Showing papers on "Esterase published in 2022"

Esterase-Activated Theranostic Prodrug for Dual Organelles-Targeted Imaging and Synergetic Chemo-Photodynamic Cancer Therapy

Abstract: Open AccessCCS ChemistryRESEARCH ARTICLE1 Mar 2022Esterase-Activated Theranostic Prodrug for Dual Organelles-Targeted Imaging and Synergetic Chemo-Photodynamic Cancer Therapy Jiabao Zhuang, Nan Li, Yaling Zhang, Baolin Li, Hanqi Wen, Xinchan Zhang, Tianyu Zhang, Na Zhao and Ben Zhong Tang Jiabao Zhuang Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Nan Li Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Yaling Zhang Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Baolin Li Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Hanqi Wen Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Xinchan Zhang Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Tianyu Zhang Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author , Na Zhao *Corresponding author: E-mail Address: [email protected] Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710019 Google Scholar More articles by this author and Ben Zhong Tang Department of Chemistry, The Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute for Advanced Study, Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100985 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Activatable prodrugs have received considerable attention in cancer therapy due to their high specificity and reduced side effects. However, the theranostic prodrug with multiple cancerous organelles targeting and combinational therapy is still rare. In this report, an esterase-responsive prodrug tetraphenylethylene functionalized quinolinium-ester-chlorambucil (TPE-QC) was developed for dual organelles-targeted and image-guided cancer therapy through synergetic chemotherapy (CT) and photodynamic therapy (PDT). TPE-QC was constructed by conjugating an anticancer drug of chlorambucil with an aggregation-induced emission active photosensitizer of tetraphenylethylene functionalized hydroxyethyl quinolinium (TPE-QO) via the hydrolyzable ester linkage. The fluorescence and photosensitization of TPE-QC were initially quenched because of the photoinduced electron transfer (PET) effect. After reacting with esterase, the ester group of TPE-QC could be selectively hydrolyzed to release chlorambucil and TPE-QO, which terminated the PET process and switched on the fluorescence and photosensitization. Benefitting from the overexpressed esterase in cancer cells, TPE-QC could be efficiently activated in cancer cells rather than in normal cells, while the restored fluorescence could precisely monitor the release of TPE-QC. Importantly, activated TPE-QC accumulated in both organelles of lysosome and mitochondria, resulting in enhanced anticancer potency. In vivo experiments demonstrated that TPE-QC displayed efficient tumor microenvironment-activatable features and excellent tumor therapeutic effects through combinational CT and PDT. Download figure Download PowerPoint Introduction Malignant solid tumors have threatened people’s lives worldwide during the past decades.1–3 Chemotherapy (CT) is the standard of care for cancer treatment, and lots of small molecular blockbuster anticancer drugs, such as taxol, doxorubicin, chlorambucil, and cisplatin have been reported for CT.4–9 Nevertheless, the lack of tumor specificity, systemic instability, and poor bioavailability of typical anticancer drugs contribute to CT’s severe side effects, including immunosuppression, myelosuppression, and significantly reduced therapeutic efficiency.10,11 This not only influences the quality of life of patients, but makes them prone to developing corresponding resistance toward specific treatments, further reducing the rate of long-term survival. Therefore, development of a facile approach for timely diagnosis and precise treatment of malignant tumors is a highly desirable and critical challenge. The combination of CT with another therapeutic modality, in particular photodynamic therapy (PDT), has been considered a promising strategy for cancer treatment.12–14 PDT is a non-invasive and remote spatiotemporal therapeutic method, in which the photosensitizer (PS) is activated by light irradiation to generate reactive oxygen species (ROS), remarkably singlet oxygen (1O2), to damage cellular functions and finally induce programmed cell death.15–22 Owing to the limited effective radius (<20 nm) with short half-life (<40 ns), the 1O2 efficiently induces cytotoxicity in the desired region with high spatiotemporal resolution.23–25 Consequently, the PS with accurate localization in subcellular organelles is desirable for improving the PDT effect and minimizing the cytotoxicity to normal organs. Additionally, in recent years PSs with aggregation-induced emission (AIE) characteristics have attracted a lot of attention. Taking advantage of their intrinsic features, AIE PSs not only display intense fluorescence but also give high ROS generation efficiency in the aggregated state, which is favorable for precise image-guided PDT.26–31 It is worth mentioning that some AIE PSs with specificity for subcellular organelles, including mitochondria, plasma membrane, and lysosome, have been fabricated to strengthen the PDT effect.32–36 