Forum Article www.acsami.org
Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Caspase‑1 Specific Light-Up Probe with Aggregation-Induced Emission Characteristics for Inhibitor Screening of CoumarinOriginated Natural Products Hao Lin,†,∧ Haitao Yang,†,∧ Shuai Huang,†,∧ Fujia Wang,†,∧ Dong-Mei Wang,†,∧ Bin Liu,*,# Yi-Da Tang,*,§ and Chong-Jing Zhang*,†,∧ †
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines and ∧Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China 100050 # Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 § Fuwai Hospital, Chinese Academy of Medical Sciences, Beijing, China 100037 S Supporting Information *
ABSTRACT: Caspase-1 is a key player in pyroptosis and inflammation. Caspase-1 inhibition is found to be beneficial to various diseases. Coumarin-originated natural products have an anti-inflammation function, but their direct inhibition effect to caspase-1 remains unexplored. To evaluate their interactions, the widely used commercial coumarin-based probe (Ac-YVAD-AMC) is not suitable, as the background signal from coumarin-originated natural products could interfere with the screening results. Therefore, fluorescent probes using a large Stokes shift could help solve this problem. In this work, we chose the fluorophore of tetraphenylethylene−thiophene (TPETH) with aggregation-induced emission characteristics and a large Stokes shift of about 200 nm to develop a molecular probe. Bioconjugation between TPETH and hydrophilic peptides (DDYVADC) through a thiol−ene reaction generated a light-up probe, C1−P3. The probe has little background signal in aqueous media and exerts a fluorescent turn-on effect in the presence of caspase-1. Moreover, when evaluating the inhibition potency of coumarin-originated natural products, the new probe could generate a true and objective result but not for the commercial probe (Ac-YVAD-AMC), which is evidenced by HPLC analysis. The quick light-up response and accurate screening results make C1−P3 very useful in fundamental study and inhibitior screening toward caspase-1. KEYWORDS: aggregation-induced emission, light-up probe, caspase-1, coumarin, natural products
■
INTRODUCTION Caspases are cysteine aspartate specific proteinases with two common features: all members of the caspase family include the catalytic cysteine residue in the active site, and they cleave their substrates on the carboxyl side of aspartate residues.1,2 Among the 12 reported human caspases, caspase-1, also called interleukin-1-converting enzyme (ICE), was the first human caspase to be identified and isolated.3,4 Active caspase-1 is involved in the cleavage and activation of interleukin 1β, interleukin 18, and interleukin 33.5 Interleukin 1β and interleukin 18 are crucial mediators of innate immunity and inflammation.6 It is known that the activation of caspase-1 © XXXX American Chemical Society
could trigger pyroptosis, a form of programmed cell death with the typical character of high inflammation.7,8 Inhibition of caspase-1 has been shown to be beneficial in many experimental animal models of human diseases, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, asthma, and sepsis, which suggests that targeting caspase-1 Special Issue: AIE Materials Received: September 29, 2017 Accepted: December 27, 2017
A
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces Scheme 1. Synthetic Route to C1−P1, C1−P2, and C1−P3
could be a novel approach to solve the problems.9,10 Recent studies also indicate that pyroptosis plays more important roles than apoptosis in the progressive depletion of CD4 T cells and development of chronic inflammation in HIV-infected individuals.11,12 As such, inhibition of caspase-1 could modulate the two signature events in HIV infection, which has the potential to form a new and exciting therapy for HIV-infected subjects. Therefore, targeting caspase-1 via small-molecule therapeutics is an active area of research. Fluorescent probes are important research tools to track13 and bind14 the targeted proteins and evaluate the enzymatic activity.15−18 The most frequently applied approach to screen small-molecule caspase-1 inhibitor is to employ the coumarin-based fluorogenic probe (Ac-YVADAMC).19,20 Active caspase-1 could cleave the probe to produce 7-amino-4-methylcoumarin, which provides an increased fluorescent signal to evaluate the inhibition potency of screened compounds. Coumarin-originated natural products have wide biological effects, such as anti-inflammatory, anticoagulant, and antibacterial activities.21 In addition, they are reported to inhibit the generation of cytokines, such as TNF-α, IL-1β, and IL-6, via suppression of the activation of caspase-1 from pro-caspase-1.22 However, it is not clear whether coumarin-originated natural products have any direct inhibition effect toward the active caspase-1. However, it should be noted that during the evaluation of their inhibition effect toward caspase-1, the conventional probe (Ac-YVAD-AMC) has potential problems, because false negative/positive results could be generated due to sharing a similar excitation and emission with 7-amino-4methylcoumarin. On the other hand, a bioluminescent assay is a promising alterative, as it does not require the usage of excited light.23 However, the potential interaction between the luciferase and screened compounds could make the results unreliable.24 To address this issue, we look into the development of light-up probes using a fluorophore with a very large Stokes shift. During the past decade, the development of fluorogens with aggregation-induced emission (AIE) characteristics has gained tremendous research interest. The AIE fluorogens (AIEgens) usually have large Stokes shifts (∼200 nm), which have been
widely used in inhibitor screening,25,26 and imaging-guided cancer therapy.27−31 The AIE property is ascribed to the restriction of intramolecular motion (RIM).32 The free molecular motions play leading roles in the consumption of the excited state energy of AIEgens when they are in a molecularly dissolved state. However, RIM plays an active role in generating the luminescence for AIEgens in the aggregate state,33 which overcomes the drawbacks of the conventional dyes with aggregation-caused quenching.34 In addition, AIEgens provide a unique approach to design light-up probes through direct binding to analytes or by virtue of the solubility change before and after the enzyme cleavage.35−40 In this study, we chose TPETH as an AIEgen to develop caspase 1-targeting light-up probes. TPETH has a large Stokes shift of about 200 nm with an orange-red emission,41−44 which could readily deplete the background signal from the coumarin-originated natural products. Based on TPETH and a hydrophilic peptide substrate, three probes were developed to screen the most suitable one with a high signal to background ratio. Probe 1 (C1−P1) and probe 2 (C1−P2) have one peptide sequence, while probe 3 (C1−P3) has two sequences. It turns out that probe 3 possesses the highest light-up effect upon incubation with caspase-1. C1−P3 was then used to evaluate the inhibition potency of coumarinoriginated natural products. As compared to the commercial probe of Ac-YVAD-AMC, C1−P3 rendered a more accurate screening result.
