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.

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.

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).

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.

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.

Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase.

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 µM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 µM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 µM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.

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.

A Chimeric ATP-Linked Nucleotide Enables Luminescence Signaling of Damage Surveillance by MTH1, a Cancer Target.

The enzyme MTH1 cleanses the cellular nucleotide pool of oxidatively damaged 8-oxo-dGTP, preventing mutagenesis by this nucleotide. The enzyme is considered a promising therapeutic target; however, methods to measure its activity are indirect and laborious and have low sensitivity. Here we describe a novel ATP-linked chimeric nucleotide (ARGO) that enables luminescence signaling of the enzymatic reaction, greatly simplifying the measurement of MTH1 activity. We show that the reporting system can be used to identify inhibitors of MTH1, and we use it to quantify enzyme activity in eight cell lines and in colorectal tumor tissue. The ARGO reporter is likely to have considerable utility in the study of the biology of MTH1 and potentially in analyzing patient samples during clinical testing.

Fluorescence Monitoring of the Oxidative Repair of DNA Alkylation Damage by ALKBH3, a Prostate Cancer Marker.

The 2-oxoglutarate-dependent iron enzyme ALKBH3 is an antitumor target and a potential diagnostic marker for several tumor types, including prostate cancer. However, there is at present no simple way to measure this enzyme's activity. Here we describe a fluorogenic probe design (MAQ) that is directly responsive to ALKBH3 repair activity. It makes use of the fluorescence-quenching properties of 1-methyladenine; removal of the alkyl group results in a >10-fold light-up signal. The probe is specific for ALKBH3 over its related homologue ALKBH2 and can be used to identify and measure the effectiveness of enzyme inhibitors. Measurements of the enzyme substrate parameters show that MAQ displays Km and kcat values essentially the same as those of the native substrate. Finally, we show that the probe functions efficiently in cells, allowing imaging and quantitation of ALKBH3 activity by microscopy and flow cytometry. We expect that MAQ probes will be broadly useful in the study of the basic biology of ALKBH3 and in clinical cancer applications as well.

Red-Shifting Azobenzene Photoswitches for in Vivo Use.

Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compounds to control specific biological targets in vivo. These azo compounds can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compounds designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near infrared region are required. This Account describes our attempts to produce such azo compounds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biological process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compounds with a number of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compounds show unusually slow thermal relaxation rates and enhanced separation of n-π* transitions of cis and trans isomers compared to analogues without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compounds, in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aqueous solutions at neutral pH. By varying the nature of ortho substituents, together with the number and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to reduction by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacology.

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.

Photoswitching azo compounds in vivo with red light.

The photoisomerization of azobenzenes provides a general means for the photocontrol of molecular structure and function. For applications in vivo, however, the wavelength of irradiation required for trans-to-cis isomerization of azobenzenes is critical since UV and most visible wavelengths are strongly scattered by cells and tissues. We report here that azobenzene compounds in which all four positions ortho to the azo group are substituted with bulky electron-rich substituents can be effectively isomerized with red light (630-660 nm), a wavelength range that is orders of magnitude more penetrating through tissue than other parts of the visible spectrum. When the ortho substituent is chloro, the compounds also exhibit stability to reduction by glutathione, enabling their use in intracellular environments in vivo.

Quantitative analysis of the effects of photoswitchable distance constraints on the structure of a globular protein.

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of folding of the Fyn SH3 domain, a well-studied model protein. We develop a theoretical framework based on an explicit-chain model of protein folding, modified to include detailed model linkers, that allows prediction of the effect of a given linker on the free energy of folding of a protein. Using this framework, we were able to quantitatively explain the experimental result that a longer, but somewhat flexible, cross-linker is less destabilizing to the folded state than a shorter more rigid cross-linker. The models also suggest how misfolded states may be generated by cross-linking, providing a rationale for altered dynamics seen in nuclear magnetic resonance analyses of these proteins. The theoretical framework is readily portable to any protein of known folded state structure and thus can be used to guide the design of photoswitchable proteins generally.

Azobenzene photoswitches for biomolecules.

The photoisomerization of azobenzene has been known for almost 75 years but only recently has this process been widely applied to biological systems. The central challenge of how to productively couple the isomerization process to a large functional change in a biomolecule has been met in a number of instances and it appears that effective photocontrol of a large variety of biomolecules may be possible. This critical review summarizes key properties of azobenzene that enable its use as a photoswitch in biological systems and describes strategies for using azobenzene photoswitches to drive functional changes in peptides, proteins, nucleic acids, lipids, and carbohydrates (192 references).

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.

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.

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.