Nevertheless, their therapeutic efficacy is limited because of the “always-on” model as well as their low specificity toward tumors. The prodrug with unique tumor microenvironment responsiveness is the ideal agent for cancer therapy due to its improved tumor specificity and minimized side effects. This kind of prodrug usually needs the help of an endogenous pathological trigger in cancer cells, including intracellular thiols,37–41 acidic pH,42,43 ROS,44–46 hypoxia,47,48 and overexpression of specific enzymes,49–53 to spatiotemporally control the progression of neoplastic diseases. Among emergent examples, the enzyme-triggered prodrug is of particular interest for cancer therapy since cancer-specific enzymes can enhance the selectivity of the prodrug for cancer cells. Esterase, one of the overexpressed enzymes within cancer cells, plays a vital role in invasion, migration and growth of malignant tumors, which makes it as a promising cancerous target.54,55 Given the combined concerns above, it is highly desirable to develop the activatable prodrug which not only targets the cancerous organelles, but also integrates both PDT and CT processes. In this report, we developed an esterase-activated prodrug tetraphenylethylene functionalized quinolinium-ester-chlorambucil (TPE-QC) for dual organelles targeted and image-guided cancer therapy by eliciting its pharmacological responses through the actions of PDT and CT. TPE-QC was synthesized with an AIE-active PS [tetraphenylethylene functionalized hydroxyethyl quinolinium (TPE-QO)], an anti-cancer drug (chlorambucil), and an ester linker (Scheme 1). The fluorescence and photosensitization of TPE-QC were efficiently quenched. However, the ester group of TPE-QC could be selectively hydrolyzed in the presence of esterase, resulting in the release of TPE-QO and chlorambucil for fluorescence imaging, PDT, and CT, respectively. Taking advantage of the high expression level of esterase in cancer cells, TPE-QC could be selectively activated in cancer cells. Meanwhile, the restored fluorescence could track the TPE-QC activation process in real time. It is interesting that the activated TPE-QC exactly accumulated in both organelles of lysosome and mitochondria, which strongly promoted its therapeutic effect on cancer cells. In vivo applications indicated that TPE-QC exhibited remarkable tumor microenvironment-activated ability and efficiently inhibited the growth of tumors through synergetic PDT and CT. Scheme 1 | Chemical structure of prodrug TPE-QC and its working mechanism in the presence of esterase. Download figure Download PowerPoint Experimental Methods Detailed materials and instruments, experimental procedures, cell imaging, and characterization data are available in the Supporting Information. Synthesis of TPE-QO The tetraphenylethylene functionalized quinoline (TPE-QN) was prepared according to the previous literature.56 TPE-QN (612 mg, 1.26 mmol) and 2-bromoethanol (788 mg, 6.30 mmol) were dissolved in 5.0 mL dimethylformamide (DMF). The mixture was stirred at 90 °C for 24 h ( Supporting Information Scheme S1). After the reaction was completed based on thin-layer chromatography (TLC) analysis, the DMF was removed under reduced pressure and the residue was purified by column chromatography on silica gel (eluent: CH2Cl2/MeOH) to give the desired product of TPE-QO (Yield: 49%). 1H NMR [600 MHz, dimethylsulfoxide (DMSO)-d6, δ] 9.29 (d, J = 6.4 Hz, 1H, Ar H), 9.01 (d, J = 8.5 Hz, 1H, Ar H), 8.60 (d, J = 8.9 Hz, 1H, Ar H), 8.50 (d, J = 6.4 Hz, 1H, Ar H), 8.24 (d, J = 16.0 Hz, 1H, vinyl CH), 8.20 (t, J = 7.6 Hz, 1H, Ar H), 8.08 (d, J = 15.9 Hz, 1H, vinyl CH), 7.98 (t, J = 7.7 Hz, 1H, Ar H), 7.79 (d, J = 8.0 Hz, 2H, Ar H), 7.19–7.08 (m, 11H, Ar H), 7.05–6.98 (m, 6H, Ar H), 5.22 (t, J = 5.6 Hz, 1H, OH), 5.11 (brs, 2H, CH2), 3.94 (d, J = 4.5 Hz, 2H, CH2). 13C NMR (151 MHz, DMSO-d6, δ) 152.72, 148.20, 145.73, 142.95, 142.87, 142.71, 142.48, 141.57, 139.92, 138.00, 134.79, 133.62, 131.27, 130.68, 130.63, 130.55, 129.00, 128.42, 127.94, 127.88, 127.77, 126.85, 126.71, 126.66, 119.76, 119.28, 116.02, 58.95, 58.76. High-resolution mass spectrometry (HRMS) [electrospray ionization time-of-flight (ESI-TOF)] m/z: [M–Br]+ calcd for C39H32NO+, 530.2478; found, 530.2478. Synthesis of TPE-QC TPE-QO (500 mg, 0.82 mmol), chlorambucil (250 mg, 0.82 mmol), dicyclohexylcarbodiimide (254 mg, 1.23 mmol), and 4-dimethylaminopyridine (500 mg, 0.82 mmol) were dissolved in 5.0 mL pyridine. The mixture was stirred at room temperature for 36 h ( Supporting Information Scheme S1). When the reaction was complete based on TLC analysis, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate to CH2Cl2/MeOH) to give the desired product of TPE-QC (Yield: 31%). 