A highly selective fluorescent probe for real-time imaging of UDP-glucuronosyltransferase 1A8 in living cells and tissues

Abstract

Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are enzymes involved in the biotransformation of important endogenous compounds such as steroids, bile acids, and hormones as well as exogenous substances including drugs, environmental toxicants, and carcinogens. Here, a novel fluorescent probe BDMP was developed based on boron-dipyrromethene (BODIPY) with high sensitivity for the detection of UGT1A8. The glucuronidation of BDMP not only exhibited a red-emission wavelength (λ_ex/λ_em = 500/580 nm), but also displayed an excellent UGT1A8-dependent fluorescence signal with a good linear relationship with UGT1A8 concentration. Based on this perfect biocompatibility and cell permeability, BDMP was successfully used to image endogenous UGT1A8 in human cancer cell lines (LoVo and HCT15) in real time. In addition, BDMP could also be used to visualize UGT1A8 in tumor tissues. These results suggested that BDMP is a promising molecular tool for the investigation of UGT1A8-mediated physiological function in humans.

Key words： UDP-glucuronosyltransferase 1A8; fluorescent probe; subtype selectivity; fluorescence imaging

1 Introduction

Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are a family of phase II metabolic enzymes that play vital role in the metabolism of endogenous substrates (e.g., bilirubin, steroids, fatty acids thyroid hormones, and bile acids) and exogenous substrates (e.g., clinic drugs and environmental toxins) [1–5]. They catalyze the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to various groups in drugs or xenobiotic such as hydroxyl, carboxyl, or amine groups [6–8]. Approximately 40%–70% of drugs can be metabolized by UGTs; moreover, the inhibition of UGTs also causes potential drug-drug interactions and disorders with endogenous substances [9]. UGTs are widely expressed in various organs in humans, animals, and plants. In humans, these species are located in the microsomal fraction of various tissues, including liver, kidney, skin, intestine, and brain [10]. To date, one hundred and seventeen members of the mammalian UGT gene superfamily has been reported; these were divided into four families: UGT1, UGT2, UGT3, and UGT8 [11,12]. Of these, UGT1 and UGT2 are the most studied families in the metabolism of endogenous and exogenous chemicals. In detail, the UGT1 family consists of nine isoforms, namely, UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, and 1A10; the catalytic functions have all been well characterized. The UGT2B family is composed of six major isoforms, namely, UGT2B4, 2B7, 2B10, 2B11, 2B15, and 2B17.

The liver is an important metabolic organ with the greatest abundance of UGTs. The gastro-intestinal tract is also a significant extra-hepatic site for drug metabolism. Intestinal UGTs play a significant role in the changes of drug pharmacokinetics and drug-drug interactions [12,13]. Among various UGTs isoforms, UGT1A8 is an extra-hepatic drug metabolic UGTs isoform mainly expressed in the small intestine and colon [14,15]. UGT1A8 is a major enzyme in the elimination of endogenous estrogen [16]. A recent study indicated that UGT1A8 was closely related to the development of various cancers such as endometrial cancer and colorectal cancer [17]. Therefore, a sensitive and efficient detection tool for endogenous UGT1A8 is urgently needed for the relational use of drugs and for disease diagnosis.

Recently, small molecule fluorescent probes have been widely used for the rapid detection of enzyme activity, ions, pH, and reactive oxygen species. They have prominent advantages such as high sensitivity, non-invasiveness, and ultrahigh imaging resolution [18–30]. In humans, various metabolic enzymes play an important role in homeostasis, and their activity and dysregulation during disease have been compared to healthy subjects. Thus, enzyme-activated fluorescent probes are increasingly common for the real-time detection and imaging of key enzymes [31–35].

UGT1A8 is a significant UGT isoform that participates in the detoxification of environmental toxins and metabolites of drugs; surprisingly, few fluorescent probes for other subtypes of UGTs have been developed [36–38]. Unfortunately, there is no selective fluorescent probe for the detection of UGT1A8. Here, based on the optimal properties of boron-dipyrromethene (BODIPY), we designed a UGT1A8-activated fluorescent probe BDMP with good selectivity and stability and that exhibited ultrahigh imaging resolution in the imaging of UGT1A8 in various living cells and tissues (Scheme 1).

