A Reactive Oxygen Species-Tyrosinase Cascade-Activated Prodrug for Selectively Suppressing Melanoma

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2022A Reactive Oxygen Species-Tyrosinase Cascade-Activated Prodrug for Selectively Suppressing Melanoma Guorui Li†, Yi Yang†, Yaya Zhang, Peiling Huang, Jiangyu Yan, Zhibin Song, Quan Yuan and Jing Huang Guorui Li† Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 , Yi Yang† Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 , Yaya Zhang Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 , Peiling Huang Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 , Jiangyu Yan Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 , Zhibin Song Key Laboratory of Functional Small Organic Molecules, Ministry of Education, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022 , Quan Yuan Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 and Jing Huang *Corresponding author: E-mail Address: [email protected] Institute of Chemical Biology and Nanomedicine (ICBN), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082 https://doi.org/10.31635/ccschem.021.202101032 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail "Off-target effect" is one of the obstacles for targeted prodrugs in chemotherapy. To circumvent this issue, herein we propose a dual biomarker cascade-activated prodrug strategy based on high levels of reactive oxygen species (ROS) and tyrosinase (TYR) in melanoma cells. A representative prodrug, Coumarin- Quinazolinone-phenyl Boronic acid pinacol ester ( CQB), was prepared. The prodrug contained Coumarin- Quinazolinone ( CQ) as mitochondria targeting and monitoring moiety and phenylboronic acid pinacol ester as a cascade-activated prowarhead. After activation by the endogenous ROS and subsequent TYR in melanoma cells, CQB can be converted to the final active reagent Coumarin- Quinazolinone-o- Quinone ( CQQ) that contains o-quinone group as the reactive species. CQQ may interact with cellular nucleophiles to exert its genotoxic effect and disrupt the cellular redox balance. This enables CQB to selectively suppress melanoma. CQB accumulates in the mitochondria and causes mitochondrial dysfunction via affecting mitochondrial DNA integrity. This cascade-activated prodrug may provide a precise strategy for melanoma theranostics. Download figure Download PowerPoint Introduction Adverse side effects are a common drawback of traditional chemotherapy, due to its nonselective damage to both normal and tumor tissues. Targeted prodrugs which exploit cancer endogenous species as the activators can release active drugs specifically to tumor sites, thereby significantly alleviating the toxic side effects.1 Prodrugs have been the focus of drug discovery since they were first proposed by Albert.2 Based on a 10-year statistic, prodrugs account for ∼10% of the drugs approved by the Food and Drug Administration (FDA), emphasizing the significance of the prodrug strategy.1 Although targeted prodrugs have been widely used in clinical cancer treatment, they have some inevitable drawbacks, one of which is the side effect caused by the "off-target effect."3 This happens all the time, even with a rationally designed "sophisticated" prodrug. The root reason for this off-target effect is that the complex biological microenvironment cannot guarantee "on site" activation of prodrugs. To circumvent the off-target effect, drug and probe design based on dual or multiple biomarkers in cancer cells is a potential approach.4–8 Prodrugs based on this approach could potentially improve the specificity of cancer therapy since they can only be converted to active drugs after activation by multiple biomarkers of a specific tumor, thereby minimizng the side effects. Herein, we propose a cascade-activated prodrug strategy that utilizes two cancer biomarkers as progressive triggers to release the active drug. As shown in Figure 1a, the prodrug I is first activated by tumor biomarker A to generate intermediate prodrug II, which is then activated by biomarker B to generate the active drug. Since neither prodrug I nor intermediate II is active, prodrug I will only have effect on specific tumors containing both biomarkers A and B, while other cells are not affected. In this paper, we select the melanoma cell as a model cell to prove this concept. Figure 1 | (a) Cascade-activated prodrug strategy. (b) The design of CQB and its