluorescein and rhodamine dyes were selected because their absorption and emission spectra do not interfere with those of the fluorophores enzymatically released from the proteasome fluorogenic substrates.30 As the compatibility of rhodamine fluorescence properties was better, we chose to introduce Ahx

The carboxylic acid functions of 8 or 9 were activated by the water-soluble carbodiimide EDC in the presence of hydroxybenzotriazole (HOBt) and reacted with tripeptide 2b19 to give 5 or 6a in 22−32% yields. Their coupling to Ahx spacers 10b,c,21 followed by introduction of tripeptide 2b, led to 6b and 6c. Target compounds 5 and 6 were characterized by NMR spectroscopy, high resolution mass spectrometry, and HPLC. UV−visible spectroscopic characteristics in water for respective maximum absorption and emission wavelengths were: 495 and 525 nm (fluorescein compound 5), 553−556 nm, and 584−585 B



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Table 1. Inhibition Profiles of Purified Human cPR and iPR at pH 8 and 37 °Ca cPR, IC50 (μM)b compd 2a 5 6a 6b 6c

ChT-L 3.2 2.59 0.43 0.48 0.49

± ± ± ± ±

0.1 0.13 0.02 0.03 0.03

PA 11.5 9.52 1.08 0.99 1.37

± ± ± ± ±

iPR, IC50 (μM)b T-L

0.5 0.41 0.08 0.04 0.06

7.1 1.93 1.63 1.24 1.28

± ± ± ± ±

ChT-L

0.2 0.05 0.07 0.02 0.02

2.53 1.49 0.42 0.56 0.67

± ± ± ± ±

PA

0.07 0,07 0.02 0.02 0.03

6.16 7.06 0.83 0.64 0.74

± ± ± ± ±

T-L 0.18 0.31 0.04 0.03 0.03

27.7 2.43 3.53 1.75 1.73

± ± ± ± ±

2.5 0.07 0.06 0.07 0.08

The inhibition was evaluated after 15 min incubation of the enzyme with the respective compound before adding the appropriate fluorogenic substrate. bThe IC50 values were calculated by fitting the experimental initial rate data to eq 1 or eq 2. They are averages of a minimum of two independent experiments, each as a technical triplicate. a

nm (Tamra derivatives 6a−c). Fluorescence quantum yields were good (0.3−0.9). The ability of compounds 5, 6a−c, and 7 to inhibit human cPR and iPR was determined in vitro using purified enzymes and the appropriate fluorogenic substrate for ChT-L, T-L, and PA activities. The rates of hydrolysis were monitored by fluorescence increase at 37 °C over 30 min. The initial rates were determined using the linear portion of the curves and the IC50 values calculated using either eq 1 or eq 2 (Table 1, SI, Figure 2S, ). The Tamra probe 7 was devoid of any significant inhibitory effect against cPR and iPR. The reference inhibitory head 2a inhibited the three activities of both cPR and iPR. The ChT-L activities were equally inhibited, whereas PA activity of cPR was poorly inhibited compared to that of iPR (factor of 1.9) and the opposite was observed for T-L (factor of 3.9). Except for the T-L activities that were better inhibited, the other inhibitions were poorly affected when the fluorescein dye had been linked to the inhibitory head (compound 5). Conversely, the Tamra group greatly increased the inhibitory power: factors of 6.6 for ChT-L, 11.6 for PA, and 2.5 for T-L (cPR), and 6, 7.4, and 7.8 for iPR, respectively. Then Ahx spacers (n = 1 or n = 3) were introduced between the Tamra group and the inhibitory head to give compounds 6b−c. Their presence did not affect the inhibitory potency and even slightly increased that of iPR T-L by a factor of 5.8. The fluorescent molecules 6b−c seemed to behave like the conjugated monomer 3 of the inhibitory head (IC50 = 0.46, 4.58, and 0.87 μM, respectively, for Ch-TL, PA, and T-L).21 Their binding within the ChT-L active site may be similarly guided by the tripeptidic moiety (SI, Figure 1S) with the benzyl group bound into S1 subsite. The binding to the iPR S1 site may be hopefully closely related, explaining why the fluorescent molecules 6a−c equally inhibited both ChT-L activities because the iPR S1 subsite is larger than that of cPR.33 The C-terminal benzyl group was as favorably accommodated in the iPR S1 subsite as in the cPR one. The subunit selectivity trend was preserved in cells as shown in SI, Figure 2S and Table 2 reporting the proteasome inhibition profile after treatment of HEK-293 cells for 1 h with the reference inhibitory head 2a and the fluorescent molecules 6a−c. As for 2a, the ChT-L activity was preferably inhibited compared to the T-L (factor of 8.6 for 6a, 8 for 6b, and 4.1 for 6c) and PA ones (factors of 3.8, 6, and 4.7, respectively). But the T-L activity itself was more efficiently inhibited by 6a (factor of 4.9), 6b, and 6c (factor of 23) than by 2a. The same was observed for the PA activity (factors of 14, 30, and 26, respectively). Finally, for a given fluorescent inhibitor, the PA and T-L activities were quasi-equally inhibited. The best potency against ChT-L activity was obtained for compound 6b (IC50 = 65 ± 5 nM). Its efficiency was close to that observed

