A series of potent BODIPY photosensitisers featuring tellurophene motifs at boron.

This article describes the synthesis and photophysical properties of a series of BODIPY photosensitisers that feature tellurophene motifs appended at the boron centre. These compounds were obtained via nucleophilic substitution of various F-BODIPYs with lithiated tellurophene. The synthetic scope, photophysical characteristics and photosensitisation properties are discussed. Structural modifications around the BODIPY core resulted in an eight-fold improvement in light IC50 values compared to previous designs.

A chloromethyl-triazole fluorescent chemosensor for O6-methylguanine DNA methyltransferase.

Fluorescent chemosensors offer a direct means of measuring enzyme activity for cancer diagnosis, predicting drug resistance, and aiding in the discovery of new anticancer drugs. O6-methylguanine DNA methyltransferase (MGMT) is a predictor of resistance towards anticancer alkylating agents such as temozolomide. Using the fluorescent molecular rotor, 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), we synthesized, and evaluated a MGMT fluorescent chemosensor derived from a chloromethyl-triazole covalent inhibitor, AA-CW236, a non-pseudosubstrate of MGMT. Our fluorescence probe covalently labelled the MGMT active site C145, producing a 18-fold increase in fluorescence. Compared to previous fluorescent probes derived from a substrate-based inhibitor, our probe had improved binding and reaction rate. Overall, our chloromethyl triazole-based fluorescence MGMT probe is a promising tool for measuring MGMT activity to predict temozolomide resistance.

Tight-Binding Small-Molecule Carboxylesterase 2 Inhibitors Reduce Intracellular Irinotecan Activation.

As the primary enzyme responsible for the activatable conversion of Irinotecan (CPT-11) to SN-38, carboxylesterase 2 (CES2) is a significant predictive biomarker toward CPT-11-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels from high CES2 activity lead to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, CES2 inhibition presents an effective strategy to directly alter the pharmacokinetics of CPT-11 conversion, ultimately controlling the amount of SN-38 produced. To address this, we conducted a high-throughput screening to discover 18 small-molecule CES2 inhibitors. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating cell permeability, and they show inhibition of CPT-11 conversion with the purified enzyme. The top five inhibitors prohibited cell death mediated by CPT-11 when preincubated in PDAC cells. Three of these inhibitors displayed a tight-binding mechanism of action with a strong binding affinity.

BODIPYs with Chalcogenophenes at Boron: Synthesis and Properties.

Reported herein are the synthesis and characterization of BODIPYs bearing heterocycles at boron. To synthesize this series, various chalcogenophenes (furan, thiophene, selenophene, and tellurophene) were lithiated and then used as nucleophiles to attack the boron center of a parent F-BODIPY. Compounds in the series were compared with respect to their photophysical and structural properties, and trends were discussed. By virtue of the "heavy atom effect", as the mass of the heterocycle appended to the BODIPY core increases, compounds exhibit a higher singlet oxygen quantum yield. The BODIPY with tellurophene at boron exhibits the highest quantum yield (ΦΔ = 0.68) in the series and reduced emission (Φf = 0.01).

Photo-Uncaging by C(sp3)-C(sp3) Bond Cleavage Restores ß-Lapachone Activity.

ß-Lapachone is an ortho-naphthoquinone natural product with significant antiproliferative activity but suffers from adverse systemic toxicity. The use of photoremovable protecting groups to covalently inactivate a substrate and then enable controllable release with light in a spatiotemporal manner is an attractive prodrug strategy to limit toxicity. However, visible light-activatable photocages are nearly exclusively enabled by linkages to nucleophilic functional sites such as alcohols, amines, thiols, phosphates, and sulfonates. Herein, we report covalent inactivation of the electrophilic quinone moiety of ß-lapachone via a C(sp3)-C(sp3) bond to a coumarin photocage. In contrast to ß-lapachone, the designed prodrug remained intact in human whole blood and did not induce methemoglobinemia in the dark. Under light activation, the C-C bond cleaves to release the active quinone, recovering its biological activity when evaluated against the enzyme NQO1 and human cancer cells. Investigations into this report of a C(sp3)-C(sp3) photoinduced bond cleavage suggest a nontraditional, radical-based mechanism of release beginning with an initial charge-transfer excited state. Additionally, caging and release of the isomeric para-quinone, α-lapachone, are demonstrated. As such, we describe a photocaging strategy for the pair of quinones and report a unique light-induced cleavage of a C-C bond. We envision that this photocage strategy can be extended to quinones beyond ß- and α-lapachone, thus expanding the chemical toolbox of photocaged compounds.