proposed mechanism of action. Download figure Download PowerPoint Melanoma, which is formed by the deterioration of melanocytes, is the most malignant disease in skin tumors, with globally increasing morbidity and mortality each year.9 The formation of melanoma cells is closely related to the melanin. As a natural catalyst for the synthesis of melanin in vivo, tyrosinase (TYR) is overexpressed in highly pigmented melanoma. Mounting evidence has proved that TYR is a significant biomarker for melanoma, making it a potential target for melanoma treatment.10 TYR is a copper-containing oxidase which can convert monophenol or catechol to the corresponding o-quinone structure in the presence of oxygen.11 In the past two decades, TYR-activated prodrugs have been developed to selectively target melanoma.12,13 The first TYR-activated prodrug developed by the Osborn group14 was termed a melanocyte-directed enzyme prodrug therapy (MDEPT). The Zhou group15,16 also reported a series of bis(catechol) derivatives which can cross-link DNA after induction by TYR. Although TYR-activated prodrugs showed some selectivity toward melanoma cells, they had side effects on normal melanocytes in view of a certain level of TYR in normal melanocytes.12,13 Thus, a second biomarker is required for precisely targeting melanoma. Besides a high level of TYR, the progression of cellular canceration is accompanied with an increase of reactive oxygen species (ROS).17 ROS has been exploited for cancer diagnosis and treatment.18 One profound strategy is ROS-activated prodrugs.19–26 A high level of ROS is also observed in melanoma cells. Considering mitochondria are the main factory for producing ROS, the prodrug with mitochondria targeting can be more effectively activated. Taking all these considerations together, we designed an antimelanoma prodrug, Coumarin- Quinazolinone-phenyl Boronic acid pinacol ester ( CQB, Figure 1b). CQB can be cascade-activated by ROS and TYR, and selectively suppress melanoma by targeting mitochondria and causing its dysfunction. Experimental Methods General information and instrumentation Chemical reagents were obtained from Aladdin (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) and used without further purification. 1H and 13C NMR spectra were recorded on a Bruker 400 MHz NMR Spectrometer (Swiss BRUKER, Switzerland) with a CryoProbe (400 MHz for 1H NMR; 100 MHz for 13C NMR). Chemical shifts were reported in δ (ppm) units using residual 13C and 1H signals from deuterated solvents as references. Mass spectra were recorded on an liquid chromatography quadrupole (LCQ)-Advantage (Thermo Finnigan, USA) instrument equipped with an electrospray ionization (ESI) source. Analytical thin-layer chromatography (TLC) was performed on silica gel HSGF254 (Shanghai Titan Technology Co., Ltd., Shanghai, China). Column chromatography was conducted on silica gel (200–300 mesh). High-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 Infinity (Agilent, USA) HPLC, equipped with a 4.6 × 250 mm, 5 μm, 100 Å C18 Aquasil column. HPLC The gradient was started at 95% H2O—5% acetonitrile (0 min) with a flow rate of 1 mL/min. The gradient was maintained for 4 min and then changed to 5% H2O—95% acetonitrile over 11 min. This gradient was maintained for 5 min and then changed back to 95% H2O—5% acetonitrile over 5 min. Total run time was 25 min followed by a minute of post-time. The liquid chromatography (LC) trace was obtained by monitoring absorbance at 260 nm. Cell culture Melanoma cell line (B16) cells were cultured in 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Liver cancer cell line (HepG2) cells and normal human hepatocytes line (LO2) cells were incubated with Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin. Primary human epidermal melanocytes (HEMs) were cultured in 254 medium with 10% FBS, 1% human melanocyte growth supplement (HMGS), and 1% penicillin-streptomycin. The LO2, HepG2, HEMs, and B16 cells used in this study were in logarithmic growth phase. MTT assay 1 × 104 cells were seeded in a 96-well plate. After the cells adhered, they were treated with various compounds for 48 h. Then the methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (0.5 mg/mL) (Sangon Biotech, Shanghai, China) was added and incubated for 4 h. Finally, all the solution was pipetted out, and 150 μL dimethyl sulfoxide (DMSO) was added to dissolve the formazan. The absorbance at 490 nm was measured by the microplate reader (TECAN, Switzerland). The cell viability was expressed as (A/A0) × 100%, where A indicates the absorbance of the prodrug-treated group and A0 represents the absorbance of the control group. Detection of TYR activity 5 × 103 cells were seeded in each well of a 96-well plate. After 12 h, the cells were washed with phosphate-buffered saline (PBS), and lysed in 1% tritionX-100 PBS buffer for 5 min. To completely lyse the cells, the cells were stored at −20 °C for 1 h, and then thawed at room temperature. 