Table 2. Inhibition of the Proteasomal ChT-L, T-L, and PA Activities of HEK-293 Cellsa IC50 (μM)b compd epoxomicinc 2ac 6a 6b 6c

ChT-L 0.026 7.97 0.289 0.065 0.125

± ± ± ± ±

0.003 0.15 0.013 0.005 0.012

PA 0.30 16 1.12 0.402 0.596

± ± ± ± ±

T-L 0.05 2 0.17 0.027 0.050

12.18 2.48 0.521 0.515

± ± ± ±

0.75 0.26 0.024 0.051

a

The proteasomal activity after 1 h incubation of cells with various concentrations of the tested molecules was detected using the appropriate proteasome Glo cell-based assay. bIC50 values were determined as indicated in Table 1. cSee ref 21.

with the epoxomicin, a reference inhibitor (IC50 = 26 ± 3 nM), and superior to that observed with purified cPR (IC50 = 480 ± 30 nM) and iPR (IC50 = 560 ± 20 nM). The proteasome forms present within cells (20S, 26S, and immune forms) may react slightly differently with the inhibitors than the purified 20S forms cPR and iPR tested in vitro. The cell penetration was possibly favored in the presence of one Ahx (6b) or three Ahx motifs (6c, IC50 = 125 ± 12 nM). Finally, the reversible character of the inhibition by our fluorescent probes was demonstrated. Unlike the known covalent irreversible epoximicin inhibitor, compound 6b is a noncovalent reversible inhibitor because a total recovery of activity was observed after inhibitor removal (SI, Figure 3S). To study the cellular entry, HEK-293 cells were treated in conditions where cell mortality was negligible and were screened using flow cytometry analysis (Figure 2) and confocal microscopy (Figure 3). The percentage of rhodamine fluorescent HEK-293 cells detected by flow cytometry was identical after 1 h treatment with the same concentration (5

Figure 2. Flow cytometry analysis of HEK-293 cells untreated (NT) and treated with the vehicle (DMSO, V), rhodamine B (Rho), and compounds 7 and 6a−c. Cells were treated for 1 h with 5 μM rhodamine or compound. *t test: p < 0.05. C