1H NMR (400 MHz, CDCl3, δ) 10.32 (d, J = 5.6 Hz, 1H, Ar H), 8.49 (t, J = 9.2 Hz, 2H, Ar H), 8.23 (d, J = 6.0 Hz, 1H, Ar H), 8.11 (t, J = 7.7 Hz, 1H, Ar H), 7.88 (t, J = 7.6 Hz, 1H, Ar H), 7.76 (d, J = 16.0 Hz, 1H, vinyl CH), 7.71 (d, J = 16.0 Hz, 1H, vinyl CH), 7.46 (d, J = 8.1 Hz, 2H, Ar H), 7.15–7.03 (m, 17H, Ar H), 6.96 (d, J = 8.3 Hz, 2H, Ar H), 6.57 (d, J = 8.4 Hz, 2H, Ar H), 5.69 (s, 2H, CH2), 4.72 (s, 2H, CH2), 3.68–3.64 (m, 4H, CH2), 3.60–3.56 (m, 4H, CH2), 2.38 (t, J = 7.4 Hz, 2H, CH2), 2.21 (t, J = 7.4 Hz, 2H, CH2), 1.75–1.68 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3, δ) 173.23, 153.71, 149.83, 147.59, 144.46, 144.00, 143.42, 143.32, 143.22, 142.76, 140.03, 138.59, 135.37, 132.96, 132.39, 131.46, 131.38, 130.24, 129.73, 129.38, 128.20, 128.07, 128.02, 127.83, 127.09, 126.94, 126.15, 125.62, 119.07, 118.51, 117.16, 112.26, 62.10, 55.45, 53.66, 40.69, 33.84, 33.23, 26.38. HRMS (ESI-TOF) m/z: [M–Br]+ calcd for C53H49Cl2N2O2+, 815.3166; found, 815.3158. Esterase-triggered emission enhancement The DMSO stock solution of TPE-QC (1 mM) was diluted into a solvent of DMSO/phosphate-buffered saline (PBS) (v/v = 1/99, pH 7.4) to give final concentration of 10 μM. Then TPE-QC (10 μM) solution was incubated with a different concentration of esterase (0–0.2 U mL−1) at 37 °C for 45 min, and the change of fluorescence was collected immediately. Activatable fluorescence imaging in cancer cells Both cancer (HeLa or MCF-7) and normal (NIH-3T3) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), and incubated at 37 °C in 5% CO2 atmosphere. The cells were seeded onto 35 mm glass-bottom dishes and allowed to grow until the confluence reached 80%. Prior to the experiments, the DMEM medium was removed, and the corresponding adherent cells were washed twice with PBS buffer. The TPE-QC stock solution (10 mM) was added into cell plates in DMEM to give the final concentration of 10 μM, and the cells were incubated for different times at 37 °C. Then the medium was removed, and the Hoechst 33342 stock solution (1 mg mL−1) was added into the cell plates in DMEM to give the final concentration of 10 μg mL−1. After incubating for 10 min at 37 °C, the cells were washed twice with PBS buffer and subsequently used for imaging. For the esterase activity inhibition, the HeLa cells were pretreated with 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) (10 mM) in PBS buffer for 30 min at 37 °C and then stained with TPE-QC and Hoechst 33342 according to the above procedures. TPE-QC: λex = 488 nm, λem = 520–620 nm. Hoechst 33342: λex = 405 nm, λem = 440–480 nm. Cell colocalization of activated TPE-QC The culture medium was removed, and adherent HeLa cells were washed with PBS buffer initially. The LysoTracker Red stock solution (750 μM) or MitoTracker DR (1 mM) was added into the cell plates in DMEM to give the final concentration of 75 or 100 nM, and the cells were incubated for 30 min at 37 °C. After that, the medium was removed, and TPE-QC stock solution (10 mM) was added into the cell plates to give the final concentration of 10 μM. The resultant cells were incubated for 15 and 45 min at 37 °C, respectively. Then the medium was removed and the Hoechst 33342 stock solution (1 mg mL−1) was added into the cell plates in DMEM to give the final concentration of 10 μg mL−1, and the cells were incubated for 10 min at 37 °C. The resultant cells were washed twice with PBS buffer and subsequently used for imaging. For lysosome destruction, the cells were pretreated with the chloroquine (CQ) solution (200 μM) for 30 min at 37 °C. For mitochondria destruction, the cells were pretreated with the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) solution (100 μM) for 30 min at 37 °C. After that, the cells were stained according to the above colocalization procedures. TPE-QC: λex = 488 nm, λem = 520–620 nm. LysoTracker Red: λex = 559 nm, λem = 570–650 nm. MitoTracker DR: λex = 635 nm, λem = 655–755 nm. Hoechst 33342: λex = 405 nm, λem = 440–480 nm. Cytotoxicity assay HeLa cells or NIH-3T3 cells were initially seeded at a density of 10,000 cells per well and incubated for 24 h at 37 °C in 5% CO2 atmosphere. TPE-QO, TPE-QC, and chlorambucil were dissolved in DMSO solution to give a 10 mM stock solution, respectively. The specific amount of the above stock solution was added into a cell culture medium to give the desired concentration and incubated for 2 h. Then the medium was replaced by fresh DMEM and selected wells were exposed to white light irradiation (25 mW cm−2, 30 min). For the inhibition group, the cells were pretreated with AEBSF (10 mM) in PBS buffer for 30 min and then incubated with different concentration of TPE-QC for 2 h. After that, all cells were further cultured for 48 h under dark. The resultant cells were incubated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) for 4 h, and the formed formazan crystals were solubilized in 100 μL of lysate buffer. Absorbance at 570 nm of each well was measured on a SpectraMax M384 (Molecular Devices, USA), and the data was recorded using Softmax Pro 6.4 software (Molecular Devices). Activatable ROS generation in cancer cells The HeLa cells were treated with 100 μM of 2′,7′-dichlorofluorescin diacetate (DCF-DA) for 15 min under dark. The resultant cells were washed twice with PBS buffer and then incubated with TPE-QC (10 μM) for 45 min under dark. After that, the cells were washed with PBS buffer and exposed to white light irradiation (25 mW cm−2), and then the images were collected with the elapse of irradiation time. For the control group, the cells were incubated with DCF-DA (100 μM) alone for 15 min, and then cells were washed with PBS buffer and exposed to the white light irradiation (25 mW cm−2) for different times. DCF-DA: λex = 488 nm, λem = 500–550 nm. Cell