Bioorthogonal Quinone Methide Decaging Enables Live-Cell Quantification of Tumor-Specific Immune Interactions.

Effective antitumor immunity hinges on the specific engagement between tumor and cytotoxic immune cells, especially cytotoxic T cells. Although investigating these intercellular interactions is crucial for characterizing immune responses and guiding immunotherapeutic applications, direct and quantitative detection of tumor-T cell interactions within a live-cell context remains challenging. We herein report a photocatalytic live-cell interaction labeling strategy (CAT-Cell) relying on the bioorthogonal decaging of quinone methide moieties for sensitive and selective investigation and quantification of tumor-T cell interactions. By developing quinone methide-derived probes optimized for capturing cell-cell interactions (CCIs), we demonstrated the capacity of CAT-Cell for detecting CCIs directed by various types of receptor-ligand pairs (e.g., CD40-CD40L, TCR-pMHC) and further quantified the strengths of tumor-T cell interactions that are crucial for evaluating the antitumor immune responses. We further applied CAT-Cell for ex vivo quantification of tumor-specific T cell interactions on splenocyte and solid tumor samples from mouse models. Finally, the broad compatibility and utility of CAT-Cell were demonstrated by integrating it with the antigen-specific targeting system as well as for tumor-natural killer cell interaction detection. By leveraging the bioorthogonal photocatalytic decaging chemistry on quinone methide, CAT-Cell provides a sensitive, tunable, universal, and noninvasive toolbox for unraveling and quantifying the crucial but delicate tumor-immune interactions under live-cell settings.

Design, synthesis and bioactivity evaluation of the combination of evodiamine and erlotinib linked by indolequinone.

Compared with single-targeted therapy, the design and synthesis of heterozygous molecules is still a significant challenge for the discovery of antitumor drugs. Quinone oxidoreductase-1 (NQO1) is a potential target for selective cancer therapy due to its overexpression in many cancer cells and its unique bioredox properties. Based on the principle of combinatorial drug design, we successfully synthesized a new hybrid molecules 13 with an indolequinone structure. We found that the synthesized compounds exhibited much higher cytotoxicity against the tested cancer cells than free drugs. Further mechanism studies confirmed that compound 13 induced cell apoptosis was achieved by regulating p53-dependent mitochondrial pathway and cell cycle arrest at the G0/G1 phase.

Synthesis and Photochemical Properties of Fluorescent Metabolites Generated from Fluorinated Benzoylmenadiones in Living Cells.

This work describes the reactivity and properties of fluorinated derivatives (F-PD and F-PDO) of plasmodione (PD) and its metabolite, the plasmodione oxide (PDO). Introduction of a fluorine atom on the 2-methyl group markedly alters the redox properties of the 1,4-naphthoquinone electrophore, making the compound highly oxidizing and particularly photoreactive. A fruitful set of analytical methods (electrochemistry, absorption and emission spectrophotometry, and HRMS-ESI) have been used to highlight the products resulting from UV photoirradiation in the absence or presence of selected nucleophiles. With F-PDO and in the absence of nucleophile, photoreduction generates a highly reactive ortho-quinone methide (o-QM) capable of leading to the formation of a homodimer. In the presence of thiol nucleophiles such as ß-mercaptoethanol, which was used as a model, o-QMs are continuously regenerated in sequential photoredox reactions generating mono- or disulfanylation products as well as various unreported sulfanyl products. Besides, these photoreduced adducts derived from F-PDO are characterized by a bright yellowish emission due to an excited-state intramolecular proton transfer (ESIPT) process between the dihydronapthoquinone and benzoyl units. In order to evidence the possibility of an intramolecular coupling of the o-QM intermediate, a synthetic route to the corresponding anthrones is described. Tautomerization of the targeted anthrones occurs and affords highly fluorescent stable hydroxyl-anthraquinones. Although probable to explain the intense visible fluorescence emission also observed in tobacco BY-2 cells used as a cellular model, these coupling products have never been observed during the photochemical reactions performed in this study. Our data suggest that the observed ESIPT-induced fluorescence most likely corresponds to the generation of alkylated products through reduction species, as demonstrated with the ß-mercaptoethanol model. In conclusion, F-PDO thus acts as a novel (pro)-fluorescent probe for monitoring redox processes and protein alkylation in living cells.

Design, synthesis and antitumor activity of potent and safe para-quinone methides derivatives in vitro and in vivo.