Brief Article

MHz) δ 17.10, 37.35, 39.71, 44.15, 51.39, 51.70, 57.58, 71.00, 76.44, 103.53, 111.47, 116.10, 123.93, 125.48, 128.08, 128.40, 128.58, 128.81, 129.43, 129.45, 130.67, 130.97, 131.36, 131.77, 137.52, 138.74, 139.64, 142.95, 159.20, 168.79, 173.19, 174.50, 174.58, 181.56. λabs = 495 nm (water), λem = 525 nm (water), ϕquant = 0.86. Anal. Calcd for C58H49N5O12·3.5HCl: C, 61.34; H, 4.66; N, 6.17. Found: C, 61.26; H, 4.93; N, 6.11. tR (HPLC) = 12.81 min (1 mL min−1, 70% MeOH/30% 0.005 M aqueous HCl, 254 nm, C18). HRMS (ESI, MeOH): calcd [M + Na]+ C58H49N5O12Na, 1030.3270; found, 1030.3270. Tetramethylrhodamine Derivative 6a. Similarly to compound 5, product 6a (12 mg, 32%) was obtained as a dark-purple solid from tripeptide 2b19 (23 mg, 0.035 mmol), DMF (0.35 mL), Et3N (14 μL, 0.10 mmol), compound 932 (15 mg, 0.035 mmol), HOBt (5 mg, 0.038 mmol), and EDC (7 μL, 0.038 mmol). 1H NMR (CD3OD, 500 MHz) δ 1.41 (d, J = 7.0 Hz, 3H), 2.51 (dd, J = 14.5 Hz, J = 9.5 Hz, 1H), 2.63 (dd, J = 14.5 Hz, J = 3.5 Hz, 1H), 3.28 (m, 13H), 4.19−4.22 (m, 2H), 4.26 and 4.37 (AB system, J = 15 Hz, 2H), 4.77 (dd, J = 10.0 Hz, J = 5.5 Hz, 1H), 5.02 (s, 2H), 6.86−7.08 (m, 12H), 7.12−7.40 (m, 18 H), 8.20 (d, J = 7.0 Hz, 1H), 8.69 (s, 1H). 13C NMR (CD3OD, 125.7 MHz) δ 17.14, 37.36, 39.71, 40.98, 44.15, 51.38, 51.70, 57.54, 71.01, 76.42, 97.47, 111.46, 114.67, 115.57, 116.07, 123.92, 125.48, 128.07, 128.39, 128.59, 128.82, 129.43, 129.45, 130.73, 130.97, 131.39, 131.64, 131.77, 131.90, 131.93, 132.64, 132.82, 137.12, 138.27, 138.73, 139.66, 142.97, 158.92, 159.00, 159.20, 160.52, 167.29, 168.72, 173.18, 174.49, 174.55, 181.52. λabs = 555 nm (water), λem = 584 nm (water), ϕquant = 0.28. Anal. Calcd for C62H59N7O10·4.5HCl: C, 60.73; H, 5.22; N, 8.00. Found: C, 60.97; H, 5.50; N, 8.29. tR (HPLC) = 14.70 min (1 mL min−1, 70% MeOH/30% 0.005 M aqueous HCl, 254 nm, C18). HRMS (ESI, MeOH): calcd [M + H]+ C62H60N7O10, 1062.4396; found, 1062.4395. Tetramethylrhodamine Derivative 6b. Step c: Aqueous 1 M LiOH (0.80 mL, 0.80 mmol) was added at 0 °C to a solution of conjugate 7 (113 mg, 0.202 mmol, see SI) in THF (0.80 mL). The mixture was stirred for 6 h at rt. Aqueous 1 M hydrochloric acid (0.90 mL) was then added. The resulting mixture was freeze-dried and purified by MPLC (C18 silica gel, 0.3 g, 0.005 M aqueous HCl/ acetonitrile gradient). The acidic conjugate (62 mg, 57%) was obtained as a dark-purple solid. HRMS (ESI, MeOH): calcd [M + H]+ C31H34N3O6, 544.2448; found, 544.2444. Step a: Similarly to 6a, product 6b (22 mg, 19%) was obtained as a dark-purple solid from tripeptide 2b19 (71 mg, 0.11 mmol) in DMF (1.1 mL), Et3N (50 μL, 0.35 mmol), the preceding acidic conjugate (53 mg, 0.097 mmol), HOBt (16 mg, 0.12 mmol), and EDC (20 μL, 0.11 mmol). 1H NMR (CD3OD, 500 MHz) δ 1.31−1.38 (m, 5 H), 1.59−1.69 (m, 4H), 2.22−2.34 (m, 2H), 2.48 (dd, J = 14.5 Hz, J = 10 Hz, 1H), 2.61 (dd, J = 14 Hz, J = 3 Hz, 1H), 2.82 (dd, J = 13.5 Hz, J = 9 Hz, 1H), 3.07 (dd, J = 14 Hz, J = 5 Hz, 1H), 3.25 (s, 12 H), 3.45 (m, 2H), 4.15 (q, J = 7.0 Hz, 1H), 4.20 (dd, J = 9.5 Hz, J = 3 Hz, 1H), 4.26 and 4.34 (AB system, J = 15.5 Hz, 2H), 4.45 (dd, J = 9 Hz, J = 5.5 Hz, 1H), 4.98 (s, 2H), 6.83−7.39 (m, 31 H), 8.26 (d, J = 7.5 Hz, 1H), 8.79 (s, 1H). 13C NMR (CD3OD, 125.7 MHz) δ 17.19, 26.39, 27.43, 30.09, 36.63, 37.46, 39.72, 41.01, 41.08, 44.14, 51.39, 51.64, 56.93, 70.96, 76.46, 97.50, 111.47, 114.72, 115.65, 115.97, 123.90, 125.50, 128.07, 128.35, 128.60, 128.85, 129.44, 129.50, 130.61, 130.99, 131.33, 131.41, 131.84, 131.93, 131.98, 132.35, 132.80, 137.80, 138.03, 138.77, 139.68, 143.02, 158.91, 159.01, 159.13, 160.56, 167.40, 168.02, 173.27, 174.57, 174.81, 176.79, 181.53. λabs = 553 nm (water), λem = 584 nm (water), ϕquant = 0.6. Anal. Calcd for C68H70N8O11·4HCl: C, 61.82; H, 5.65; N, 8.48. Found: C, 61.63; H, 5.85; N, 8.29. tR (HPLC) = 12.81 min (1 mL min−1, 70% MeOH/30% 0.005 M aqueous HCl, 254 nm, C18). HRMS (ESI, MeOH): calcd [M + H]+ C68H71N8O11, 1175.5237; found, 1175.5238. Tetramethylrhodamine Derivative 6c. Step d: To a solution of 10c21 (137 mg, 0.33 mmol) in anhydrous DMF (3 mL) and CH3CN (3 mL) at 0 °C was successively added Et3N (158 μL, 1.01 mmol), carboxyrhodamine 9 (145 mg, 0.34 mmol), HOBt (49 mg, 0.37 mmol), and EDC (65 μL, 0.37 mmol).The resulting mixture was stirred at rt for 16 h and then concentrated in vacuo (0.1 mbar). The residue was purified twice by MPLC (C18 silica gel, 13 g, 0.005 M aqueous HCl/acetonitrile gradient). The Tamra conjugate (96 mg,