■
RESULTS AND DISCUSSION Probe 1 (C1−P1) was the initial design. Its peptide has a sequence of DDYVAD, in which YVAD is the substrate sequence of caspase-1, and the two aspartic (D) residues help to increase the water solubility. Probe 2 (C1−P2) with a sequence of DDDDYVADF was the second design. The additional phenylalanine (F) and the additional two D residues were employed to improve the probe solubility and enlarge the polarity difference before and after the enzyme cleavage. Probe 3 (C1−P3) with two sequences of DDYVAD is the third design. The three probes were synthesized according to Scheme 1, through the thiol−ene reaction between interB
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces
Figure 1. Optical properties of three probes and their reactivity toward caspase-1. (A) Absorption spectra of three probes (5 μM) in DMSO/HEPES buffer (1:99); (B) emission spectra of three probes (5 μM) in DMSO/HEPES buffer (1:99), Ex: 420 nm. The insets are the fluorescent photos of probes in DMSO/HEPES buffer (1:99), 1 for TPETH-2OMe, 2 for C1−P2, 3 for C1−P1, 4 for C1−P3; (C−H) HPLC profiles of three probes (10 μM) before (C,E,G) or after (D,F,H) incubation with caspase-1 (100 pM), (C,D) for C1−P1, (E,F) for C1−P2; (G,H) for C1−P3.
caspase-1. Only C1−P3 was observed to produce an obvious increase (about 10-fold) in the measurement of the fluorescent quantum yield (Table S1). We then carried out a comprehensive study to understand the C1−P3 response to caspase-1. As shown in Figure 2A, within the first 20 min, C1−P3 (10 μM) shows a steady fluorescence increase upon incubation with caspase-1 (100 pM). After 20 min of incubation, the light-up fluorescence becomes saturated, and the final signal is more than 4-fold higher than the background signal. However, due to the high background signal, C1−P1 and C1−P2 did not show an obvious light-up effect under the same conditions. To optimize the probe concentration in the caspase-1 activity study, six different concentrations ranging from 0.5 to 20 μM were employed. As shown in Figure 2B, higher concentration of C1− P3 generated stronger fluorescent intensity. And the light-up effect is the highest when the concentration of C1−P3 is 2 μM, and an 8-fold fluorescence increase was observed under this condition (Figure S10). However, the results could be easily interfered with as they fluctuate considerably during the course of incubation. The results showed a stable fluorescence increase when the probe concentration was higher than 5 μM. Thus, most of the following experiments were carried out using the probe of C1−P3 at 5 or 10 μM. We also investigated the effect of different concentrations of caspase-1 on the probe fluorescence change. The probe concentration is fixed as 10 μM, and the enzyme concentration changes from 10 to 200 pM. The fluorescence intensity change showed a linear relationship with the increasing concentration of caspase-1 (Figure 2C). To evaluate the specificity of C1−P3, several other proteins (pepsin, bovine serum albumin, trypsin, and lysozyme) were also incubated with the probe, respectively.