Fig.1 Scheme 1 The glucuronidation reaction of BDMP mediated by UGT1A8.

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2 Experimental

2.1 Materials and reagents

The enzymes include UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17. These were all obtained from Corning Gentest (NY, USA). Bovine serum albumin, glutathione, serine, tryptophan, tyrosine, glutamate, glycine, arginine, cysteine, lysine, glutamine, myristic acid, and glucose were purchased from Shanghai Yuanye (Shanghai, China). Metal ions including Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺, Sn⁴⁺, K⁺, Cu²⁺, Fe³⁺, Na⁺, Ba²⁺, Cr⁶⁺, NO₃^–, CO₃^2–, and SO₄^2– were purchased from Shanghai Yuanye (Shanghai, China). Phenylbutazone, nilotinib, fluconazole, diclofenac, β-estradiol, deoxyschizandrin, hecogenin, magnolol, and propofol were purchased from J&K Chemicals (Beijing, China). LoVo cells and HCT15 cell lines were purchased from ATCC (Manassas, VA).

Nuclear magnetic resonance (NMR) spectra were analyzed on Bruker Avance II (400 MHz) spectrometer. All of the fluorescence tests were analyzed on Synergy H1 Microplate Reader (Bio-Tek). The reaction samples were analyzed by a Waters e2695 HPLC (high performance liquid chromatography) equipped with a photo-diode array detector. High-resolution mass data were measured on G6224A TOF MS. All of the other reagents and solvents used were of the highest grade commercially available.

2.2 Design and synthesis of BDMP for UGT1A8

Synthesis of compound 1. Compound 1 was synthesized according to the literature [39]. The ¹H NMR data (400 MHz, CDCl₃) include: δ 7.64 (s, 1H), 7.20 (s, 1H), 6.93 (d, J = 2.9 Hz, 1H), 6.43 (s, 1H), 6.16 (s, 1H), 2.59 (s, 3H), and 2.28 (s, 3H) (Scheme S1, cf. Electronic Supplementary Material, ESM).

Synthesis of BDMP. Piperidine (170.3 mg, 2.0 mmol) and AcOH (120.1 mg, 2.0 mmol) were sequentially added to a dry toluene solution (20 mL) of compound 1 (44.0 mg, 0.2 mmol) and 2-bromo-3-hydroxy-4-methoxybenzaldehyde (55.2 mg, 0.24 mmol) under an argon atmosphere; the mixture was refluxed for 2 h. After removing the solvent, the residues were further purified by a silica gel column chromatograph using CH₂Cl₂/MeOH (100/1 v/v) as the mobile phase to afford BDMP as a dark red solid (69.7 mg, yield: 80.7%). ¹H NMR (400 MHz, DMSO-d₆, δ_ppm): 9.73 (s, 1H), 7.82 (t, J = 8.1 Hz, 2H), 7.73 (s, 1H), 7.33 (d, J = 16.1 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.12 (dd, J = 8.8, 4.0 Hz, 3H), 6.53 (dd, J = 3.8, 2.0 Hz, 1H), 3.90 (s, 3H), 2.35 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆, δ_ppm): 157.52, 149.63, 145.59, 144.17, 138.86, 138.53, 137.26, 132.97, 127.94, 126.70, 124.91, 118.14, 118.04, 117.47, 116.62, 112.31, 111.16, 56.25, and 11.06. HRMS (ESI negative) (high resolution mass spectrometry (electrospray ionization negative)): calcd. [M − H]^– 431.0384, found 431.0392 (Figs. S1–S4, cf. ESM).