Photocaged DNA-Binding Photosensitizer Enables Photocontrol of Nuclear Entry for Dual-Targeted Photodynamic Therapy.

Photodynamic therapy (PDT) is a clinically approved cancer treatment that requires a photosensitizer (PS), light, and molecular oxygen─a combination which produces reactive oxygen species (ROS) that can induce cancer cell death. To enhance the efficacy of PDT, dual-targeted strategies have been explored where two photosensitizers are administered and localize to different subcellular organelles. To date, a single small-molecule conjugate for dual-targeted PDT with light-controlled nuclear localization has not been achieved. We designed a probe composed of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen). Illumination with 480 nm light removes WinterGreen from the conjugate and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes to nuclei, binds DNA, and produces ROS using one- or two-photon illumination. We observe synergistic photocytotoxicity in MCF7 breast cancer cells, and a reduction in size of three-dimensional (3D) tumor spheroids, demonstrating that nuclear/cytosolic photosensitization using a single agent can enhance PDT efficacy.

Selective detection of carboxylesterase 2 activity in cancer cells using an activity-based chemiluminescent probe.

Carboxylesterase 2 (CES2) has crucial roles in both xenobiotic metabolism and formation of pathogenic states including cancer. Thus, it is highly critical to monitor intracellular CES2 activity in living cancer cells. Here, we report a CES2 activatable phenoxy 1,2-dioxetane based chemiluminescent agent (CL-CES2). The probe exhibited a selective turn-on response in the presence of CES2 enzyme and enabled detection of CES2 activity in three different cancer cells that possess varying enzyme concentrations with high signal to noise ratios. In contrast no signal was obtained with CES1, an isoform of CES2 enzyme. CL-CES2 marks the first ever example of a CES2-responsive chemiluminescent luminophore and holds a great potential in further understanding the roles of CES2 activity in tumorogenesis.

Two-Photon Photodynamic Therapy Targeting Cancers with Low Carboxylesterase 2 Activity Guided by Ratiometric Fluorescence.

Human carboxylesterase 2 (hCES2) converts anticancer prodrugs, such as irinotecan, into their active metabolites via phase I drug metabolism. Owing to interindividual variability, hCES2 serves as a predictive marker of patient response to hCES2-activated prodrug-based therapy, whereby a low intratumoral hCES2 activity leads to therapeutic resistance. Despite the ability to identify nonresponders, effective treatments for resistant patients are needed. Clinically approved photodynamic therapy is an attractive alternative for irinotecan-resistant patients. Here, we describe the application of our hCES2-selective small-molecule ratiometric fluorescent chemosensor, Benz-AP, as a single theranostic agent given its discovered functionality as a photosensitizer. Benz-AP produces singlet oxygen and induces photocytotoxicity in cancer cells in a strong negative correlation with hCES2 activity. Two-photon excitation of Benz-AP produces fluorescence, singlet oxygen, and photocytotoxicity in tumor spheroids. Overall, Benz-AP serves as a novel theranostic agent with selective photocytotoxicity in hCES2-prodrug resistant cancer cells, making Benz-AP a promising agent for in vivo applications.

Dark Dynamic Therapy: Photosensitization without Light Excitation Using Chemiluminescence Resonance Energy Transfer in a Dioxetane-Erythrosin B Conjugate.

Reactive oxygen species (e.g., singlet oxygen) are the primary cytotoxic agents used in the clinically approved technique photodynamic therapy (PDT). Although singlet oxygen has high potential to effectively kill tumor cells, its production via light excitation of a photosensitizer has been limited by the penetration depth and delivery of light in tissue. To produce singlet oxygen without light excitation, we describe the use of Schaap's chemiluminescent scaffold comprising an adamantylidene-dioxetane motif. Functionalizing this scaffold with a photosensitizer, Erythrosin B, resulted in spontaneous chemiluminescence resonance energy transfer (CRET) leading to the production of singlet oxygen. We show that this compound is cell permeable and that the singlet oxygen produced via CRET is remarkably efficient in killing cancer cells at low micromolar concentrations. Moreover, we demonstrate that protection of the phenol on the chemiluminescent scaffold with a nitroreductase-responsive trigger group allows for cancer-selective dark dynamic cell death. Here, we present the concept of dark dynamic therapy using a small cell-permeable molecule capable of producing the effects of PDT in cells, without light.

Introducing the Tellurophene-Appended BODIPY: PDT Agent with Mass Cytometry Tracking Capabilities.