mediates 1 or 2 with the relative thiol-containing peptides (Scheme 1). All the probes were purified with high-pressure liquid chromatography (HPLC), and their structures were verified by mass and NMR spectra (Figures S1−S6) to reveal the right structures with high purity. As shown in Figure 1A, C1−P1, C1−P2, and C1−P3 have roughly the same absorption spectra as that of TPETH-2OMe with an absorption maximum of 450 nm. However, they show completely different emission spectra. C1−P1 and C1−P2 have lower fluorescence intensities than TPETH-2OMe in water, but they still show a clear background signal (Figure 1B and the inset). Two arms of the peptide make C1−P3 almost nonfluorescent in HEPES buffer. Incubation of all three probes with caspase-1 generated products that had a slightly different retention time from that of the respective probe (Figure 1C− H). Specifically, the retention times of C1−P1, C1−P2, and C1−P3 in the absence of caspase-1 is 8.435/8.734 min, 8.483/ 8.737 min, and 6.717 min, while the addition of caspase-1 shifted their retention times to 8.428/8.745 min, 8.657/8.944 min, and 6.529 min, respectively. Notably, C1−P1 and C1−P2 have two peaks in HPLC, because they have one arm of a peptide sequence and contain a mixture of cis/trans isomers, while two arms of a peptide sequence make C1−P3 symmetric with only one peak in HPLC. In addition, for all three probes, the cleavage of amide bonds by caspase-1 was further validated by LC−MS results (Figures S7−S9). Thus, both the HPLC and mass spectra indicated that all three probes were successfully cleaved by caspase-1. However, as the background signal is very high for C1−P1 and C1−P2, the light-up property is only observed for C1−P3 after incubation with caspase-1 (see inset photos in Figure 1C−H). We also measured fluorescent quantum yields of three probes before and after incubation with C
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces
Figure 2. Probe P1−C3 could be specifically lit up by caspase-1. (A) Time dependent fluorescent change of three probes (10 μM) upon incubation with caspase-1 (100 pM), Ex: 420 nm; Em: 630 nm; (B) the fluorescent change of various concentrations of C1−P3 upon incubation with caspase-1 (100 pM) for 20 min, Ex: 420 nm; Em: 630 nm; (C) linear relationship between fluorescent intensity increase of C1−P3 (10 μM) and concentration of caspase-1. The insets are the fluorescent photos of probes in the presence of various concentrations of caspase-1. The concentrations of the enzyme from the right side to left side are 0, 10, 30, 50, 70, 100, 130, 160, and 200 pm, respectively. (D) PL enhancement of probe C1−P3 incubated with different enzymes for 1 h. BSA, bovine serum albumin; Lyso, lysozyme; Cas-1, caspase-1. I and I0 are the fluorescence intensities of the incubation mixture with or without enzymes.
The fluorescence intensity remains the same as that of the probe only, indicating that C1−P3 could not be cleaved by pepsin, bovine serum albumin, trypsin, or lysozyme (Figure 2D). Interestingly, C1−P3 became moderately fluorescent in the presence of caspase-3, and the fluorescence intensity of mixtures remained almost unchangeable after incubation for 1 h (Figure 3). Meanwhile, the HPLC profile indicates that the probe remains intact during the incubation with caspase-3 (Figure S11). Also, after the addition of caspase-1 to the mixture of caspase-3 and C1−P3, the probe became more emissive with the increased incubation time (Figure 3). These
results suggest that C1−P3 shows high affinity toward caspase3, but it cannot be cleaved by this enzyme. This high affinity restricted the rotation of TPETH in the probe so as to turn on its fluorescence. Apparently, the interaction between C1−P3 and caspase-3 is so fast that the curve of fluorescence intensity over time is almost flat during the course of incubation (Figure 3). We also incubated C1−P3 with other members (caspase-7 and caspase-8) of the caspase family. Caspase-7 had a similar interaction with the probe as caspase-3, while caspase-8 only slightly lit up the fluorescence (Figure S12). Together, these results from the incubation assays and HPLC spectra indicated that C1−P3 shows the highest preference toward caspase-1 among the tested enzymes. One potential application of the C1−P3 is to evaluate the inhibition effect of screened compounds toward caspase-1. As shown in Figure S13, caspase-1 preincubated with a known inhibitor (Ac-YVAD-CMK, see compound 57 in Table S2) could not light up C1−P3, which indicates that the probe could clearly report the inhibition potency of the inhibitor toward caspase-1. We then employed C1−P3 to evaluate the inhibition potency of coumarin-originated compounds toward caspase-1. The commercial probe (Ac-YVAD-AMC) is also employed as a comparison to evaluate their inhibition effect. A total of 56 coumarin-originated natural products (Table S2) were obtained from commercial sources. To test their inhibition effect, the compounds were first incubated with caspase-1 for 30 min followed by the addition of C1−P3 or Ac-YVAD-AMC. In Figure 4, E0 is the first data acquisition of the fluorescence intensity upon addition of the probe, while IE is the
Figure 3. C1−P3 showed high affinity toward caspase-3. The fluorescent intensity of C1−P3 (10 μM) upon incubation with caspase-3 (100 pM) for 21 min followed by addition of caspase-1 (100 pM) was measured by a microplate reader. Ex = 420 nm. Em = 630 nm. D
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces
Figure 4. Inhibition evaluation of coumarin-originated natural products toward caspase-1. (A) Screening results by commercial probe Ac-YVADAMC; (B) screening results by C1−P3. Compounds from 1 to 56 are coumarin-originated natural products. Compound 57 (green color) is AcYVAD-CMK, a reference inhibitor to caspase-1. C1 and C2 are the controls (pink color) with probes and caspase-1 only. E0 is the first acquisition of fluorescence intensity after the addition of C1−P3 or Ac-YVAD-AMC. E is the fluorescence intensity of the mixture at 10 min after addition of C1− P3 or Ac-YVAD-AMC. The value of E − E0 below the blue dashed line indicates the corresponding compound possessed potential inhibition toward caspase-1.