2.3 Incubation system for the in vitro assay

In the in vitro assay, all of the evaluations of UGT1A8 activity were performed in a standard incubation system including: 50 mmol·L^–1 tris-HCl (pH 7.4), 50 mmol·L^–1 MgCl₂, 40 mmol·L^–1 UDPGA, fluorescent probe and recombinant UGT1A8 with a final incubation volume of 0.2 mL. After 3 min pre-incubation of fluorescent probe and enzyme, UDPGA was added as a cofactor to start the glucuronidation reaction, and incubated at 37 °C for another 30 min. Finally, the reaction was terminated upon addition of 100 μL ice-cold acetonitrile. The mixture was centrifuged at 4 °C at 20000 g for 20 min to remove the precipitate and retain the supernatant for further fluorescence experiments. The reaction samples were then analyzed by HPLC and mobile phases including A: 0.03% trifluoroacetic acid water; B: 100% MeOH. The gradient method was as follows: 0–10 min 80% A; 10– 30 min 80%–10% A; 30–35 min 10% A; 35–40 min 10%–80% A; and 40–45 min 80% A. The flow rate was set to 0.8 mL·min^–1. Control incubations without enzyme were performed to ensure the metabolite formation was enzyme dependent.

2.4 Selectivity assays of BDMP

A selectivity assay was next performed to investigate whether BDMP possessed optimal selectivity toward UGT1A8. Briefly, BDMP (10 μmol·L^–1) was added into the assay system in the presence of 13 different isoforms of UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17. The concentration of UGTs isoforms was set at 25 μg·mL^–1 and incubated at 37 °C for 30 min, and then 100 μL ice-cold acetonitrile was added to stop the reaction for the fluorescence assay. To investigate the stability of BDMP, the influence of common metal ions (Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺, Sn⁴⁺, K⁺, Cu²⁺, Fe³⁺, Na⁺, Ba²⁺, Cr⁶⁺, NO₃^–, CO₃^2–, and SO₄^2–) were evaluated on BDMP along with glutathione, serine, tryptophan, tyrosine, glutamate, glycine, arginine, cysteine, lysine, glutamine, myristic acid, glucose; the concentration of metal ions and amino acids was 10 μmol·L^–1.

2.5 Chemical inhibition

To further confirm whether the fluorescence changes were selectively mediated by UGT1A8, different kinds of inhibitors of UGTs including phenylbutazone (500 μmol·L^–1), nilotinib (0.5 μmol·L^–1), gluconazole (2.5 mmol·L^–1), diclofenac (50 μmol·L^–1), β-estradiol (100 μmol·L^–1), deoxyschizandrin (20 μmol·L^–1), hecogenin (10 μmol·L^–1), magnolol (10 μmol·L^–1), and propofol (200 μmol·L^–1) were added to the reaction system [40–43]. The inhibition curve of different concentrations of nilotinib toward UGT1A8-mediated glucuronidation of BDMP was performed for a IC₅₀ value. The inhibition activity was obtained by comparing the fluorescence intensity of the inhibitor group; the control group used blank solvent to replace the inhibitors.

2.6 Kinetic study

A kinetic study was performed to estimate the glucuronidation metabolism of BDMP. Here, BDMP (0– 50 μmol·L^–1) was incubated with UGT1A8 for 30 min with a UGT1A8 concentration of 12.5 μg·mL^–1. Both incubation time and protein concentrations were selected within the linear interval to ensure that less than 20% of the substrate was metabolized and the formation of the glucuronidation metabolite was in the linear range [1,44,45]. Finally, the kinetic curve was fitting into the substrate inhibition formula (Eq. (1)) with the following formula:

(1)v={\displaystyle \frac{V_{\max }\left[S\right]}{K_{\mathrm{m}}+\left[S\right]+{\left[S\right]}^{\mathrm{2}}/K_{\mathrm{i}}}}

Here, V_max represents the maximum velocity; K_m is the substrate concentration at half-maximal rate, and K_i represents the dissociation constant of substrate binding to the inhibitory site. Kinetic curves were fitted using GraphPad Prism 6 and represent the mean ± SD.