100 μL 0.1% Levodopa (l-DOPA) was added in each well. The absorbance of the reaction mixture at 475 nm was measured immediately. Then the 96-well plate was incubated at 37 °C for 2 h, and the absorbance at 475 nm was measured again using the microplate reader. Cell cycle analysis Briefly, 1 × 105 B16 cells were seeded in a 12-well plate. Then the cells were treated with 20 μM CQB or Coumarin-Quinazolinone-phenyl Boronic acid ( CQBA) for 24 h. After washing the cells with cold PBS, they were fixed with 70% alcohol at 4 °C. The fixed cells were stained with propidium iodide (PI; BD, NJ, USA) and supplied with 100 μg/mL RNase (Solarbio, Beijing, China) and 0.2% Triton-100 (Solarbio) at 37 °C for 30 min in the dark. Finally, the stained cells were subjected to flow cytometry (BD C6 Plus) and analyzed by FlowJo software (FlowJo v10, Treestar, USA). Annexin V-FITC/PI assay The apoptosis assay was performed with an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) based on the manufacturer's instructions. Briefly, 1 × 105 B16 cells were seeded in a 12-well plate. The cells were treated with or without 20 μM CQB or CQBA for 24 and 48 h. Then the cells were collected and stained with Annexin V-FITC and PI in the dark for 15 min. Finally, the cells were subjected to flow cytometry (BD Accuri C6 Plus) and analyzed by FlowJo software (FlowJo v10). Determination of intracellular ROS level Intracellular ROS levels were measured using an oxidation-sensitive probe, 2,7-dichlorodi -hydrofluorescein diacetate (DCFH-DA) (Aladdin) by flow cytometry (BD Accuri C6 Plus). First of all, 1 × 105 cells were seeded in a 12-well plate. Briefly, the cells were exposed to 20 μM of prodrugs for 24 h. After washing with cold PBS for three times, 10 μM DCFH-DA probes (dissolved in DMEM, 1640 and 254) were added and incubated for 30 min in the dark. Then the cells were washed three times with cold PBS and collected for flow cytometry analysis. Measurement of GSH and GSSG 1 × 105 cells were seeded in a 12-well plate. The cells were cultured in the presence or absence of 20 μM prodrugs for 24 h. Then the cells were harvested for glutathione/oxidized glutathione disulfide (GSH/GSSG) determination using a GSH/GSSG Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The protein concentration was determined by a BCA Protein Assay Kit (TaKaRa, Beijing, China). The relative GSH contents were determined to be (2*GSH-2*GSSG)/Protein. Cellular localization 2 × 104 B16 cells were seeded on a 3.5 cm confocal dish. After 16 h for adherence, the cells were incubated with 100 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA) for 30 min and washed twice with PBS. Then 5 μM CQB was added for 3 h. After rinsing the samples three times with PBS, they were immediately imaged with a fluorescence confocal microscope (Nikon [Tokyo, Japan] A1 R MP, λex = 405 nm, λem = 500–550 nm). Detection of mitochondrial membrane potential 1 × 105 B16 cells were seeded in a 12-well plate. Then the cells were incubated with 20 μM CQB or CQBA for 24 h. A mitochondrial membrane potential (MMP) detection kit (Beyotime) was used to detect MMPs according to the manufacturer's instructions. For flow cytometry detection, 1 × 104 cells were collected in each sample. Every sample had three replicate wells, and the cells cultured without drug treatment were used as negative controls. For fluorescence imaging, the cells treated and untreated with prodrugs were stained with JC-1 probe for 20 min in a 37 °C incubator. After washing the samples three times, they were immediately imaged by a fluorescence confocal microscope (Nikon A1 R MP). For the green channel, the excitation wavelength was set at 488 nm, and the emission wavelength was collected from 500 to 550 nm. For the red channel, the excitation wavelength was set at 561 nm, and the emission wavelength was collected from 