Figure 5. Optical properties of selected coumarin compounds and AIEgen (TPETH-2OMe). (A) Absorption spectra of compounds 22, 32, 42, 47, 55, NH2−coumarin, and TPETH-2OMe in HEPES buffer (50 μM). (B) Normalized PL intensity of compounds 22, 32, 42, 47, 55, NH2−coumarin, and TPETH-2OMe in HEPES buffer.
fluorescence intensity of the probe after it was added for 10 min. The higher the value of (E−E0) is, the lower is the inhibition potency of the screened compound toward caspase-1 will be. As shown in Figure 4, the result by Ac-YVAD-AMC is quite different from that by C1−P3. In the screening by AcYVAD-AMC, several compounds showed potent inhibition on the enzyme. However, in the screening by C1−P3, none of the screened compounds showed detectable inhibition on caspase1. To verify whether these “hits” from the commercial probe showed true inhibition on the enzyme, HPLC was employed as a third method to analyze the mixture. As shown in Figures S14−18, the commercial probe was completely cleaved in the presence of these “hits” but remained intact in the presence of a positive inhibitor (Ac-YVAD-cmk). This result implied that the enzyme could still perform its function as usual in the presence of these “hits”. In other words, these “hits” actually could not inhibit the enzyme’s function, and they belong to false positive results. The generation of these false positive results could be
ascribed to the optical similarity between coumarin compounds and NH2-AMC. As shown in Figure 5, the absorption and emission spectra of “hits” overlapped well with NH2-AMC, whereas the overlap between “hits” and TPETH-2OMe is negligible. Thus, the background signal from coumarinoriginated compounds exerts little interference to the screening results generated by C1−P3. Therefore, our newly developed probe could render a more accurate screening result than the commercial probe. This result further implies that compound 22 could not inhibit the function of active caspase-1 but prevent the generation of active caspase-1 to reduce the production of IL-1β and IL-6.
■
CONCLUSION In conclusion, a caspase-1 specific light-up probe (C1−P3) was developed by decoration of an AIEgen (TPETH-2OMe) with DDYVADC through a thiol−ene reaction. Compared with one sequence of peptide, two sequences of peptide endow C1−P3 with a much lower background signal in aqueous media. This E
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces
solid (2.97 mg, 16.37% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.25−8.27 (t, J = 6, 1.35H), 8.19−8.20 (d, J = 6, 1.77H), 8.13−8.16 (m, 1.49H), 7.98−8.05 (m, 5.39H), 7.74−7.76 (m, 1.49H), 7.66 (m, 1.01), 7.59−7.60 (m, 1.41H), 7.36−7.38(t, J = 6,1.23H), 7.33−7.35 (d, 1.97H), 7.08−7.26 (m,13H), 7.02−7.03 (d, J = 6, 1.97H),6.96− 6.97(d, J = 6, 2.16H), 6.89−6.92 (m, 2.42H), 6.83−6.86 (m, 2.37H), 6.67−6.72 (m, 4.59H), 6.61−6.62 (d, J = 6, 2.13H), 4.47−4.56 (m, 6.04H), 4.41−4.4.44 (m, 3.82H), 4.28−4.30 (m, 2.02H), 4.11−413 (m, 1.96H), 4.01−4.03 (m, 1.19H), 3.93−3.94 (m, 1.21H), 3.87−3.90 (m, 2.40H), 3.67−3.69 (d, 3.0H), 3.50−3.52 (m, 2.64H), 3.09−3.15 (m, 4.09H), 2.93−3.03(m, 5.08H), 2.80−2.89 (m, 4.47H), 2.61− 2.77(m, 9.42H), 2.38−2.45(m, 4.66H), 1.87−2.02 (m,7.76H), 1.83 (s, 3.31H), 0.81−0.87 (m, 6.46H); HR-MS (Q-TOF), calcd for [M + H]+: 1891.6296, found: 1891.6318. Synthesis of C1−P3. To the solution of 2 (4.4 mg, 5.5 μM) in DMSO (0.4 mL) was added Ac-DDYVADCF-NH2 (12.6 mg, 15 μM, triphenylphosphine (4.27 mg, 16.3 μM) and DIEA (1.94 mg, 15 μM). The resulting mixture was stirred at room temperature for 2 h. The mixture was separated with HPLC using a reverse C18 column and a mixture of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) as the elution buffer. The separation was carried out using the constant acetonitrile fraction of 40%. C1−P3 was obtained as a red solid (5.0 mg, 