High-contrast NIR fluorescent probes for selective detection of NQO1 in breast cancer.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a potential biomarker for breast cancer (BC) diagnosis and prognosis. However, existing fluorescent probes for NQO1 detection have limitations such as short emission wavelength, weak fluorescence response, or large background interference. Here, we developed two novel near-infrared (NIR) fluorescent probes, DCl-Q and DCl2-Q, that selectively detect NQO1 activity in BC cells and tissues. They consist of a trimethyl-locked quinone as the recognition group and a donor-π-acceptor structure with halogen atoms as the reporter group. They exhibit strong fluorescence emission at around 660 nm upon binding to NQO1. We demonstrated that they can distinguish BC cells with different NQO1 expression levels and image endogenous NQO1 in tumor-bearing mice. Our probes provide a convenient and highly sensitive tool for BC diagnosis and prognosis based on NQO1 detection.

Structural dynamics at the active site of the cancer-associated flavoenzyme NQO1 probed by chemical modification with PMSF.

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates ß-amyloid aggregation.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-ß peptide (Aß1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aß1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aß1-42 peptide, the aggregation kinetics of Aß1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aß1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aß1-42. Aß1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aß1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aß1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aß1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.

Application of cation-π interactions in enzyme-substrate binding: Design, synthesis, biological evaluation, and molecular dynamics insights of novel hydrophilic substrates for NQO1.

Cation-π interaction is a type of noncovalent interaction formed between the π-electron system and the positively charged ion or moieties. In this study, we designed a series of novel NQO1 substrates by introducing aliphatic nitrogen-containing side chains to fit with the L-shaped pocket of NQO1 by the formation of cation-π interactions. Molecular dynamics (MD) simulation indicated that the basic N atom in the side chain of NQO1 substrates, which is prone to be protonated under physiological conditions, can form cation-π interactions with the Phe232 and Phe236 residues of the NQO1 enzyme. Compound 4 with a methylpiperazinyl substituent was identified as the most efficient substrate for NQO1 with the reduction rate and catalytic efficiency of 1263 ± 61 µmol NADPH/min/µmol NQO1 and 2.8 ± 0.3 × 106 M-1s-1, respectively. Notably, compound 4 exhibited increased water solubility (110 µg/mL) compared to that of ß-lap (43 µg/mL), especially under acidic condition (pH = 3, solubility > 1000 µg/mL). Compound 4 (IC50/A549 = 2.4 ± 0.6 µM) showed potent antitumor activity against NQO1-rich cancer cells through ROS generation via NQO1-mediated redox cycling. These results emphasized that the application of cation-π interactions by introducing basic aliphatic amine moiety is beneficial for both the water solubility and the NQO1-substrate binding, leading to promising NQO1-targeting antitumor candidates with improved druglike properties.

Enzyme-Triggered Disassembly of Polymeric Micelles by Controlled Depolymerization via Cascade Cyclization for Anticancer Drug Delivery.

The high activity of specific enzymes in cancer has been utilized in cancer diagnosis, as well as tumor-targeted drug delivery. NAD(P)H:quinone oxidoreductase-1 (NQO1), an overexpressed enzyme in certain tumor types, maintains homeostasis and inhibits oxidative stress caused by elevated reactive oxygen species (ROS) in tumor cells. The activity of NQO1 in lung and liver cancer cells is increased compared to that in normal cells. Interestingly, NQO1 reacts with trimethyl-locked quinone propionic acid (QPA) and produces a lactone-based group via intramolecular cyclization. Toward this objective, we synthesized an amphiphilic block copolymer (QPA-P) composed of NQO1 enzyme-triggered depolymerizable QPA-locked polycaprolactone (PCL) and poly(ethylene glycol) (PEG) as hydrophobic and hydrophilic constituents, respectively. This QPA-P formed self-assembled micelles in aqueous conditions. It was observed that NQO1 catalyzed the depolymerization of QPA-locked PCL via a cascade two-step cyclization process, which eventually induced the dissociation of micellar structure and triggered the release of loaded drugs at the target cancer cells. Compared to the control group, the NQO1-responsive micelle showed NQO1-triggered intracellular drug release and enhanced anticancer effects. These results indicate that the NQO1-responsive polymeric micelles present a promising potential for improving therapeutic efficacy of an anticancer drug delivery system.

Developing Push-Pull Hydroxylphenylpolyenylpyridinium Chromophores as Ratiometric Two-Photon Fluorescent Probes for Cellular and Intravital Imaging of Mitochondrial NQO1.