Figure 3. Imaging of HEK-293 cells by confocal microscopy. Cell cultures were preincubated for 1 h with 5 μM rhodamine B or 5 μM fluorescent inhibitor (6a, 6b, or 6c).

μM) of rhodamine alone or compounds 6a and 7, whereas it was decreased with compounds 6b and 6c by factors of 1.4 and 3.3, respectively. The cell survival was 67.6 (7), 90.1 (6a), 78.2 (6b), and 100% (6c). The cell penetration was evidenced by treatment of HEK293 cells by rhodamine and compounds 6a−c in identical experimental conditions. All molecules clearly entered the cells (Figure 3). The fluorescent DNA marker DAPI was used to visualized the nucleus (λex = 405 nm; λem = 416−480 nm). It accumulated in the nucleus.

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CONCLUSION We have designed and synthesized noncovalent fluorescent probes of cPR and iPR activities derived from linear mimics of compound 1 to evaluate cell penetration of the mimics inhibitory head. The newly synthesized molecules inhibit quasi-equally activities of cPR and iPR and are even more efficient in cells. Because of high expectation toward noncovalent inhibitors,8,10,13 noncovalent probes that are less prone to displace all noncovalent proteasome inhibitors may constitute an advantage to detect proteasomal cellular inhibition due to reversible inhibitors. They can be used on purified proteasomes, whole cell lysates, and eukaryotic cells. Cells can also be assessed by confocal microscopy or flow cytometry, allowing a coevaluation of cell penetration of the tested inhibitors. By inhibiting both cPR and iPR, they may be useful to examine the selectivity of novel inhibitors for iPR or cPR in a competitive manner and allow the potential reuse of the treated cells.

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EXPERIMENTAL SECTION

General methods are described in Supporting Information (SI). Tripeptide 2b19 and Ahx derivatives 10b−c21 were previously described. 5-Carboxyfluoresceine 831 and 5-carboxytetramethyl rhodamine 932 were prepared as described in the literature. Purity of compounds 5 and 6a−c was checked by elemental analysis. Data for C, H, and N were within 0.4% of the theoretical values. Purity was also determined by reverse phase HPLC using a C18 column (5 μm, 4.6 mm × 250 mm, ACE) in conditions described below for each compound. Purity of fluorescent proteasome inhibitors 5 and 6a−c was higher than 95%. Fluorescein Derivative 5. To a solution of tripeptide 2b19 (48 mg, 0.074 mmol) in anhydrous DMF (0.7 mL) at 0 °C was successively added Et3N (30 μL, 0.22 mmol), 5-carboxyfluorescein 831 (28 mg, 0.074 mmol), HOBt (11 mg, 0.081 mmol), and EDC (14 μL, 0.80 mmol).The resulting mixture was stirred at rt for 16 h and then concentrated in vacuo (0.1 mbar). The residue was purified twice by medium pressure liquid chromatography (MPLC) using C18 silica gel (5 g) and a 0.005 M aqueous HCl/MeOH gradient. Product 5 (16 mg, 22%) was obtained as a dark-green solid. 1H NMR (CD3OD, 500 MHz) δ 1.39 (d, J = 7.0 Hz, 3H), 2.50 (dd, J = 14.0 Hz, J = 9.5 Hz, 1H), 2.62 (dd, J = 14.0 Hz, J = 3 Hz, 1H), 3.03 (dd, J = 14.0 Hz, J = 9.5 Hz, 1H), 3.25 (dd, J = 14.0 Hz, J = 5.5 Hz, 1H), 4.19−4.22 (m, 2H), 4.25 and 4.37 (AB system, J = 15 Hz, 2H), 4.73 (dd, J = 9.5 Hz, J = 5.5 Hz, 1H), 5.03 (s, 2H), 6.81−7.05 (m, 12H), 7.21−7.39 (m, 18 H), 8.17 (d, J = 8.0 Hz, 1H), 8.52 (s, 1H). 13C NMR (CD3OD, 125.75 D