The synthesis and characterization of the first BODIPY appended to the five-membered heterocylic tellurophene [Te] moiety is reported. By incorporating tellurophene at the meso position, the tellurophene-appended boron-dipyrromethene dye (BODIPY) acts as a multimodal agent, becoming a potent photosensitizer with a mass cytometry tag. To synthesize the compound, we developed a method to enable late-stage Suzuki-Miyaura coupling by preparing and isolating tellurophene-2-BPin in a one-step procedure from the parent tellurophene. Coupling to a meso-substituted BODIPY functionalized with a pendant aryl bromide provides the desired tellurophene-appended BODIPY. This compound demonstrated a singlet oxygen quantum yield of 0.26 ± 0.01 and produced a light dose-dependent cytotoxicity with nanomolar IC50 values against 2D cultured HeLa cells and high efficacy against 3D cultured HeLa tumor spheroids, proving to be a strong photosensitizer. The presence of the tellurophene moiety could be detected using mass cytometry, thus showcasing the ability of a tellurophene-appended BODIPY as a novel photodynamic-therapy-mass-cytometry theranostic agent.

Highly Potent Photoinactivation of Bacteria Using a Water-Soluble, Cell-Permeable, DNA-Binding Photosensitizer.

Antimicrobial photodynamic therapy (APDT) employs a photosensitizer, light, and molecular oxygen to treat infectious diseases via oxidative damage, with a low likelihood for the development of resistance. For optimal APDT efficacy, photosensitizers with cationic charges that can permeate bacteria cells and bind intracellular targets are desired to not limit oxidative damage to the outer bacterial structure. Here we report the application of brominated DAPI (Br-DAPI), a water-soluble, DNA-binding photosensitizer for the eradication of both Gram-negative and Gram-positive bacteria (as demonstrated on N99 Escherichia coli and Bacillus subtilis, respectively). We observe intracellular uptake of Br-DAPI, ROS-mediated bacterial cell death via one- and two-photon excitation, and selective photocytotoxicity of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at submicromolar concentrations (IC50 = 0.2-0.4 µM) and low light doses (5 min irradiation times, 4.5 J cm-2 dose), making it superior to commonly employed APDT phenothiazinium photosensitizers such as methylene blue. Given its high potency and two-photon excitability, Br-DAPI is a promising novel photosensitizer for in vivo APDT applications.

A Green-Absorbing, Red-Fluorescent Phenalenone-Based Photosensitizer as a Theranostic Agent for Photodynamic Therapy.

Phenalenone is a synthetically accessible, highly efficient photosensitizer with a near-unity singlet oxygen quantum yield. Unfortunately, its UV absorption and lack of fluorescence has made it unsuitable for fluorescence-guided photodynamic therapy against cancer. In this work, we synthesized a series of phenalenone derivatives containing electron-donating groups to red-shift the absorption spectrum and bromine(s) to permit good singlet oxygen production via the heavy-atom effect. Of the derivatives synthesized, the phenalenone containing an amine at the 6-position with bromines at the 2- and 5-positions (OE19) exhibited the longest absorption wavelength (i.e., green) and produced both singlet oxygen and red fluorescence efficiently. OE19 induced photocytotoxicity with nanomolar potency in 2D cultured PANC-1 cancer cells as well as light-induced destruction of PANC-1 spheroids with minimal dark toxicity. Overall, OE19 opens up the possibility of employing phenalenone-based photosensitizers as theranostic agents for photodynamic cancer therapy.

An Activatable Photosensitizer Targeting Human NAD(P)H: Quinone Oxidoreductase†1.

Human NAD(P)H: Quinone Oxidoreductase 1 (hNQO1) is an attractive enzyme for cancer therapeutics due to its significant overexpression in tumors compared to healthy tissues. Its unique catalytic mechanism involving the two-electron reduction of quinone-based compounds has made it a useful target to exploit in the design of hNQO1 fluorescent chemosensors and hNQO1-activatable-prodrugs. In this work, hNQO1 is exploited for an optical therapeutic. The probe uses the photosensitizer, phenalenone, which is initially quenched via photo-induced electron transfer by the attached quinone. Native phenalenone is liberated in the presence of hNQO1 resulting in the production of cytotoxic singlet oxygen upon irradiation. hNQO1-mediated activation in A549 lung cancer cells containing high levels of hNQO1 induces a dose-dependent photo-cytotoxic response after irradiation. In contrast, no photo-cytotoxicity was observed in the normal lung cell line, MRC9. By targeting hNQO1, this scaffold can be used to enhance the cancer selectivity of photodynamic therapy.