apoptosis detection The HeLa cells were initially incubated with 10 μM of TPE-QO or TPE-QC for 45 min and washed twice with PBS. Then the cells were exposed to white light irradiation (25 mW cm−2) for 30 min. In the parallel experiment, the cells were treated with or without the esterase inhibitor of AEBSF (10 mM) for 30 min at 37 °C in PBS. After removing the inhibitor by PBS washing, the cells were incubated with TPE-QC, TPE-QO, or chlorambucil for 45 min, respectively. The cells were washed with PBS buffer to remove the remaining reagents, and the corresponding cells were incubated under dark for another 30 min. All resultant cells were cultured for 1 h and then stained with both Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) following the protocols of the manufacturer (Life Technologies, USA), and the images were collected immediately. Annexin V-FITC: λex = 488 nm, λem = 500–550 nm. PI: λex = 559 nm, λem = 570–670 nm. Activatable fluorescence imaging in vivo All animal experiments were carried out with the approval of the specialized scientific ethics committee of Shaanxi Normal University. The 5 weeks female BALB/c mice were housed at 25 °C with 50% humidity and subjected to 12 h light/12 h dark cycles. Mice were used for experiments after being acclimatized for 1 week. During the imaging and treatment, mice were anesthetized with a 0.5 L/min oxygen/isoflurane stream. At the end of this research, mice were sacrificed by diethyl ether inhalation. 100 μL of CT26 cells (1 × 106) in saline suspension was injected into BALB/c (female) mice subcutaneously. The mice were used for fluorescence imaging when the volume of tumor reached 100 mm3. BALB/c mice bearing CT26 tumors were intratumorally injected with 100 μL of TPE-QC (5 mmol/L in PBS). Fluorescence images were captured at different times after injections using a Bruker In-Vivo Xtreme II imaging system (Bruker, Germany). The excitation wavelength was 450 nm, and the emission was collected at 650 nm. The Bruker MI SE Image software (Bruker, Germany) was employed to quantify the imaging results. Antitumor efficiency in vivo BALB/c mice bearing CT26 tumors were randomly divided into four groups of three mice each. The four groups were separately treated with saline, saline with light irradiation, TPE-QC, and TPE-QC with light irradiation. For the group treated with TPE-QC, 100 μL of TPE-QC (5 mmol/L in PBS) was intratumorally injected into the mice twice a week. For the group treated with saline, 100 μL of saline solution was intratumorally injected into the mice twice a week. For the groups with light irradiation, the mice were exposed to white light (100 mW cm−2) for 15 min after 24 h of postinjection. For the groups without light irradiation, the mice were kept in the dark after 24 h of postinjection. The tumor size and bodyweight of each mouse after treatment was measured daily by using a caliper, and the tumor volume was calculated according to the following equation: volume = W2 × L/2 (L and W represent the longer diameter and shorter diameters of the tumor, respectively). The mice in different groups were sacrificed on day 21. The major organs and tumors were separated to make slices for hematoxylin and eosin (H&E) staining. Major organs were fixed in 4% paraformaldehyde and then embedded into paraffin, sliced at a thickness of 4 μm. Slices were stained with H&E and imaged by optical microscopy. Results and Discussion Synthesis and photophysical properties The synthesis of prodrug TPE-QC is shown in Supporting Information Scheme S1. By adopting the condensation reaction of formal substituted tetraphenylethylene (TPE- CHO) and lepidine, the TPE-QN was facilely prepared with reasonable yield. The nitrogen atom of TPE-QN was then alkylated by 2-bromoethanol to form TPE-QO, which was further reacted with chlorambucil to give the desired product of TPE-QC. The TPE-QC and related key intermediates were fully characterized by 1H , 13C NMR, and high-resolution mass spectroscopies. The corresponding data were well consistent with the desired structures ( Supporting Information Figures S14–S17). TPE-QC showed an absorption peak centered at 445 nm in DMSO solution and gave extremely weak emission with the quantum yield (Φ) of 0.4% in both DMSO and DMSO/PBS (v/v = 1/99, pH 7.4) solution ( Supporting Information Figures S1 and S2a). The particle size distribution revealed that TPE-QC formed nanoaggregates with the average particle size of 428 nm in DMSO/PBS (v/v = 1/99, pH 7.4) solution ( Supporting Information Figure S2b). Based on a previous report,57 the fluorescence quenching of TPE-QC might be caused by the photoinduced electron transfer (PET) process between chlorambucil and TPE-QO units. Esterase-activated emission enhancement The emission response of TPE-QC in DMSO/PBS (v/v = 1/99, pH 7.4) solution toward esterase was subsequently investigated. Upon addition of esterase (0.1 U mL−1), the emission of TPE-QC lit up with the prolongation of incubation time while the emission intensity at 572 nm enhanced up to approximately 32-fold (Φ = 12.6%) after incubation for 45 min ( Supporting Information Figure S3). As shown in Figure 1a, with the increase of esterase concentration (0 to 0.2 U mL−1), the emission of TPE-QC was gradually