This work highlights the use of push-pull hydroxylphenylpolyenylpyridinium fluorophores coupled with trimethyl lock quinone to engineer the ratiometric two-photon probes for cellular and intravital imaging of mitochondrial NAD(P)H:quinone oxidoreductase 1 (NQO1), a critical antioxidant enzyme responsible for detoxifying quinones. As a typical representative, QBMP showed favorable binding with NQO1 with a Michaelis constant of 12.74 µM and exhibited a suite of superior properties, including rapid response (4 min), large Stokes shift (162 nm), ultralow detection limit (0.9 nM), favorable two-photon cross section for the released fluorophore (70.5 GM), and deep tissue penetration (225 µm) in fixed brain tissues. More importantly, this probe was successfully applied for distinguishing different NQO1-expressing cancer and normal cells, revealing decreased NQO1 activity in a cellular Parkinson's disease model, screening NQO1 inducers as neuroprotective agents, and imaging of NQO1 in live mouse brain.

Structural insights into vesicle amine transport-1 (VAT-1) as a member of the NADPH-dependent quinone oxidoreductase family.

Vesicle amine transport protein-1 (VAT-1) has been implicated in the regulation of vesicular transport, mitochondrial fusion, phospholipid transport and cell migration, and is a potential target of anticancer drugs. Little is known about the molecular function of VAT-1. The amino acid sequence indicates that VAT-1 belongs to the quinone oxidoreductase subfamily, suggesting that VAT-1 may possess enzymatic activity in unknown redox processes. To clarify the molecular function of VAT-1, we determined the three-dimensional structure of human VAT-1 in the free state at 2.3 Å resolution and found that VAT-1 forms a dimer with the conserved NADPH-binding cleft on each protomer. We also determined the structure of VAT-1 in the NADP-bound state at 2.6 Å resolution and found that NADP binds the binding cleft to create a putative active site with the nicotine ring. Substrate screening suggested that VAT-1 possesses oxidoreductase activity against quinones such as 1,2-naphthoquinone and 9,10-phenanthrenequinone.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

Targeting Lipid Peroxidation for Cancer Treatment.

Cancer is one of the highest prevalent diseases in humans. The chances of surviving cancer and its prognosis are very dependent on the affected tissue, body location, and stage at which the disease is diagnosed. Researchers and pharmaceutical companies worldwide are pursuing many attempts to look for compounds to treat this malignancy. Most of the current strategies to fight cancer implicate the use of compounds acting on DNA damage checkpoints, non-receptor tyrosine kinases activities, regulators of the hedgehog signaling pathways, and metabolic adaptations placed in cancer. In the last decade, the finding of a lipid peroxidation increase linked to 15-lipoxygenases isoform 1 (15-LOX-1) activity stimulation has been found in specific successful treatments against cancer. This discovery contrasts with the production of other lipid oxidation signatures generated by stimulation of other lipoxygenases such as 5-LOX and 12-LOX, and cyclooxygenase (COX-2) activities, which have been suggested as cancer biomarkers and which inhibitors present anti-tumoral and antiproliferative activities. These findings support the previously proposed role of lipid hydroperoxides and their metabolites as cancer cell mediators. Depletion or promotion of lipid peroxidation is generally related to a specific production source associated with a cancer stage or tissue in which cancer originates. This review highlights the potential therapeutical use of chemical derivatives to stimulate or block specific cellular routes to generate lipid hydroperoxides to treat this disease.

Synthesis and biological evaluation of NQO1-activated prodrugs of podophyllotoxin as antitumor agents.

Podophyllotoxin (PPT), a toxic polyphenol derived from the roots of genus Podophyllum, had been reported with strong inhibition on both normal human cells and tumor cells, which hindered the development of PPT as the candidate antitumor agent. In the present work, multiple NQO1-activatable PPT prodrugs were synthesized for reducing normal cell toxicity and keeping tumor cell toxicity. The antiproliferative activities in vitro showed prodrug 3 was greatly selectively toxic to tumor cells over-expressing NQO1, taxol-resistant A549, hypoxia A549 and HepG2, and lower damage to normal cells in comparison with podophyllotoxin, prodrug 1 and 2. As elucidated by further mechanistic research, prodrug 3 was activated via NQO1 to efficiently while gently produce cytotoxic PPT units and kill tumor cells. In additions, in vivo study revealed that 3 significantly suppressed cancer growth in HepG2 xenograft models without obvious toxicity. Therefore, this NQO1-activatable prodrug delivery system exhibits good biosafety and provides a novel strategy for the development of drug delivery systems.

NQO1-selective activated prodrugs of combretastatin A-4: Synthesis and biological evaluation.