36.8% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.25− 8.26 (d, 2.1H), 8.16−8.21 (m, 6.05H), 8.05−8.06 (m, 2.99H), 7.99− 8.00 (m, 3.12H), 7.73 (m, 3.51H), 7.66 (d, 2.54H), 7.54−7.64 (m, 6.83H), 7.36−7.38 (t, 1.86H), 7.28−7.35 (m, 6.38H), 7.18−7.20 (t, J = 6, 3.80H), 7.13−7.14 (d, 1.42H), 7.10−7.11 (d, 2.54H), 7.01−7.02 (d, 2.29H), 6.97−6.99 (d, J = 12, 3.52H), 6.89−6.90 (d, 2.32H), 6.82− 6.85 (d, 2.42H), 6.66−6.70 (t, 3.93H), 6.61−6.62 (d, J = 6, 3.01H), 4.52−4.56 (m, 5.03H), 4.45−4.4.48 (m, 3.08H), 4.35−4.41 (m, 6.92H), 4.26−4.28 (m, 2.92H), 4.12 (m, 3.71H), 4.01−4.02 (m, 2.19H), 3.96−3.98 (m, 2.06), 3.87−3.90 (m, 4.07H), 3.50−3.56 (m, 5.15H), 3.01−3.06 (m, 6.07H), 2.84−2.90 (m, 7.07H), 1.83 (s, 5.91H), 0.81−0.87 (m, 12H); HR-MS (Q-TOF), calcd for [M + Na]+: 2499.8173, found: 2499.8256.
property endows the probe with a fluorescence-enhancement effect in the presence of caspase-1, which specially cleaved the amide bond between D and C. The probe also shows high selectivity to caspase-1, which has been successfully used to detect the caspase-1 inhibition of a commercial inhibitor (AcYVAD-CMK). When the probe was employed to evaluate the inhibition effect of coumarin-originated natural products toward caspase-1, the TPETH-2OMe probe indicates that none of the screened compounds could inhibit caspase-1, while the commercial probe finds several potent inhibitors. Further HPLC analysis demonstrates that the “hits” were false positive results, which were generated due to the high optical similarity between coumarin compounds and NH2-AMC. As coumarin derivatives represent a large family in natural products, our study indicates that AIEgen-based probes could offer objective evaluation of their bioactivities in various applications.
■
EXPERIMENTAL SECTION
Materials and Instruments. Caspase-1 and caspase-3 were purchased from Biovison Biochemical Company; amino acids and peptides were purchased from GL Biochem (Shanghai) Ltd; pepsin, trypsin, and BSA were purchased from Absin Biochemical Company; lysozyme was purchased from Sigma-Aldrich, and other chemicals were all purchased from 3A Chemicals and Innochem Ltd. Highperformance liquid chromatography (HPLC) was performed using an Agilent 1260 series HPLC with acetonitrile containing trifluoroacetic acid (0.1%) and water containing trifluoroacetic acid (0.1%) as the elution buffers. NMR spectra were recorded on a JEOL ECZ-400S/ 600S NMR spectrometer. Chemical shifts were recorded in parts per million referenced according to residual solvent ((CD3)2SO = 2.50 ppm and CDCl3 = 7.26 ppm) in 1H NMR. Mass spectra were reported on the Thermo Exactive Plus for ESI. Compounds 1 and 2 were synthesized according to procedures in the previous published reference.41 Synthesis of C1−P1. To the solution of 1 (4.62 mg, 6.86 μM) in DMSO (0.4 mL) was added Ac-DDYVADC-NH2 (7.49 mg, 8.91 μM), triphenylphosphine (2.5 mg, 9.54 μM), and DIEA (1.086 mg, 8.4 μM). The resulting mixture was stirred at room temperature for 4 h. The mixture was separated with HPLC using a reverse C18 column and a mixture of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) as the elution buffer. The fraction of acetonitrile was 30% from 0 to 7 min, 30%−98% from 7 to 12 min, 98% from 12.0 to 12.5 min, and then, it was gradually decreased to 30% from 12.5 to 15 min. C1−P1 was obtained as a red solid (6.0 mg, 53.4% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.26 (t, 1.48H), 8.17−8.22 (m, 3.83H), 8.05 (m, 1.68H), 7.98−8.01 (t, 1.60H), 7.72 (d, 1.66H), 7.64−7.67 (m, 2.71H), 7.32−7.38 (m, 3.76H), 7.28−7.29 (m, 1.70H), 7.19 (t, 2.71H), 7.10−7.15 (m, 3.46H), 7.02 (d, J = 6, 2.2H), 6.96−6.98 (d, J = 12, 2.39H), 6.90−6.92 (t, 2.41H), 6.83−6.86 (m, 2.49H), 6.67−6.72 (m, 4.90H), 6.61−6.62 (d, J = 6, 2.39H), 4.52−4.56 (m, 2.88H), 4.45−4.4.49 (m, 1.60H), 4.39−4.43(m, 1.66H), 4.35−4.38 (m, 1.73H), 4.26−4.29 (m, 1.72H), 