2.7 Cell culture and fluorescence imaging

LoVo cells and HCT15 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained in 5% CO₂ at 37 °C. The cells were seeded on a 20 mm confocal dish and then cultured overnight. The media was then replaced with FBS-free media containing 2 μmol·L^–1 BDMP and incubated for another 1 h. In the inhibition group, 20 μmol·L^–1 nilotinib was pre-added into the cells and incubated with cells for 30 min in 5% CO₂ at 37 °C. The media was replaced with FBS-free media containing 2 μmol·L^–1 BDMP and 20 μmol·L^–1 nilotinib incubated with the remaining living cells for another 1 h under the same conditions. The culture media was then discarded, and the cells were washed with phosphate buffer saline (PBS) three times and imaged on a confocal microscope (Leica TCS SP8).

2.8 Fluorescent imaging of cancer tissues slices

The tissue slices from colon tumors were prepared from frozen sections. The slices were incubated with 2 μmol·L^–1 BDMP at 37 °C for 1 h, washed with PBS for 3 times, and then incubated with 4% paraformaldehyde at room temperature for 10 min. Finally, the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI)-containing Vecta shield solution (Vector Laboratories Inc.). The expression of UGT1A8 in tumors was imaged on a confocal microscope (Leica TCS SP8), and the conditions were consistent with the methods above.

3 Results and discussion

3.1 Spectrum response of BDMP toward UGT1A8

The spectroscopic properties change of BDMP after incubating with UGT1A8 were evaluated. As shown in Figs. 1(a) and 1(b), after metabolizing by UGT1A8, the absorption at 558 nm exhibited a significant enhancement, and a remarkable fluorescence signal was observed at 580 nm. In addition, in the HPLC analysis, we observed a new chromatographic peak in the incubation sample of BDMP in the presence of UGT1A8. The HRMS (ESI negative) was calculated for C₂₅H₂₃BBrF₂N₂O₈^– that was found at m/z 607.0707 [M − H]^–, which corresponds to the glucuronidation product of BDMP (Fig. S5, cf. ESM). These results further confirmed the glucuronidation reaction for BDMP catalyzed by UGT1A8.

Fig.2 (a) Absorption and (b) fluorescence emission spectra of BDMP (10 μmol·L^–1) before and after incubating with UGT1A8; (c) the HPLC analysis of BDMP and the incubation sample with UGT1A8.

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3.2 Fluorescence response of BDMP toward UGT1A8 at different concentrations

In order to accurately determine the activity of the target enzyme, it is necessary to investigate the relationship between fluorescence intensity and enzyme concentration. As shown in Fig. 2, the fluorescence intensity changes of BDMP mediated by UGT1A8 gradually increased and exhibited a good linear relationship with the increase of the concentration of UGT1A8 from 0 to 25 μg·mL^–1 range. These results indicated that BDMP could serve as a novel tool for the real-time activity assaying of UGT1A8 in complex biosamples.

Fig.3 (a) The fluorescence emission spectrum of BDMP (10 μmol·L^–1) after incubating with different concentrations of UGT1A8; (b) the linear regression of fluorescence intensity with UGT1A8 concentration.

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3.3 Selectivity study of BDMP toward UGT1A8

To investigate the selectivity of BDMP toward UGT1A8, the probe was incubated with 13 UGTs isoforms including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17. As shown in Fig. 3, UGT1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 could trigger the fluorescence enhancement of BDMP (Fig. 3(a)). Of these, UGT1A8 exhibited a relative prominent activity. In addition, as shown in Fig. 3(b), common endogenous substances including metal ions and amino acids did not have any influence on the fluorescence signal of BDMP. Next, the effects of different chemical inhibitors on UGT1A8-mediated reactions were evaluated as shown in Fig. 3(c). Nilotinib had the strongest inhibitory effect on the reaction. The IC₅₀ value of Nilotinib toward BDMP glucuronidation was 1.863 ± 0.037 μmol·L^–1 (Fig. 3(d)). These results demonstrated that BDMP displayed a high selectivity toward UGT1A8 and could be used to determine the real biological activity of UGT1A8 in complex biological systems.