Tumor-specific prodrug treatment renders the exclusive delivery of antitumor agents with the lowest untoward effects. In this work, we reported the synthesis and biological assessment of four NQO1-activatable combretastatin A-4 prodrugs constituted by active drug CA-4, different self-immolating linkers, and NQO1-responsive trigger groups. The in vitro antiproliferative activities showed that prodrug 4 displayed greater selective toxicity toward the tumor cells that overexpressed NQO1, taxol-resistant A549 cells, hypoxia-exposed A549 and HepG2 cells, and incurred lower damage to normal cells in comparison with combretastatin A-4, prodrugs 1, 2, and 3. Moreover, based on a mechanistic study, NQO1 triggered prodrug 4 to effectively liberate the parent drug combretastatin A-4 and kill tumor cells. Furthermore, we also demonstrated that prodrug 4 exerted a stronger anticancer effect and greater safety than combretastatin A-4 under in vivo conditions. Hence, from the above results, NQO1 can be used as a specific delivery system for releasing anticancer agents; besides, prodrug 4 can serve as a candidate lead for developing specific anticancer agents.

Potential Modulation of Human NAD[P]H-Quinone Oxidoreductase 1 (NQO1) by EGCG and Its Metabolites-A Systematic Computational Study.

At high doses, green tea extracts and green tea's major active constituent, (-)-epigallocatechin gallate (EGCG), despite their generally perceived health benefits, have been suspected to cause hepatotoxicity in certain human populations. It has been reported that o-quinone metabolites of gallic acid or EGCG are causative agents for this hepatotoxicity. However, no experimental information is available at the molecular level on the possible role of NQO1 in the detoxification of EGCG and its metabolites, including reactive intermediates. In the present study, we investigated the possibility of NQO1 inhibition by EGCG and its metabolites by studying their interaction profiles and binding mechanism at the active site of NQO1 using molecular docking, binding free energy calculations, and molecular dynamics (MD) simulations. The binding free energy calculations showed that some metabolites exhibited strong predicted binding affinity and found that the binding orientation of the EGCG metabolites overlapped with that of dicoumarol found in an NQO1 X-ray crystal structure. The results suggest that these metabolites may act as strong NQO1 inhibitors, highlighting the need for experimental validation of this with appropriate biological methods. The Prime MM-GBSA computed average binding free energies after MD simulations of compounds 1, 2, 24, 31, and 33 revealed that these compounds highly favored van der Waals (VdW) and Coulombic interactions with NQO1. In addition, the MD results revealed that selected EGCG metabolites formed a stable and strong complex with NQO1, with amino acids W105, Y126, Y128, H161, F178, H194, F232, and F236 being critical for potential NQO1 binding. The current results together with experimental data as well as studies of the polymorphisms of NQO1 (especially C609T) may explain the observed idiosyncratic hepatotoxicity caused by the consumption of green tea and its constituents.

Diaphorase-Catalyzed Formation of a Formazan Precipitate and Its Electrodissolution for Sensitive Affinity Biosensors.

Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (∼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.

Structural and spectral characterisation of 2-amino-2H-[1,2,3]triazolo[4,5-g]quinoline-4,9-dione polymorphs. Cytotoxic activity and molecular docking study with NQO1 enzyme.

Depending on temperature, the 2-amino-2H-[1,2,3]triazolo[4,5-g]quinoline-4,9-dione forms two polymorphic structures, which differ in the spatial arrangement of the amine group. Both polymorphs were investigated using different experimental methods as well as various quantum chemical calculations in order to characterise their molecular structures. We used X-ray diffraction, FT-IR and NMR (solid-state and liquid) methods supplemented by the density functional theory (DFT) calculations, molecular electrostatic potential (MEP) and molecular orbital (HOMO, LUMO) analyses. It was found that the arrangement of the amine group affected the crystal structure, formation of H-bonds, the amine and carbonyl vibration bands in the FT-IR spectra, chemical shift of amine group in 15N CP/MAS NMR and chemical shift of amine protons in 1H NMR spectra. Both polymorphs were tested on anticancer activity against a panel of human cancer cell lines. Comparing the activity of both compounds showed that activity against MCF-7, MDA-MB-231 and Caco-2 lines depend on the arrangement of the amine group. Moreover, both polymorphs exhibited the highest activity against cell line with high NQO1 protein level, such as: A549, MCF-7 and Caco-2. The molecular docking was used to examine the probable interaction between the ligand of the tested polymorphs and the NQO1 enzyme. The analysis showed that ligands formed a hydrophobic interaction with tryptophan (Trp105), phenylalanine (Phe126 and Phe178) and tyrosine (Tyr 126).

Cytosolic NQO1 Enzyme-Activated Near-Infrared Fluorescence Imaging and Photodynamic Therapy with Polymeric Vesicles.