enhanced. The emission intensity of TPE-QC reached to plateau when the concentration of esterase was 0.1 U mL−1, illustrating that the hydrolysis process was complete. It is notable that the rate of emission enhancement at 572 nm exhibited good linear relationship (R2 = 0.9984) with the concentration of esterase from 0 to 0.07 U mL−1 (Figure 1b). The detection limit of TPE-QC for esterase was calculated to be 2.38 × 10−5 U mL−1 based on the 3δ/k rule, which was lower than most of the esterase-specific probes.58–60 This high sensitivity toward esterase ensured the further theranostic applications of TPE-QC both in vitro and in vivo. Figure 1 | (a) Emission spectra of TPE-QC (10 μM) treated with esterase (0 to 0.2 U mL−1) for 45 min. (b) Plot of I572 versus the different concentrations of esterase (0 to 0.07 U mL−1). (c) Emission spectra of mixture of TPE-QC (10 μM) and esterase (0.1 U mL−1) with various concentrations of AEBSF (from 0 to 10 mM). (d) The emission intensity of TPE-QC (10 μM) at 572 nm with different types of biological-relevant species: KCl (1 mM), NaCl (1 mM), MgCl2 (1 mM), Na2SO4 (1 mM), Na2CO3 (1 mM), NaNO3 (1 mM), HSA (1 mg mL−1), BSA (1 mg mL−1), β-Gal (0.1 U mL−1), DNase I (0.1 U mL−1), Lyso (0.1 U mL−1), pepsin (0.1 U mL−1), AchE (0.1 U mL−1), ALP (0.1 U mL−1), and esterase (0.1 U mL−1). Inset in (b): photographs of visual emission color of TPE-QC without (left) or with (right) esterase (0.1 U mL−1) under the irradiation of 365 nm UV lamp. Download figure Download PowerPoint To further validate that the emission of TPE-QC was boosted by esterase, an esterase inhibitor of AEBSF was employed to investigate the inhibition effect. A different concentration of AEBSF (0 to 10 mM) was incubated with the mixed solution of TPE-QC (10 μM) and esterase (0.1 U mL−1). As depicted in Figure 1c, the fluorescence intensity at 572 nm was dramatically reduced along with the raising of AEBSF concentration, and almost no emission was observed after treatment with 10 mM of AEBSF. The above results clearly demonstrated that the fluorescence turn-on of TPE-QC in the presence of esterase arose from the esterase catalyzed hydrolysis. The selectivity of TPE-QC to esterase was evaluated by employing different types of biological-relevant substance. As shown in Figure 1d and Supporting Information Figure S4, compared to the esterase, negligible emission enhancement was observed when nonspecific inorganic salt (KCl, NaCl, MgCl2, Na2SO4, Na2CO3, NaNO3) or other type of enzyme [including human serum albumin (HSA), bovine serum albumin (BSA), β-galactosidase (β-Gal), deoxyribonuclease I (DNase I), lysozyme (Lyso), pepsin, acetylcholinesterase (AchE), and alkaline phosphatase (ALP)] was added into the TPE-QC solution with fixed concentration. Obviously, TPE-QC possessed high selectivity for esterase over other biological species. Hydrolyzed product and mechanism investigation The release of TPE-QO and chlorambucil from esterase hydrolyzed TPE-QC was verified by high-performance liquid chromatography (HPLC) and MS analysis. As illustrated in Figures 2a–2e, TPE-QC gave a unique peak with retention time at 11 min. After treatment with esterase for 60 min, the original peak of TPE-QC almost disappeared, while two new peaks with retention times at 4.4 min and 9.1 min appeared, which was well consistent with the retention time of TPE-QO and chlorambucil, respectively. Additionally, the mass peak at m/z = 815 ([M–Br]+) was observed for unhydrolyzed TPE-QC. After incubation with esterase for 60 min, however, two significant new peaks at m/z = 304 ([M]+) and 530 ([M–Br]+) were detected, which were assigned to the chlorambucil and TPE-QO, respectively. Both HPLC and MS analysis indicated that TPE-QO and chlorambucil were successfully liberated from TPE-QC upon esterase catalyzed enzymatic cleavage reaction. Figure 2 | HPLC analysis of (a) TPE-QC, (b) TPE-QO, (c) chlorambucil, and (d) TPE-QC with esterase. (e) MS analysis of TPE-QC and TPE-QC with esterase. Download figure Download PowerPoint The mechanism of esterase catalyzed TPE-QC hydrolysis was computationally elucidated through molecular docking by using SYBYL2.0 (Cetara, USA). The cholesterol esterase from Bos Taurus was selected as the template enzyme.61 The active domain of cholesterol esterase was automatically predicted, and the best protein model selected for docking analysis was achieved by removing water, while adding the hydrogen atoms and charges. The optimal binding model based on the lowest energy after docking revealed that TPE-QC could enter into the enzyme active cavity to give the total score of 7.1598 (Figure 3a). However, the hydrolyzed products of TPE-QO and chlorambucil that located in the enzymatic domain presented a lower total score of 5.4511 and 4.8514, respectively (Figures 3b and 3c). The higher total score of TPE-QC was essential for its retention on the catalytic luminal site of the cholesterol esterase, which was favorable for following the hydrolysis process. When the hydrolyzed reaction was complete, the decreased interaction of TPE-QO and chlorambucil facilitated their escape from the enzyme surface to give their activity. Figure 3 | Cholesterol esterase (PDB entry 2BCE) in complex with TPE-QC and its hydrolyzed p

