Andrew A. Beharry

Azobenzene Photoswitching without Ultraviolet Light

Most azobenzene-based photoswitches use UV light for photoisomerization. This can limit their application in biological systems, where UV light can trigger unwanted responses, including cellular apoptosis. We have found that substitution of all four ortho positions with methoxy groups in an amidoazobenzene derivative leads to a substantial (~35 nm) red shift of the n-π* band of the trans isomer, separating it from the cis n-π* transition. This red shift makes trans-to-cis photoswitching possible using green light (530-560 nm). The cis state is thermally stable with a half-life of ~2.4 days in the dark in aqueous solution. Reverse (cis-to-trans) photoswitching can be accomplished with blue light (460 nm), so bidirectional photoswitching between thermally stable isomers is possible without using UV light at all.

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.

Fluorescence Imaging of Azobenzene Photoswitching In Vivo

The photoisomerization of azobenzene has been used to control a wide variety of molecular processes. It has been applied to the photocontrol of biomolecular targets (peptides, proteins, and nucleic acids) in vitro and in cell extracts. Recently it has been applied to the photocontrol of coiled-coil proteins in living cells in culture and to the photocontrol of ion channels in vivo. 8] These studies highlight the promise of azobenzene-modified biomolecules as general agents for the remote control of biomolecular function using light. In order to function as agents for controlling molecular events in complex living systems, however, the azobenzene-based photoswitches must be chemically stable in a variety of intracellular environments. At least for certain azobenzene photoswitches used for conformational control, glutathione in the intracellular environment can reduce and inactivate the photoswitch. Enzyme-mediated reduction or other modification, as occurs with numerous azo dyes, 11] is also possible. Even if not inactivated, azobenzene photoswitches may exhibit different isomerization rates in a cellular environment. For instance glutathione has been found to catalyze thermal cis-to-trans isomerization of certain azobenzenes. Fluorescence imaging of azobenzene photoswitching would enable a direct test of the feasibility of using azobenzene for intracellular photocontrol in a living organism. Since azobenzenes used for conformational control are not intrinsically fluorescent, we developed a fluorescence reporter for azobenzene photoswitching. Peptides bearing pairs of Cys residues can be intramolecularly cross-linked with thiol reactive azobenzene-based photoswitches such as 1 (Figure 1). These p-amido substituted azobenzene derivatives are relatively electron-rich compounds among those that have been used for conformational control in vitro 8,9, 13] and are resistant to reduction by glutathione in vitro (see Supporting Information). The metabolism of azo dyes has been found to be sensitive to their redox potentials, but also to the pattern and nature of ring substituents. Photoisomerization of the azobenzene cross-linker 1 alters the conformation of the peptide depending on the location of the Cys residue attachment points (Figure 1). Since a substantial conformational change occurs (Figure 2a,b), we reasoned that attachment of a fluorescent dye near the photoswitch may result in a fluorescence change upon isomerization. We explored a variety of fluorescent dyes with both sulfonated and non-sulfonated photoswitches (see Supporting Information). Peptide 2, shown in Figure 1, produced the largest change (a 40% decrease, Figure 2c) in fluorescence emission intensity in vitro upon trans-to-cis isomerization. The dark-adapted reporter has the azobenzene switch in the trans state; incubation in the dark restores the trans isomer with a half life of 10.7 min at 25 8C. Irradiation in the UV range (350–390) causes trans-to-cis isomerization of the photoswitch as well as excitation of fluorescein. Irradiation with blue light (440–490) causes cis-to-trans isomerization as well as fluorescein excitation. Since the cis isomer of the peptide (2a-cis) has a lower quantum yield for fluorescence than the trans isomer (2a-trans), irradiation of a darkadapted solution of reporter peptide with UV light produces a time-dependent fluorescence decrease (Figure 2d). Conversely, irradiation of 2a-cis with blue light causes a timedependent fluorescence increase (Figure 2d). The rates of these switching processes depend on the intensity and the wavelength of irradiation; action spectra are shown in the Supporting Information, Figure S1. A variety of mechanisms for the decrease in fluorescence intensity observed upon trans-to-cis isomerization are possible. Partial protonation of the fluorescein moiety due to an increase in its pKa upon trans-to-cis isomerization is unlikely since the fluorescence response is unchanged between pH 7 Figure 1. Structure of the photoswitches and the fluorescent peptide reporter.

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 enzymes 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.

Site-Selective Affinity Labelling of Maltose Binding Protein in Bacterial Cells

Carbohydrates play diverse roles in biological processes through selective binding interactions to their cognate carbohydrate binding protein. This important class of biomolecular interactions is exemplified by the interactions of Siglecs, galectins and selectins, which control many aspects of the immune response. The development of selective probes for carbohydrate binding proteins would provide useful tools to further understand their complex biological roles. Early labelling strategies for lectins focused on photoaffinity techniques. More recently, elegant examples of catalytic affinity-based labelling approaches have been developed. These approaches use a combination of a N,N-dialkyl-4aminopyridine catalyst and a thioester-derivatized fluorophore to facilitate the selective acylation of the lectin of interest. For other classes of ligand binding proteins, selective chemistries involving alternate electrophiles, such as primary tosylates have been developed. Here, we demonstrate that readily synthesised carbohydrate thioester conjugates can be used directly as site-selective labelling agents for the maltose binding protein (MBP) both in vitro and in intact Escherichia coli. Nature uses thioester chemistry extensively for selective acylation reactions. Perhaps the best-known example is from intein-mediated protein splicing, which inspired the development of native chemical ligation, now extensively used in protein synthesis. Thioesters have also shown promise as ligand-directed protein labelling agents. Glutathione transferase has been sitespecifically modified at tyrosine and lysine residues by using glutathione-based thioesters containing aryl substituents. Antiviral compounds that target the HIV nucleocapsid protein include p-mercaptobenzamide thioesters that have been shown to selectively acylate the protein. Recently, the amyloidogenic transthyretin protein was selectively labelled with fluorescent stilbene aryl thioesters at a pKa-perturbed lysine residue, and the Flag-tag peptide has been labelled with a thioester-derived nickel chelator. The labelling construct developed here is composed of a carbohydrate ligand and a fluorescent thioester acyl donor. Maltose binding protein was chosen for evaluation of the labelling reaction. MBP is an E. coli periplasmic protein that binds maltodextrins with micromolar affinity (1–80 mm) and is commonly used as a fusion partner for the expression of poorly behaved proteins. 11] MBP also acts as a convenient purification tag as it displays high affinity for amylose resin. It was hypothesised that recognition of a maltosyl thioester would facilitate delivery of the fluorescent acyl group to a local nucleophilic residue in the MBP binding site, thereby allowing site-selective labelling (Scheme 1).

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.

Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response.

Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment.

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.

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.