The utilization of enzymes as a triggering module could endow responsive polymeric nanostructures with selectivity in a site-specific manner. On the basis of the fact that endogenous NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) is overexpressed in many types of tumors, we report on the fabrication of photosensitizer-conjugated polymeric vesicles, exhibiting synergistic NQO1-triggered turn-on of both near-infrared (NIR) fluorescence emission and a photodynamic therapy (PDT) module. For vesicles self-assembled from amphiphilic block copolymers containing quinone trimethyl lock-capped self-immolative side linkages and quinone-bridged photosensitizers (coumarin and Nile blue) in the hydrophobic block, both fluorescence emission and PDT potency are initially in the "off" state due to "double quenching" effects, that is, dye-aggregation-caused quenching and quinone-rendered PET (photoinduced electron transfer) quenching. After internalization into NQO1-positive vesicles, the cytosolic NQO1 enzyme triggers self-immolative cleavage of quinone linkages and fluorogenic release of conjugated photosensitizers, leading to NIR fluorescence emission turn-on and activated PDT. This process is accompanied by the transformation of vesicles into cross-linked micelles with hydrophilic cores and smaller sizes and triggered dual drug release, which could be directly monitored by enhanced magnetic resonance (MR) imaging for vesicles conjugated with a DOTA(Gd) complex in the hydrophobic bilayer. We further demonstrate that the above strategy could be successfully applied for activated NIR fluorescence imaging and tissue-specific PDT under both cellular and in vivo conditions.

Sensitive detection of low-concentration sulfide based on the synergistic effect of rGO, np-Au, and recombinant microbial cell.

With the aggravation of sulfide pollution, more and more attention has been paid to the detection of sulfide in the environment. However, the detection of low-concentration sulfide is still a technical bottleneck to be solved urgently. In this study, a synergistic effect strategy that combines the co-catalysis of nanoporous gold (np-Au) and recombinant microbial cell with the excellent electrical conductivity of reduced graphene oxide (rGO) was proposed for the sensitive detection of low-concentration sulfide. A rGO/np-Au composite was fabricated and then used as an immobilization support for the bio-recognition element of recombinant Escherichia coli (E. coli) over-expressed sulfide: quinone oxidoreductase (SQR). A microbial biosensor (E. coliSQR/rGO/np-Au/GCE) was successfully constructed for the sensitive detection of low-concentration sulfide. Due to the synergistic effect of rGO, np-Au, and E. coliSQR cells, the sensitivity of the proposed microbial biosensor towards sulfide reached 400.42 µA mM-1 cm-2 with a wide linear response ranging from 100 nM to 7 mM, as well as a low detection limit of 98.5 nM using amperometric i-t curve method. Furthermore, the microbial biosensor was successfully applied to the detection of sulfide in wastewater with strong anti-interference ability, high reproducibility, and strong stability. These results confirmed that the proposed microbial biosensor was ideal for the detection of low-concentration sulfide in a reliable, specific, and sensitive way.

A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.

The oligomeric state of the Caldivirga maquilingensis type III sulfide:Quinone Oxidoreductase is required for membrane binding.

Sulfide:quinone oxidoreductase (SQR) is a monotopic membrane flavoprotein present in all domains of life, with multiple roles including sulfide detoxification, homeostasis and energy generation by providing electrons to respiratory or photosynthetic electron transport chains. A type III SQR from the hyperthermophilic archeon Caldivirga maquilingensis has been previously characterized, and its C-terminal amphipathic helices were demonstrated to be responsible for membrane binding. Here, the oligomeric state of this protein was experimentally evaluated by size exclusion chromatography, native gels and crosslinking, and found to be a monomer-dimer-trimer equilibrium. Remarkably, mutant and truncated variants unable to bind to the membrane are able to maintain their oligomeric association. Thus, unlike other related monotopic membrane proteins, the region involved in membrane binding does not influence oligomerization. Furthermore, by studying heterodimers between the WT and mutants, it was concluded that membrane binding requires an oligomer with at least two copies of the protein with intact C-terminal amphipathic helices. A structural homology model of the C. maquilingensis SQR was used to define the flavin- and quinone-binding sites. CmGly12, CmGly16, CmAla77 and CmPro44 were determined to be important for flavin binding. Unexpectedly, CmGly299 is only important for quinone reduction despite its proximity to bound FAD. CmPhe337 and CmPhe362 are also important for quinone binding apparently by direct interaction with the quinone ring, whereas CmLys359, postulated to hydrogen bond to the quinone, seems to have a more structural role. The results presented differentiate the Type III CmSQR from some of its counterparts classified as Type I, II and V.

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.