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

Abstract: Abstract Engineering dual‐function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2E2, with efficient native transaminase (k cat: 69.49±1.77 min−1) and artificial esterase (k cat: 3908–0.41 min−1) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2E2 (pHopt: 8.0–9.5; T opt: 60–65 °C) efficiently converts methyl 3‐oxo‐4‐(2,4,5‐trifluorophenyl)butanoate into 3‐(R)‐amino‐4‐(2,4,5‐trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β‐keto ester into the β‐keto acid at the hydrolytic site and subsequently into the β‐amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2E2 PluriZyme was proven with a set of β‐keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Amino Acid-Mediated Synthesis of the ZIF-8 Nanozyme That Reproduces Both the Zinc-Coordinated Active Center and Hydrophobic Pocket of Natural Carbonic Anhydrase

Abstract: The zeolitic imidazolate framework-8 (ZIF-8) nanozyme has been synthesized using hydrophobic amino acid (AA) to regulate crystal growth. The as-synthesized ZIF-8 reproduces both the structural and functional properties of natural carbonic anhydrase (CA). Structurally, Zn2+/2-methylimidazole coordinated units mimic very well the active center of CA while the hydrophobic microdomains of the adsorbed AA simulate the CA hydrophobic pocket. Functionally, the nanozymes show excellent CA-like esterase activity by giving specific enzyme activity of 0.22 U mg-1 at 25 °C in the case of Val-ZIF-8. More strikingly, such nanozymes are superior to natural CA by having excellent hydrothermal stability, which can give highly enhanced esterase activity with increasing temperature. The specific enzyme activity of Val-ZIF-8 at 80 °C is about 25 times higher than that at 25 °C. In addition, AA-ZIF-8 also shows an excellent catalytic efficiency toward carbon dioxide (CO2) hydration. This study puts forward the important role of hydrophobic microdomains in biomimetic nanozymes for the first time and develops a facile and mild method for the synthesis of nanozymes with controlled morphology and size to achieve excellent catalytic efficiency.