Measuring human carboxylesterase 2 activity in pancreatic cancer patient-derived xenografts using a ratiometric fluorescent chemosensor.

Irinotecan-based therapy is a common treatment for pancreatic cancer. To elicit its anticancer activity, the drug requires first the hydrolysis action of the enzyme human carboxylesterase 2 (hCES2). It has been established that pancreatic cancer patients have various levels of hCES2, whereby patients having low levels respond poorer to Irinotecan than patients with higher levels, suggesting that hCES2 can be used to predict response. However, current methods that measure hCES2 activity are inaccurate, complex or lengthy, thus being incompatible for use in a clinical setting. Here, we developed a small molecule ratiometric fluorescent chemosensor that accurately measures hCES2 activity in a single-step within complex mixtures. Our chemosensor is highly selective for hCES2 over hCES1, cell permeable and can measure hCES2 activity in pancreatic cancer patient-derived xenografts. Given the simplicity, accuracy and tissue compatibility of our assay, we anticipate our chemosensor can be used to predict patient response to Irinotecan-based therapy.

DNA directed damage using a brominated DAPI derivative.

Photodynamic therapy (PDT) is a clinically approved cancer treatment that uses light, oxygen and a photosensitizer to produce localized reactive oxygen species (ROS). Due to the short lifetime of ROS, the location of the photosensitizer in the cell is believed to be the key determinant governing the outcome of PDT. To explore the effect of direct association between a photosensitizer and DNA a well know DNA-binding dye, DAPI, was converted into a photosensitizer. Br-DAPI - unlike native DAPI - upon irradiation produces ROS. We demonstrate that the ROS are only effective in inducing dsDNA breaks when Br-DAPI is bound to DNA. In cancer cells (A549) Br-DAPI causes rapid light dependent cell death. This work supports the design of photosensitizers which bind with high affinity to the DNA of target cells for potentially more effective PDT.

Fluorescent reporter assays provide direct, accurate, quantitative measurements of MGMT status in human cells.

The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) strongly influences the effectiveness of cancer treatment with chemotherapeutic alkylating agents, and MGMT status in cancer cells could potentially contribute to tailored therapies for individual patients. However, the promoter methylation and immunohistochemical assays presently used for measuring MGMT in clinical samples are indirect, cumbersome and sometimes do not accurately report MGMT activity. Here we directly compare the accuracy of 6 analytical methods, including two fluorescent reporter assays, against the in vitro MGMT activity assay that is considered the gold standard for measuring MGMT DNA repair capacity. We discuss the relative advantages of each method. Our data indicate that two recently developed fluorescence-based assays measure MGMT activity accurately and efficiently, and could provide a functional dimension to clinical efforts to identify patients who are likely to benefit from alkylating chemotherapy.

Exploiting the Cellular Redox-Control System for Activatable Photodynamic Therapy.

Photodynamic therapy (PDT) has been successfully used to treat a variety of cancers. However, one drawback has been the adverse side effects experienced by patients during therapy, as a result of the destruction of normal tissues upon irradiation. Herein, we describe the design, synthesis and characterisation of a photosensitiser to overcome this issue that, in addition to light, is also dependent on the overactive redox system present in cancer cells for its activation. Our probe consists of the photosensitiser, protoporphyrin IX, and a FRET-based quencher dye, BHQ-3, on a scaffold containing a disulfide bond. The close proximity of BHQ-3 to protoporphyrin IX quenches its ability to fluoresce and produce reactive oxygen species, whereas nonenzymatic or enzymatic reduction can recover its native properties. We further demonstrate its ability to be activated in cancer cells in a thiol-dependent manner and destroy breast and lung cancer cells upon red-light irradiation.

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses.

The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 in vitro or in biological samples. Herein, we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both in vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and western blots. Furthermore, the probe provides a viable high-throughput assay for drug discovery.

Fluorescent Chemosensors as Future Tools for Cancer Biology.

It is well established that aberrant cellular biochemical activity is strongly linked to the formation and progression of various cancers. Assays that could aid in cancer diagnostics, assessing anticancer drug resistance, and in the discovery of new anticancer drugs are highly warranted. In recent years, a large number of small molecule-based fluorescent chemosensors have been developed for monitoring the activity of enzymes and small biomolecular constituents. These probes have shown several advantages over traditional methods, such as the ability to directly and selectively measure activity of their targets within complex cellular environments. This review will summarize recently developed fluorescent chemosensors that have potential applications in the field of cancer biology.