Fig.4 (a) Fluorescence intensity of BDMP (10 μmol·L^–1) incubating with 13 isoforms of UGTs; (b) the stability of BDMP among metal ions and amino acids; (c) chemical inhibition of different inhibitors on UGT1A8 mediating the glucuronidation reaction; (d) the inhibition curve of Nilotinib toward the BDMP glucuronidation.

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3.4 Kinetic study

To clarify the metabolism progress of BDMP, the kinetic behavior of BDMP glucuronidation in the presence of UGT1A8 was analyzed as shown in Fig. 4. The glucuronidation reaction had a substrate inhibition model kinetic for UGT1A8, and the dynamic parameters K_i and K_m were calculated to be 27.04 μmol·L^–1 and 1.35 μmol·L^–1, respectively (Fig. 4). These results indicated that BDMP possessed excellent affinity toward UGT1A8, which provided assistance for the activity of UGT1A8 in complex biosamples.

Fig.5 The kinetic analysis of BDMP in the presence of UGT1A8.

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3.5 Fluorescence imaging of BDMP in living cells and cancer tissues

UGT1A8 is an extrahepatic enzyme that is especially highly expressed in intestine, and plays a significant role in the detoxification of environmental carcinogens. Herein, BDMP was applied to image the activity of UGT1A8 in different colon cancers. First, a CCK8 cytotoxicity test showed that BDMP had no cytotoxicity to LoVo cells and HCT15 cells (Fig. S6, cf. ESM). Figure 5 showed that the fluorescence signal was detected in HCT15 cells after incubating with BDMP, which indicated that BDMP had excellent cell permeability; its biotransformation was mediated by endogenous UGT1A8. Furthermore, the UGTs inhibitor nilotinib could significantly suppress the fluorescence signal in living cells (Fig. 5(i)). The UGT1A8 could also be imaged in LoVo cells with BDMP (Fig. S7, cf. ESM). BDMP is a novel fluorescent probe for UGT1A8 and it has good imaging capabilities in the detection of intracellular UGT1A8. We next investigated cancer tissue imaging capabilities. Figure 6 showed that after incubating with BDMP for 1 h, the fluorescence signal was detected in the cytoplasm of cancer tissue slices. These results indicated that BDMP could be successfully used to detect endogenous UGT1A8 in tumor tissues and living cells.

Fig.6 (a, e, i) The fluorescence of HCT-15 cells; (b, f, j) staining of nuclei by Hoechst 33342; (c, g, k) bright field images of HCT15 cells; (d, h, l) merge of the confocal fluorescence images (The scale bar is 50 μm. BDMP: λ_ex 514 nm; λ_em 550–610 nm).

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Fig.7 (a, d, g) Fluorescence microscope imaging of UGT1A8 for the tissue slices of normal LoVo tumor tissues; (b, e, h) nuclear staining with DAPI; (c, f, i) merged confocal fluorescence images (The scale bars are 50 and 25 μm. λ_ex 514 nm; λ_em 550–610 nm).

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4 Conclusions

In conclusion, a novel fluorescent probe BDMP for the detection of endogenous UGT1A8 was successfully developed using a BODIPY skeleton. BDMP could be metabolized by UGT1A8 to form a single glucuronidation metabolite, and possessed high sensitivity toward UGT1A8 on the basis of the isoform screening and chemical inhibition assay. Furthermore, BDMP exhibited excellent biocompatibility and could be further applied to real-time imaging of the endogenous UGT1A8 in various living cells and tissues. These findings demonstrate that BDMP is a promising tool for further investigation of the biological functions of endogenous UGT1A8 in many physiological and pathological processes.

Declaration of competing interest

All the authors declare no competing financial interest.

Acknowledgments

The authors thank the Natural Science Foundation of Liaoning Province 2020-MS-252 and the National Key R&D Program of China (Grant No. 2018YFC1603001).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://dx.doi.org/10.1007/s11705-021-2064-8 and is accessible for authorized users.

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