4.12−4.14 (m, 1.54H), 4.00− 4.03(m, 1.07H), 3.96−3.98 (m, 1.02H), 3.87−3.90 (m, 2.61H), 3.67−3.69 (d, 3.0H), 3.50−3.53(m, 2.51H), 3.13−3.22 (m, 3.62H), 3.01−3.04 (m, 2.20H), 2.84−2.90 (m, 2.64H), 2.59−2.74 (m, 7.56H), 2.38−2.49 (m, 1.96H), 1.86−2.02 (m, 7.27H), 1.83(s, 3.01 H), 0.81− 0.87(m, 6.01H); HR-MS (Q-TOF), calcd for [M + Na]+: 1536.4893, found: 1536.4831. Synthesis of C1−P2. To the solution of 1 (6.5 mg, 9.6 μM) in DMSO (0.4 mL) was added Ac-DDDDYVADCF-NH2 (14.0 mg, 11.52 μM), triphenylphosphine (3 mg, 11.52 μM), and N,Ndiisopropylethylamine (1.45 mg, 11.52 μM). The resulting mixture was stirred at room temperature for 2 h. The mixture was separated using a reverse C18 column and a mixture of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) as the elution buffer. The fraction of acetonitrile was 30% from 0 to 7 min, 30%−98% from 7 to 12 min, 98% from 12.0 to 12.5 min, and then, it was gradually decreased to 30% from 12.5 to 15 min. C1−P2 was obtained as a red
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b14845. High-resolution mass spectra and NMR spectra; the structures of 56 coumarin-originated natural products, AC-YVAD-CMK, and NH2−coumarin; LC profiles of the incubation mixture of caspase-3 and C1−P3; LC profiles of the incubation mixture of selected coumarinoriginated natural products, caspase-1, and YVAD-AMV. UV−vis and PL spectra of selected coumarin-originated natural products and NH2−coumarin (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (B.L.) *E-mail:
[email protected] (Y.T.) *E-mail:
[email protected] (C.-J.Z.) ORCID
Bin Liu: 0000-0002-0956-2777 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
We thank the start-up funding from the Institute of Materia Medica, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Peking Union Medical College, CAMS Innovation Fund for F
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces
(15) Razgulin, A.; Ma, N.; Rao, J. Strategies for in vivo imaging of enzyme activity: an overview and recent advances. Chem. Soc. Rev. 2011, 40, 4186−4216. (16) Leung, C.-H.; Chan, D. S.-H.; Man, B. Y.-W.; Wang, C.-J.; Lam, W.; Cheng, Y.-C.; Fong, W.-F.; Hsiao, W.-L. W.; Ma, D.-L. Simple and Convenient G-Quadruplex-Based Turn-On Fluorescence Assay for 3′→5′ Exonuclease Activity. Anal. Chem. 2011, 83, 463−466. (17) Leung, K.-H.; He, H.-Z.; Ma, V. P.-Y.; Zhong, H.-J.; Chan, D. SH.; Zhou, J.; Mergny, J.-L.; Leung, C.-H.; Ma, D.-L. Detection of base excision repair enzyme activity using a luminescent G-quadruplex selective switch-on probe. Chem. Commun. 2013, 49, 5630. (18) Messerli, S. M.; Prabhakar, S.; Tang, Y.; Shah, K.; Cortes, M. L.; Murthy, V.; Weissleder, R.; Breakefield, X. O.; Tung, C.-H. A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe. Neoplasia 2004, 6, 95−105. (19) Le, G. T.; Abbenante, G.; Madala, P. K.; Hoang, H. N.; Fairlie, D. P. Organic Azide Inhibitors of Cysteine Proteases. J. Am. Chem. Soc. 2006, 128, 12396−12397. (20) Garcia-Calvo, M.; Peterson, E. P.; Leiting, B.; Ruel, R.; Nicholson, D. W.; Thornberry, N. A. Inhibition of Human Caspases by Peptide-based and Macromolecular Inhibitors. J. Biol. Chem. 1998, 273, 32608−32613. (21) Venugopala, K. N.; Rashmi, V.; Odhav, B. Review on Natural Coumarin Lead Compounds for Their Pharmacological Activity. BioMed Res. Int. 2013, 2013, 963248. (22) Lee, N. Y.; Chung, K.-S.; Jin, J. S.; Lee, Y.-C.; An, H.-J. The Inhibitory Effect of Nodakenin on Mast-Cell-Mediated Allergic Inflammation Via Downregulation of NF-kB and Caspase-1 Activation. J. Cell. Biochem. 2017, 118, 3993. (23) Fan, F.; Wood, K. V. Bioluminescent Assays for HighThroughput Screening. Assay Drug Dev. Technol. 