570 to 620 nm. Detection of DNA damage by quantitative PCR Four different kinds of cells were seeded in a six-well plate at the density of 2 × 105. After being adhered, the cells were incubated with or without 20 μM CQB for 24 h. The genomic DNA of these cells was extracted with a Genomic DNA Mini Preparation Kit (Beyotime) and quantified with PicoGreen dye (Invitrogen). The PrimeSTAR GXL DNA Polymerase (TaKaRa) was used for PCR amplification according to the manufacturer's protocol. The primers used in the PCR amplification are shown below. Human-mito-sense: TCT AAG CCT CCT TAT TCG AGC CGA; human-mito-antisense: TTT CAT CAT GCG GAG ATG TTG GAT GG; human-control-sense: CGA GTA AGA GAC CAT TGT GGC AG; human-control-antisense: GCA CTG GCT TAG GAG TTG GAC T; mouse-mito-sense: GCC AGC CTG ACC CAT AGC CAT ATT AT; mouse-mito-antisense: GAG AGA TTT TAT GGG TGT ATT GCG G; mouse-control;sense: TTG AGA CTG TGA TTG GCA ATG CCT; mouse-control-antisense: CCT TTA ATG CCC ATC CCG GAC T. Cell fractionation The separation of the cytoplasm and mitochondria of B16 cells was performed using digitonin and triton lysis buffer. After 20 μM CQB or CQBA treatment for 24 h, the B16 cells were collected by certification and washed with cold PBS. Then the cells were lysed by digitonin lysis buffer on ice for 10 min. After certification at 14,000 rpm for 5 min, the supernatant was collected as cytoplasm fraction, and the pellets were lysed by triton lysis buffer supplied with benzonase (HaiGene, Haerbin, China) on ice for 1 h. After certification at 14,000 rpm for 5 min, the supernatant was collected as a fraction of the mitochondria. Western blot analysis B16 cells were seeded in a six-well plate at a density of 2 × 105. After treatment with or without 20 μM CQB/ CQBA for 24 h, the cells were lysed in radio-immunoprecipitation assay (RIPA) Buffer (Sorlabio) with protease inhibitor cocktail (Bimake, TX, USA) on ice for 20 min. The total proteins were extracted by 14,000g certification for 10 min and determined by a bicinchoninic acid (BCA) Protein Assay Kit (TaKaRa). After being denatured by an SDS-loading buffer and boiled for 5 min, the samples were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and transferred to poly(vinylidene difluoride) (PVDF) membranes. Then the membrane was blocked with 5% milk (BD), and incubated with the indicated primary antibodies and the secondary antibodies (caspase3: 9662s, CST (Cell Signaling Technology, Inc., Boston, MA, USA); BCL2-Associated X (BAX): 2772S, CST; a beta-lactamase gene family (BCL)-2: CAS7511, BioWorld (Bioworld Technology, Inc., Bloomington, MN, USA); Tubulin: BS1482M, BioWorld; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GB12002, Servicebio). Total RNA extraction The B16 cells in the T25 tissue flask were pretreated with 20 μM CQB or CQBA for 24 h, and untreated cells were used as reference controls. Each set of groups consisted of three biological replicates. Each sample collected 1 × 107 cells. Total RNA was extracted from the B16 cells using TRIzol (Invitrogen, Carlsbad, CA). Then the cells were quickly frozen by liquid nitrogen, homogenized, and rested horizontally for 5 min. After being centrifuged at 12,000g, the supernatant was dissolved in chloroform/isoamyl alcohol (24:1), then shaken vigorously and centrifuged at 12,000g at 4 °C for 10 min. Then the supernatant was mixed with an equal volume of isopropyl alcohol and centrifuged at 13,600 rpm at 4 °C for 20 min. The RNA pellet was washed twice with 75% ethanol and dried by air in the biosafety cabinet for 5–10 min. Finally, the total RNA was dissolved by diethyl pyrocarbonate(DEPC)-treated water, qualified and quantified using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, Waltham, MA). mRNA library construction The extracted mRNA was purified by Oligo(dT)-attached magnetic beads and fragmented into small pieces with fragment buffer. Then cDNA was generated by reverse transcription and amplified by PCR. After purification by Ampure XP Beads and validated on the Agilent 2100 bioanalyzer for quality control, the PCR products were denatured and circularized by the splint oligo sequence to get the final library. The DNA nanoballs were made by final library amplification and loaded into the patterned nanoarray. Finally, the 50 bases reads were generated on a MGI2000 platform (TSINGKE, Beijing, China). Genetic difference analysis The sequencing data