Brief Article

added. An equivalent volume of DMSO was used in controls for a final DMSO concentration of 1% (v/v). After an incubation period of 2 h, 25 μL of assay buffer containing a recombinant firefly luciferase and the luminogenic proteasome substrate Suc-LLVY-Glo (ChT-L activity), Z-nLPnLD-Glo (PA activity), or Z-LRR-Glo (T-L activity) was added. After a further incubation at room temperature for 10 min, chemioluminescence expressed as relative light units (RLU) was measured on a BMG Labtech Fluostar Optima plate reader with a measuring time of 12 s for each well. The final concentrations of tested compounds varied between 2 and 20 μM. Remaining ChT-L, PA, or T-L activities were determined according to eq 3:

36%) was obtained as a dark-purple solid. Step c: Similarly as for 6b, the acidic conjugate (55 mg, 58%) was obtained as a dark-purple solid from aqueous 1 M LiOH (0.60 mL, 0.60 mmol), the preceding Tamra conjugate (96 mg, 0.12 mmol) in THF (0.80 mL). HRMS (ESI, MeOH): calcd [M + H]+ C43H56N5O8, 770.4123; found, 770.4118. Step a: Similarly to 6b, product 6c (17 mg, 18%) was obtained as a dark-purple solid from tripeptide 2b19 (44 mg, 0.067 mmol) in DMF/ CH3CN (1/1 mixture, 1.4 mL), Et3N (28 μL, 0.20 mmol), the preceding acidic conjugate (52 mg, 0.067 mmol), HOBt (10 mg, 0.074 mmol), and EDC (13 μL, 0.074 mmol). 1H NMR (CD3OD, 500 MHz) δ 1.18−1.24 (m, 2H), 1.32−1.37 (m, 5H), 1.42−1.53 (m, 8H), 1.58−1.64 (m, 2H), 1.69−1.74 (m, 4H), 2.15−2.25 (m, 6H), 2.47 (dd, J = 14 Hz, J = 9.5 Hz, 1H), 2.60 (dd, J = 14 Hz, J = 3 Hz, 1H), 2.78 (dd, J = 14 Hz, J = 9.5 Hz, 1H), 3.06−3.19 (m, 5H), 3.31 (s, 12H), 3.49 (t, J = 7.5 Hz, 2H), 4.15 (q, J = 7 Hz, 1H), 4.19 (dd, J = 9.5 Hz, J = 3 Hz, 1H), 4.27 and 4.39 (AB system, JAB = 15.1 Hz, 2H), 4.48 (dd, J = 9.5 Hz, J = 5 Hz, 1H), 5.01 (s, 2H), 6.85−7.53 (m, 33 H), 8.27 (d, J = 7.5 Hz, 1H), 8.79 (s, 1H). 13C NMR (CD3OD, 125.7 MHz) δ 17.20, 26.42, 26.74, 26.80, 27.46, 27.59, 27.68, 30.13, 30.17, 30.22, 36.66, 37.02, 37.04, 37.46, 39.74, 40.26, 40.99, 41.14, 44.18, 51.38, 51.59, 56.67, 71.01, 76.47, 97.52, 111.46, 114.78, 115.64, 115.97, 123.92, 125.52, 128.13, 128.45, 128.63, 128.89, 129.47, 129.53, 130.66, 130.99, 131.31, 131.35, 131.53, 131.87, 131.97, 132.35, 132.87, 137.88, 138.08, 138.81, 139.72, 143.05, 159.01, 159.09, 159.16, 160.68, 167.39, 168.12, 173.25, 174.56, 174.79, 175.99, 176.07, 176.67, 181.55. λabs = 556 nm (water, 1% CH3CN), λem =585 nm (water, 1% CH3CN), ϕquant = 0.6. Anal. Calcd for C80H92N10O13·5HCl: C, 60.66; H, 6.17; N, 8.84. Found: C, 60.49; H, 6.30; N, 8.72. tR (HPLC) = 14.35 min (1 mL min−1, 70% MeOH/30% 0.005 M aqueous HCl, 254 nm, C18). HRMS (ESI, MeOH): calcd [M + Na]+ C80H92N10O13Na, 1426.6737; found, 1423.6738. Biological Assays. Enzyme Activity and Inhibition Assays Using Purified Proteasomes. Proteasome activities were determined by monitoring the hydrolysis of the appropriate substrate (λexc = 360 nm and λem = 460 nm for AMC substrates; λexc = 340 nm and λem = 404 nm for the βNA substrate) (see SI). The enzyme-catalyzed fluorescence emission was followed for 45 min at 37 °C in microplates using the FLUOstar Optima (BMG Labtech). The enzymes were either untreated (controls) or treated by various concentrations of the tested compounds. Before use, substrates and compounds were previously dissolved in DMSO. The percentage of this cosolvent was maintained equal to 2% (v/v) in all experiments. In addition to DMSO, the buffers (pH 8) used to measure ChT-L and PA activities contained 20 mM Tris-HCl, 10% glycerol, and 0.01% (w/v) SDS, whereas the buffer used for T-L activity was devoid of SDS. The enzyme concentrations were 0.3 nM in both cases; the substrate concentrations were 20 μM for Suc-LLVY-AMC (cPR and iPR for ChT-L activity), 100 μM for Z-LLE-βNA (cPR and iPR for PA activity), and 100 μM (cPR) or 50 μM (iPR) Boc-LRR-AMC for T-L activity. To determine the IC50 values (inhibitor concentration giving 50% inhibition), the enzyme (cPR or iPR) and inhibitors (0.1−100 μM) were incubated for 15 min before the measurement of the remaining activity. They were obtained by fitting the experimental data to eq 1 or eq 2.