Biochemical and molecular identification of root-knot nematodes in greenhouse vegetable areas of Eastern Mediterranean Region (Turkey)

Abstract: Root-knot nematode surveys were conducted during the growing seasons of 2017 and 2018 to cover the greenhouse vegetable areas in Mersin, Hatay and Adana Provinces in the Eastern Mediterranean Region (Turkey). A total of 46 root-knot nematode populations were characterized using biochemical and molecular diagnostic techniques. DNA extraction was done from second-stage juvenile and in molecular tests using SCAR primers, Meloidogyne arenaria (Neal, 1889) Chitwood, 1949, Meloidogyne javanica (Treub) Chitwood, 1949, Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949, Meloidogyne hapla Chitwood, 1949 and Meloidogyne ethiopica Whitehead, 1968 (Tylenchida: Meloidogynidae) species were screened. Samples obtained from thirty-nine regions were identified as M. incognita (85%) and seven as M. javanica (15%). In addition, young females obtained from all regions were biochemically analyzed using the polyacrylamide gel electrophoresis diagnostic method. The esterase enzyme profile was examined to identify the M. incognita , M. javanica and M. ethiopica groups. Esterase phenotype I1 band was observed in 62% of M. incognita populations and esterase phenotype I2 band was observed in 39%. Esterase phenotype J3 band was detected in all M. javanica populations. M. javanica and M. incognita were verified and also supported by molecular and biochemical methods in the Eastern Mediterranean Region.

Coumarins inhibit η-class carbonic anhydrase from Plasmodium falciparum

Abstract: Abstract Coumarins were discovered to act as inhibitors of α-carbonic anhydrases (CAs, EC 4.2.1.1) after undergoing hydrolysis mediated by the esterase activity of the enzyme to the corresponding 2-hydroxycinnamic acids. Other classes of CAs among the eight currently known do not possess esterase activity or this activity was poorly investigated. Hence, we decided to look at the potential of coumarins as inhibitors of the η-CA from the malaria-producing protozoan Plasmodium falciparum, PfaCA. A panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system acted as low to medium micromolar PfaCA inhibitors, whereas their affinities for the cytosolic off-target human isoforms hCA I and II were in a much higher range. Thus, we confirm that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective PfaCA inhibitors.

Sustainable Biosynthesis of Esterase Enzymes of Desired Characteristics of Catalysis for Pharmaceutical and Food Industry Employing Specific Strains of Microorganisms

Abstract: Reactions catalysed by sustainably produced enzymes can contribute to the bioeconomy supporting several industries. Low-value compounds can be transformed into added-value products or high-resolution chemicals could be prepared in reactions catalysed by biocatalyst esterase enzymes. These enzymes can be synthesised by purposely isolated or genetically modified strains of microorganisms. Enzymes belonging to the hydrolase family catalyse the formation and hydrolysis of ester bonds to produce the desired esterified molecule. The synthesis of homo-chiral compounds can be accomplished either by chemical or biocatalytic processes, the latter being preferred with the use of microbial esterases. For varied applications, esterases with high stability and retained activity at lower and higher temperatures have been produced with strains isolated from extreme environments. For sustainable production of enzymes, higher productivity has been achieved by employing fast-growing Escherichia coli after incorporating plasmids of required characteristics from specific isolates. This is a review of the isolated and engineered strains used in the biosynthesis of esterase of the desired property, with the objective of a sustainable supply of enzymes, to produce products of industrial importance contributing to the economy.

Molecular and Biochemical Analyses of a Novel Trifunctional Endoxylanase/Endoglucanase/Feruloyl Esterase from the Human Colonic Bacterium Bacteroides intestinalis DSM 17393.

Abstract: A novel enzyme Bi76 comprising GH10, E_set_Esterase_N, and CE1 modules was identified, with the highest homology (62.9%) with a bifunctional endoxylanase/feruloyl esterase among characterized enzymes. Interestingly, Bi76 hydrolyzed glucan substrates besides xylans and feruloylated substrates, suggesting that it is the first characterized trifunctional endoxylanase/endoglucanase/feruloyl esterase. Analyses of truncation variants revealed that GH10 and E_set_Esterase_N + CE1 modules encoded endoxylanase/endoglucanase and feruloyl esterase activities, respectively. Synergism analyses indicated that endoxylanase, α-l-arabinofuranosidase, and feruloyl esterase acted cooperatively in releasing ferulic acid (FA) and xylooligosaccharides from feruloylated arabinoxylan. The interdomain synergism of Bi76 overmatched the intermolecular synergism of TM1 and TM2. Importantly, Bi76 exhibited good capacity in producing FA, releasing 5.20, 4.38, 2.12, 1.35, 0.46, and 0.19 mg/g from corn bran, corn cob, wheat bran, corn stover, rice husk, and rice bran, respectively. This study expands the trifunctional endoxylanase/endoglucanase/feruloyl esterase repertoire and demonstrates the great potential of Bi76 in agricultural residue utilization.