2007, 5, 127−136. (24) Thorne, N.; Shen, M.; Lea, W. A.; Simeonov, A.; Lovell, S.; Auld, D. S.; Inglese, J. Inglese, Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter. Chem. Biol. 2012, 19, 1060−1072. (25) Xue, W.; Zhang, G.; Zhang, D.; Zhu, D. A New Label-Free Continuous Fluorometric Assay for Trypsin and Inhibitor Screening with Tetraphenylethene Compounds. Org. Lett. 2010, 12, 2274−2277. (26) Wang, H.; Huang, Y.; Zhao, X.; Gong, W.; Wang, Y.; Cheng, Y. A Novel Aggregation-Induced Emission based Fluorescent Probe for An Angiotensin Converting Enzyme (ACE) Assay and Inhibitor Screening. Chem. Commun. 2014, 50, 15075−15078. (27) Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B. Image-Guided Combination Chemotherapy and Photodynamic Therapy Using a Mitochondria-Targeted Molecular Probe with Aggregation-Induced Emission Characteristics. Chem. Sci. 2015, 6, 4580−4586. (28) Liang, J.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobes based on AIEgen Conjugates. Chem. Soc. Rev. 2015, 44, 2798−2811. (29) Wu, W.; Feng, G.; Xu, S.; Liu, B. A Photostable Far-Red/NearInfrared Conjugated Polymer Photosensitizer with AggregationInduced Emission for Image-Guided Cancer Cell Ablation. Macromolecules 2016, 49, 5017−5025. (30) Wu, W.; Mao, D.; Hu, F.; Xu, S.; Chen, C.; Zhang, C.-J.; Cheng, X.; Yuan, Y.; Ding, D.; Kong, D. A Highly Efficient and Photostable Photosensitizer with Near-Infrared Aggregation-Induced Emission for Image-Guided Photodynamic Anticancer Therapy. Adv. Mater. 2017, 29, 1700548. (31) Fan, Z.; Ren, L.; Zhang, W.; Li, D.; Zhao, G.; Yu, J. AIE Luminogen-Functionalised Mesoporous Silica Nanoparticles as Nanotheranostic Agents for Imaging Guided Synergetic Chemo-/Photothermal Therapy. Inorg. Chem. Front. 2017, 4, 833−839. (32) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718−940. (33) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-Induced Emission: The Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429−5479.
Medical Sciences (CIFMS) (2017-I2M-4−005), CAMS Fund of the Nonprofit Central Research Institutes (2017RC31009), the Singapore National Research Foundation (R279−000− 444−281 and R279−000−483−281), and the National University of Singapore (R279−000−482−133) for financial support. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Prof. Zhang Tiantai and Prof. Li Li for their help in the usage of the microplate reader and luminescence spectrometer (JΛSCO FP-6500).
■
REFERENCES
(1) Alnemri, E. S.; Livingston, D. J.; Nicholson, D. W.; Salvesen, G.; Thornberry, N. A.; Wong, W. W.; Yuan, J. Human ICE/CED-3 Protease Nomenclature. Cell 1996, 87, 171. (2) Earnshaw, W. C.; Martins, L. M.; Kaufmann, S. H. Mammalian Caspases: Structure, Activation, Substrates and Functions During Apoptosis. Annu. Rev. Biochem. 1999, 68, 383−424. (3) Thornberry, N. A.; Bull, H.; Calaycay, J.; Chapman, K.; Howard, A.; Kostura, M.; Miller, D.; Molineaux, S.; Weidner, J.; Aunins, J.; Elliston, K.; Ayala, J.; Casano, F.; Chin, J.; Ding, G.; Egger, L.; Gaffney, E.; Limjuco, G.; Palyha, O.; Raju, S.; Rolando, A.; Salley, J.; Yamin, T.; Lee, T.; Shively, J.; MacCross, M.; Mumford, R.; Schmidt, J.; Tocci, M. A Novel Heterodimeric Cysteine Protease is Required for Interleukin1Bold Betaprocessing in Monocytes. Nature 1992, 356, 768−774. (4) Cerretti, D.; Kozlosky, C. J.; Mosley, B.; Nelson, N.; Van Ness, K.; Greenstreet, T.; March, K.; Kronheim, S. M.; Druck, T.; Cannizzaro, L.; et al. Molecular Cloning of the Interleukin-1 beta Converting Enzyme. Science 1992, 256, 97−100. (5) Howley, B.; Fearnhead, H. O. Caspases as Therapeutic Targets. J. Cell. Mol. Med. 2008, 12, 1502−1516. (6) Mariathasan, S.; Monack, D. M. Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat. Rev. Immunol. 2007, 7, 31−40. (7) Bergsbaken, T.; Fink, S. L.; Cookson, B. T. Pyroptosis: Host Cell Death and Inflammation. Nat. Rev. Microbiol. 