⎛ 100 × [I ]0 V⎞ %Inhibition = 100 × ⎜1 − i ⎟ = IC50 + [I ]0 V0 ⎠ ⎝ %Inhibition =

100 × [I ]n0 ICn50 + [I ]n0

⎛ ⎛ 100 × [I ]n0 ⎞ Activityi ⎞ ⎟⎟ = 100 − ⎜ n %Activity = 100 × ⎜⎜1 − n⎟ Activity0 ⎠ ⎝ IC50 + [I ]0 ⎠ ⎝ (3) Reversibility Study. HEK-293 cells were cultured in DMEM medium as described above. They were treated 1 h either with 3 μL of DMSO or by epoxomicin (1 μM) or compound 6b (1−5 μM) in a final volume of 3 mL [0.001% DMSO (v/v)]. The intracellular proteasome activity (2500 cells/well) was immediately measured using the proteasome GloTM cell-based assay. In parallel, cells were harvested and washed with PBS twice. They were homogenized in a lysis buffer (CellLyticTMM cell lysis reagent, Sigma) for 15 min at 4 °C. Cell lysates were centrifuged at 20000g for 15 min at 4 °C. The supernatant was collected as whole cell extract and total protein concentration was determined (BCA assay). The ChT-L activity was measured for 5 μg of total proteins using the fluorogenic substrate SucLLVY-AMC (see above). Flow Cytometry Analysis. HEK-293 cells (1 million) were incubated with compounds 6a, 6b, or 6c (5 μM) for 1 h. They were before equilibrated at 37 °C, 5% CO2 during 2 h. These experimental conditions were previously established by testing Rhodamine B (Sigma) at 5, 10, 20, 50, and 100 μM on 1 million HEK293 cells, and compound 7 at 1 and 5 μM concentrations. The flow cytometric analysis of fluorescent cells was performed using MACSQuant TEN. DAPI staining was omitted, and PE signal was detected using a 488 nm bandpass filter behind a 585/40 nm long-pass with a laser power of 25 mW. For each histogram, the cell distribution was calculated using the MACSQuantify software program. HEK-293 cells were cultured. Confocal Microscopy. Microscopy experiments were performed using 4 × 105 cells seeded on Lab-Tek Plate. The cells were cultivated overnight in cell culture conditions before incubation during 1 h with rhodamine B (5, 10, 20, 50, and 100 μM) or the fluorescent inhibitors (5 μM). The cells were washed and visualized by confocal microscopy (Leica TC SP5, inverse) with an excitation at 561 nm and a UV laser at 405 nm. Leica Software LASAF was used for image processing. Rhodamine B and inhibitors were excited at 570 nm and detected at 620 nm. All experiments were done using identical acquisition settings for each probe. Cytotoxicity. Cell viability was assessed by XTT assay. 104 cells were seeded in 96-well plates and cultured at 37 °C for up to 24 h, 95% humidity and 5% CO2. Fluorescent inhibitors (or DMSO for control) were added 1 h before adding the XTT. This salt ([2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)]; Sigma) was incubated during 3 h, and reduction product of XTT salt (formazan) was measured by spectrophotometer at 485 nm with a BMG Labtech Fluostar Optima plate reader.