The Potential Binding Interaction and Hydrolytic Mechanism of Carbaryl with the Novel Esterase PchA in Pseudomonas sp. PS21.

Abstract: Microbial bioremediation is a very potent and eco-friendly approach to alleviate pesticide pollution in agricultural ecosystems, and hydrolase is an effective element for contaminant degradation. In the present study, a novel Mn2+-dependent esterase, PchA, that efficiently hydrolyzes carbamate pesticides with aromatic structures was identified from Pseudomonas sp. PS21. The hydrolytic activity was confirmed to be related closely to the core catalytic domain, which consists of six residues. The crucial residues indirectly stabilized the position of carbaryl via chelating Mn2+ according to the binding model clarified by molecular simulations, and the additional hydrophobic interactions between carbaryl with several hydrophobic residues also stabilized the binding conformation. The residue Glu398, by serving as the general base, might activate a water molecule and facilitate PchA catalysis. This work offers valuable insights into the binding interaction and hydrolytic mechanism of carbaryl with the hydrolase PchA and will be crucial to designing strategies leading to the protein variants that are capable of degrading related contaminants.

A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis

Abstract: Esterase enzymes catalyze diverse hydrolysis reactions with important biological, commercial, and biotechnological applications. For the improvement of these biocatalysts, there is a need for widely accessible, inexpensive, and adaptable activity screening assays that identify enzymes with particular substrate specificities. Natural systems for biopolymer bioconversion, and likely those designed to mimic them, depend on cocktails of enzymes, each of which specifically targets the intact material as well as water-soluble subunits of varying size. In this work, we have adapted a UV/visible assay using pH-sensitive sulfonphthalein dyes for the real-time quantification of ester hydrolysis of bis-(2-hydroxyethyl) terephthalate (BHET), a subunit of polyethylene terephthalate (PET) plastic. We applied this method to a diverse set of known PET hydrolases and commercial esterases in a microplate format. The approach identified four PET hydrolases and one commercial esterase with high levels of specificity for BHET hydrolysis. Five additional PET hydrolases and three commercial esterases, including a thermophilic enzyme, effectively hydrolyzed both BHET and its monoester product MHET (mono-(2-hydroxyethyl) terephthalate). Specific activities were discernible within one hour and reactions reached an unequivocal endpoint well within 24 hours. The results from the UV/visible method correlated well with conventional HPLC analysis of the reaction products. We examined the suitability of the method toward variable pH, temperature, enzyme preparation method, mono- and multi-ester substrate type, and level of sensitivity versus stringency, finding the assay to be easily adaptable to diverse screening conditions and kinetic measurements. This method offers an accurate, easily accessible, and cost-effective route towards high-throughput library screening to support the discovery, directed evolution, and protein engineering of these critical biocatalysts.

An easily available endoplasmic reticulum targeting near-infrared fluorescent probe for esterase imaging in vitro and in vivo.

Abstract: Here, we report an easily available endoplasmic reticulum-targeting near-infrared fluorescent probe (ER-CE), which can detect esterase in the endoplasmic reticulum and monitor the changes in the esterase amount in tumors in mice in real time. These results indicate that ER-CE is expected to become a powerful analysis tool for the research of endoplasmic reticulum esterase-related diseases.

Identification and technological characterization of endogenous yeast isolated from fermented black carrot juice, shalgam

Abstract: Shalgam is a fermented beverage obtained by lactic acid fermentation and its quality depends on flora. Therefore, it is essential to be able to characterize yeasts, in order to improve the quality and safety controls of final products. This study aimed to identify endogenous yeasts found during the fermentation of shalgam and to determine their technological properties. 172 yeast isolates were obtained and the DNA fingerprints were constituted by iPBS-PCR method. Sequencing of ITS regions of different groups revealed the species of Pichia kudriavzevii, P. fermentans, Saccharomyces cerevisiae, Candida oleophila, Kazachstania bulderi, and Geotrichum candidum. Interestingly, the prevalence of P. kudriavzevii in the flora increased during the fermentation, while the other species decreased. To determine the preliminary technological properties of yeasts, their development at different conditions was examined and 31 technologically superior strains were determined by Principal Component Analysis (PCA). Among these, 17 P. kudriavzevii strains had a vigorous fermentation rate and selected to assess their enzymatic activity. The isolates had diverse enzyme activities, including esterase, esterase lipase, and leucine arylamidase, which may have crucial roles for aroma formation. According to the results, P. kudriavzevii 3-3S9, 3-3S2, and 3-3Y1 strains were selected to be candidate starters for shalgam production.