2009, 7, 99−109. (8) Miao, E. A.; Rajan, J. V.; Aderem, A. Aspase-1-Induced Pyroptotic Cell Death. Immunol. Rev. 2011, 243, 206−214. (9) Cornelis, S.; Kersse, K.; Festjens, N.; Lamkanfi, M.; Vandenabeele. Inflammatory Caspases: Targets for Novel Therapies. Curr. Pharm. Des. 2007, 13, 367−385. (10) Boost, K. A.; Hoegl, S.; Hofstetter, C.; Flondor, M.; Stegewerth, K.; Platacis, I.; Pfeilschifter, J.; Muhl, H.; Zwissler, B. Targeting Caspase-1 by Inhalation-Therapy: Effects of Ac-YVAD-CHO on IL-1β, IL-18 and Downstream Proinflammatory Parameters as Detected in Rat Endotoxaemia. Intensive Care Med. 2007, 33, 863−871. (11) Doitsh, G.; Galloway, N. L. K.; Geng, X.; Yang, Z.; Monroe, K. M.; Zepeda, O.; Hunt, P. W.; Hatano, H.; Sowinski, S.; Munoz-Arias, A.; et al. Cell Death by Pyroptosis Drives CD4 T-cell Depletion in HIV-1 Infection. Nature 2014, 505, 509−514. (12) Monroe, K. M.; Yang, Z.; Johnson, J. R.; Geng, X.; Doitsh, G.; Krogan, N. J.; Greene, W. C. IFI16 DNA Sensor Is Required for Death of Lymphoid CD4 T Cells Abortively Infected with HIV. Science 2014, 343, 428−432. (13) Wang, W.; Yang, C.; Lin, S.; Vellaisamy, K.; Li, G.; Tan, W.; Leung, C.-H.; Ma, D.-L. First Synthesis of an Oridonin-Conjugated Iridium(III) Complex for the Intracellular Tracking of NF-κB in Living Cells. Chem. - Eur. J. 2017, 23 (2017), 4929−4935. (14) Li, X.; Kim, C.-Y.; Lee, S.; Lee, D.; Chung, H.-M.; Kim, G.; Heo, S.-H.; Kim, C.; Hong, K.-S.; Yoon, J. Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy. J. Am. Chem. Soc. 2017, 139, 10880−10886. G
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Forum Article
ACS Applied Materials & Interfaces (34) Birks, J. B., Ed. Photophysics of Aromatic Molecules; WileyInterscience: London, 1970. (35) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. Real-Time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-Up Probe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 17972−17981. (36) Yuan, Y.; Zhang, C.-J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2015, 54, 1780−1786. (37) Zhang, R.; Zhang, C.-J.; Feng, G.; Hu, F.; Wang, J.; Liu, B. Specific Light-Up Probe with Aggregation-Induced Emission for Facile Detection of Chymase. Anal. Chem. 2016, 88, 9111−9117. (38) You, X.; Li, L.; Li, X.; Ma, H.; Zhang, G.; Zhang, D. A New Tetraphenylethylene Derived-Fluorescence Probe for Nitroreductase Detection and Hypoxic Tumor Cell Imaging. Chem. - Asian J. 2016, 11, 2918−2923. (39) Jiang, G.; Zeng, G.; Zhu, W.; Li, Y.; Dong, X.; Zhang, G.; Fan, X.; Wang, J.; Wu, Y.; Tang, B. Z. A Selective and Light-Up Fluorescent Probe for β-Galactosidase Activity Detection and Imaging in Living Cells based on An AIE Tetraphenylethylene Derivative. Chem. Commun. 2017, 53, 4505−4508. (40) Cao, F.-Y.; Long, Y.; Wang, S.-B.; Li, B.; Fan, J.-X.; Zeng, X.; Zhang, X.-Z. Fluorescence Light-Up AIE Probe for Monitoring Cellular Alkaline Phosphatase Activity and Detecting Osteogenic Differentiation. J. Mater. Chem. B 2016, 4, 4534−4541. (41) Zhang, C.-J.; Feng, G.; Xu, S.; Zhu, Z.; Lu, X.; Wu, J.; Liu, B. Structure-Dependent cis/trans Isomerization of Tetraphenylethene Derivatives: Consequences for Aggregation-Induced Emission. Angew. Chem., Int. Ed. 2016, 55, 6192−6196. (42) Yuan, Y.; Zhang, C.-J.; Kwok, R. T. K.; Xu, S.; Zhang, R.; Wu, J.; Tang, B. Z.; Liu, B. Light-Up Probe for Targeted and Activatable Photodynamic Therapy with Real-Time In Situ Reporting of Sensitizer Activation and Therapeutic Responses. Adv. Funct. Mater. 2015, 25, 6586−6595. (43) Zhang, R.; Feng, G.; Zhang, C.-J.; Cai, X.; Cheng, X.; Liu, B. Real-Time Specific Light-Up Sensing of Transferrin Receptor: Image Guided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration. Anal. Chem. 2016, 88, 4841−4848. (44) Feng, G.; Zhang, C.-J.; Lu, X.; Liu, B. Zinc(II)-TetradentateCoordinated Probe with Aggregation-Induced Emission Characteristics for Selective Imaging and Photoinactivation of Bacteria. ACS Omega. 2017, 2, 546−553.
H
DOI: 10.1021/acsami.7b14845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