(1)

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(2)

Proteasome Glo Cell-Based Assays. HEK-293 cells were cultured at 37 °C, 95% humidity, and 5% CO2 in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen). The effects of the compounds on ChT-L, PA, and T-L activities of HEK293 cells were determined using the chemiluminescent assays from Promega. HEK-293 cells were dispensed into white opaque 384-well microtiter plates (2500 cells/well, 25 μL culture medium per well). Then 0.5 μL of each compound previously dissolved in DMSO was

ASSOCIATED CONTENT

S Supporting Information *

Figure showing compound 3 in complex with the ChTL active site; IC50 curves corresponding to Tables 1 and 2; Figure illustrating reversibility; general methods; synthesis of compound 7; NMR spectra for compounds 5, 6a−c, and 7; absorption and emission spectra for 5 and 6c. This material is available free of charge via the Internet at http://pubs.acs.org. E



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cious Reversible Lead Motif. Angew. Chem., Int. Ed. Engl. 2014, 53, 1679−1683. (13) Beck, P.; Dubiella, C.; Groll, M. Covalent and non-covalent reversible proteasome inhibition. Biol. Chem. 2012, 393, 1101−1120. (14) Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.; Ohnuki, T.; Komatsubara, S. TMC-95A, B, C, and D, novel proteasome inhibitors produced by Apiospora montagnei Sacc. TC 1093. Taxonomy, production, isolation, and biological activities. J. Antibiot. 2000, 53, 105−109. (15) Kaiser, M.; Groll, M.; Renner, C.; Huber, R.; Moroder, L. The core structure of TMC-95A is a promising lead for reversible proteasome inhibition. Angew. Chem., Int. Ed. 2002, 41, 780−783. (16) Berthelot, A.; Piguel, S.; Le Dour, G.; Vidal, J. Synthesis of Macrocyclic Peptide Analogues of Proteasome Inhibitor TMC-95A. J. Org. Chem. 2003, 68, 9835−9838. (17) Yang, Z.-Q.; Kwok, B. H. B.; Lin, S.; Koldobskiy, M. A.; Crews, C. M.; Danishefsky, S. J. Simplified synthetic TMC-95A/B analogues retain the potency of proteasome inhibitory activity. ChemBioChem 2003, 4, 508−513. (18) Basse, N.; Piguel, S.; Papapostolou, D.; Ferrier-Berthelot, A.; Richy, N.; Pagano, M.; Sarthou, P.; Sobczak-Thepot, J.; ReboudRavaux, M.; Vidal, J. Linear TMC-95-Based Proteasome Inhibitors. J. Med. Chem. 2007, 50, 2842−2850. (19) Groll, M.; Gallastegui, N.; Marechal, X.; Le Ravalec, V.; Basse, N.; Richy, N.; Genin, E.; Huber, R.; Moroder, L.; Vidal, J.; ReboudRavaux, M. 20S Proteasome inhibition: designing noncovalent linear peptide mimics of the natural product TMC-95A. ChemMedChem 2010, 5, 1701−1705. (20) Maréchal, X.; Pujol, A.; Richy, N.; Genin, E.; Basse, N.; ReboudRavaux, M.; Vidal, J. Noncovalent inhibition of 20S proteasome by pegylated dimerized inhibitors. Eur. J. Med. Chem. 2012, 52, 322−327. (21) Desvergne, A.; Genin, E.; Maréchal, X.; Gallastegui, N.; Dufau, L.; Richy, N.; Groll, M.; Vidal, J.; Reboud-Ravaux, M. Dimerized Linear Mimics of a Natural Cyclopeptide (TMC-95A) Are Potent Noncovalent Inhibitors of the Eukaryotic 20S Proteasome. J. Med. Chem. 2013, 56, 3367−3378. (22) Blackburn, C.; Barrett, C.; Blank, J. L.; Bruzzese, F. J.; Bump, N.; Dick, L. R.; Fleming, P.; Garcia, K.; Hales, P.; Hu, Z. G.; Jones, M.; Liu, J. X.; Sappal, D. S.; Sintchak, M. D.; Tsu, C.; Gigstad, K. M. Optimization