were filtered with SOAPnuke (Beijing Genomics Institute, Beijing, China; v1.5.2), mapped to the reference genome using HISAT2 (Center for Computational Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; v2.0.4), and aligned to the reference coding gene set by StringTie (Department of Computer Sciences, University of Wisconsin-Madison, Madison, WI, USA; v2.1.2). Differential expression analysis was performed by the DESeq2 (Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany; v1.4.5) with Q value The was made by for analysis of common by CQB/ CQBA and the control group. and the of and analysis were performed using The significant levels were by with Q value quantitative PCR 2 × 105 B16 cells were seeded in 3.5 cm cell culture After being adhered, the cells were incubated with 20 μM for 24 h. The total RNA from each sample was extracted by Total RNA Kit China) and converted into cDNA using reagent Kit with (TaKaRa). Then was performed using China) and PCR Waltham, MA, USA). The gene expression level was to and analyzed by the The was three times for biological The primers were designed in ( and are in In the for the in were approved by the on at Hunan University, the The B16 model was using at 5 of B16 cells × in 100 μL were into the of the were to 100 The were into control and 50 μL of the drug was into the The CQB was The tumor and of the were other analysis for all in was with of were to be of and tumor were collected from the after 2 of The samples were into and the of the was Then they were stained with and for analysis. and and synthesis of CQB As the prodrug CQB contained two moiety A as a mitochondria targeting and monitoring group and moiety B as a cascade-activated 1b). we a Coumarin- Quinazolinone ( CQ) as a with fluorescence and derivatives in the Thus, was used as moiety For moiety B, we the which is well to be by ROS that CQB can be first activated by ROS to and then as the TYR to generate the active reagent CQQ which contains o-quinone group as the reactive species 1b). is well that o-quinone can with as DNA or CQB may exert genotoxic effects after The synthesis of CQB was by a reaction of with pinacol ester these compounds were by NMR and the Considering both and products may be the of a NMR was As shown in Figure the of in the B moiety and in the moiety was In the 13C of in the B moiety was which was in the of reported in the the that CQB can be as the 1 | (a) The of 12 acid pinacol 3 h, then 24 h. (b) The of CQB to and CQQ by ROS and TYR. Download figure Download PowerPoint CQB can be cascade-activated by ROS and TYR in buffer first the of CQB by via HPLC analysis. As shown in Figure CQB a at time of min. the of of two at of and min, which were as the CQBA and the The two compounds had also been and by NMR and the is that ( was and not be further at the assay for The of both CQB and CQBA by treatment with different of at different times were The showed both compounds can be activated by to and the were and and the the activation of CQB was more CQBA for CQB for CQBA by treatment with for 1 The was also by As shown in Figure after treatment, the at δ the of pinacol a at δ the formation of a group. This was further by ( Figure also CQB was selectively activated by CQB was treated with different and and analyzed by As shown in Figure CQB can be selectively converted to by In other reagents had effect on Figure 2 | (a) of CQB to CQBA and (b) HPLC analysis of the reaction of CQB with 100 μM CQB was incubated with μM in solution for different 1H NMR spectra of CQB and Download figure Download PowerPoint the activation of CQB by only the ester while the group is This is different from that the group be after the and the have shown that the on o-quinone formation and In the of CQB, we the of the as well as the of with at the its we a of Quinazolinone-phenyl Boronic acid pinacol ester ( ( which not the group after treatment with further ( Figure for the ( treatment with the group and as the ( Figure Although the mechanism of the further it is that the to the its and in from this of CQB, the group can as a TYR which a cascade-activated strategy in this then the can be further converted to Coumarin- Quinazolinone-o- Quinone ( CQQ) by TYR Considering that are and to the were performed to the TYR activation the activation of with TYR was by As shown in Figure after with TYR at room

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