of a series of dipeptides with a P3 threonine residue as non-covalent inhibitors of the chymotrypsin-like activity of the human 20S proteasome. Bioorg. Med. Chem. Lett. 2010, 20, 6581−6586 and references cited therein.. (23) Furet, P.; Imbach, P.; Noorani, M.; Koeppler, J.; Laumen, K.; Lang, M.; Guagnano, V.; Fürst, P.; Roesel, J.; Zimmermann, J.; Garcia Echeverria, C. Entry into a new class of potent proteasome inhibitors having high antiproliferative activity by structure-based design. J. Med. Chem. 2004, 47, 4810−4813 and references cited therein.. (24) Bordessa, A.; Keita, M.; Marechal, X.; Formicola, L.; Lagarde, N.; Rodrigo, J.; Bernadat, G.; Bauvais, C.; Soulier, J. L.; Dufau, L.; Milcent, T.; Crousse, B.; Reboud-Ravaux, M.; Ongeri, S. alpha- and beta-Hydrazino acid-based pseudopeptides inhibit the chymotrypsinlike activity of the eukaryotic 20S proteasome. Eur. J. Med. Chem. 2013, 70, 505−524 and references cited therein.. (25) Basse, N.; Papapostolou, D.; Pagano, M.; Reboud-Ravaux, M.; Bernard, E.; Felten, A.-S.; Vanderesse, R. Development of lipopeptides for inhibiting 20S proteasomes. Bioorg. Med. Chem. Lett. 2006, 16, 3277−3281. (26) Gallastegui, N.; Beck, P.; Arciniega, M.; Huber, R.; Hillebrand, S.; Groll, M. Hydroxyureas as Noncovalent Proteasome Inhibitors. Angew. Chem., Int. Ed. 2012, 51, 247−249. (27) Basse, N.; Montes, M.; Maréchal, X.; Qin, L.; Bouvier-Durand, M.; Genin, E.; Vidal, J. l.; Villoutreix, B. O.; Reboud-Ravaux, M. l. Novel Organic Proteasome Inhibitors Identified by Virtual and in Vitro Screening. J. Med. Chem. 2010, 53, 509−513. (28) Maréchal, X.; Genin, E.; Qin, L.; Sperandio, O.; Montes, M.; Basse, N.; Richy, N.; Miteva, M. A.; Reboud-Ravaux, M.; Vidal, J.; Villoutreix, B. O. 1,2,4-Oxadiazoles identified by virtual screening and

AUTHOR INFORMATION

Corresponding Authors

*For M.R.R.: phone, 33 (0) 1 44 27 50 78; E-mail, michele. [email protected]. *For E.G.: phone, 33 (0) 5 40 00 67 34; E-mail, e.genin@ism. u-bordeaux1.fr. *For J.V.: phone, 33 (0) 2 23 23 57 33; E-mail, joelle.vidal@ univ-rennes1.fr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The “Association Française contre les Myopathies” (AFM) (grant 14999) is acknowledged. A.D. and X.M. received financial support from the Ministère de l′Enseignement Supérieur et de la Recherche. E.G. and S.G. received financial support from “Région Bretagne” (SAD grant). We thank Annie Munier, Jean-François Gilles, and Richard Schwartzmann from the Cell Imaging and Flow Cytometry Facility of the IFR83 (Paris, France) for precious help in microscopy/cytometry/ image analysis. The facilities are supported by the Conseil Régional Ile-de-France.

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ABBREVIATIONS USED Ahx, aminohexanoic residue; BODIPY, 4,4-difluoro-4-bora3a,4a-diaza-s-indacene; cPR, 20S constitutive proteasome; iPR, 20S immunoproteasome; DAPI, 4′,6-diamidino-2-phenylindole; EDC: 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide; HOBt, hydroxybenzotriazole; Tamra, tetramethylrhodamine

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REFERENCES

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Noncovalent Fluorescent Probes of Human Immuno- and Constitutive

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