Compounds containing Michael acceptor units display a wide variety of biological effects, and have attracted much attention in medicinal chemistry. In this paper, we designed and synthesized a panel of para-quinone methides (p-QMs) derivatives, classified as electron-deficient alkenes, and evaluated their cytotoxicity against cancer cells. These results revealed that drawing substituents into the ortho-position of the phenyl ring could obviously strengthen the cytotoxicity of p-QMs derivatives compared with that of meta- and para-substituents. Further biological studies demonstrated that the cytotoxicity of p-QMs derivatives originated from their ROS-generation abilities, which could further disrupt the redox balance, lipid peroxidation, the loss of MMP, cell cycle arrest at G0/G1 phase and apoptosis. 1h also exhibited potent antitumor activity through inhibiting TrxR and activating Bax and caspase 3 expression in vitro and in vivo, and 1h had certain safety in vivo. Moreover, the electrophilicity of the Michael acceptor, which could covalently modify with the TrxR, play a potent role in the ROS generation. From the perspective of chemistry, we affirmed that p-QMs derivatives could rapidly covalent binding with cysteamine, and the addition product was characterized by 1H NMR. Together, these new p-QMs derivatives may possess potential as leads for development of effective antitumor agents.

Metal-Free Reductive Coupling of para-Quinone Methides with 4-Cyanopyridines Enabled by Pyridine-Boryl Radicals: Access to Pyridylated Diarylmethanes with Anti-Cancer Activity.

Reported herein is a streamlined protocol to produce pyridylated diarylmethanes through pyridine-boryl radical induced reductive coupling between para-quinone methides (p-QMs) and 4-cyanopyridines using bis(pinacolato)diboron (B2 pin2 ) as a templated reagent. The metal-free process is characterized by an operationally simple approach, excellent chemoselectivity (1,2- vs. 1,6-selectivity), and a broad substrate scope with good functional group compatibility. The mechanistic studies provided important insights into the reductive cross-coupling process between diarylmethyl radical and pyridine-boryl radical. Moreover, part of the obtained pyridylated diarylmethane products were screened against a panel of cancer cell lines, and 3 v was confirmed to significantly inhibit the proliferation of head and neck squamous cell carcinoma (HNSCC) cells. This method offers a platform for the preparation of new lead compounds with antitumor activity.

Harnessing ortho-Quinone Methides in Natural Product Biosynthesis and Biocatalysis.

The implementation of ortho-quinone methide (o-QM) intermediates in complex molecule assembly represents a remarkably efficient strategy designed by Nature and utilized by synthetic chemists. o-QMs have been taken advantage of in biomimetic syntheses for decades, yet relatively few examples of o-QM-generating enzymes in natural product biosynthetic pathways have been reported. The biosynthetic enzymes that have been discovered thus far exhibit tremendous potential for biocatalytic applications, enabling the selective production of desirable compounds that are otherwise intractable or inherently difficult to achieve by traditional synthetic methods. Characterization of this biosynthetic machinery has the potential to shine a light on new enzymes capable of similar chemistry on diverse substrates, thus expanding our knowledge of Nature's catalytic repertoire. The presently known o-QM-generating enzymes include flavin-dependent oxidases, hetero-Diels-Alderases, S-adenosyl-l-methionine-dependent pericyclases, and α-ketoglutarate-dependent nonheme iron enzymes. In this review, we discuss their diverse enzymatic mechanisms and potential as biocatalysts in constructing natural product molecules such as cannabinoids.

Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity.

Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10-2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10-5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105-106 M-1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M-1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

Caspase-3: A primary target for natural and synthetic compounds for cancer therapy.

Caspases, a group of protease enzymes (cysteine proteases), exist as inactive zymogens in the cells and execute apoptosis (programmed cell death). Caspase-3, an executioner caspase, plays an imperative role in apoptosis and becomes a primary target for cancer treatment. A number of analogues of quinazoline, quinazolinone, indoloquinazolines, quinone, naphthoquinones, pyrroloiminoquinones, styrylquinolines, tetheredtetrahydroquinoline, fluoroquinolone, thiosemicarbazones, benzotriazole, pyrimidines, chalcone, and carbazoles have been reported till date, representing caspase-3 mediated apoptosis for cancer therapy. Simultaneously, plant isolates, including lysicamine, podophyllotoxin, and majoranolide, have also been claimed for caspase-3-mediated apoptosis-induced cytotoxicity. Procaspase-activating compound-1 (PAC-1) is the first FDA approved orphan drug, and its synthetic derivative WF-208 also showed fascinating caspase-3 mediated anticancer activity. Till date, a large number of compounds have been reported and patented for their caspase-3-mediated cytotoxicity and now scientist is also focusing to introduce new compounds in market to encompass anticancer activity.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Synthesis, anticancer evaluation and mechanism studies of novel indolequinone derivatives of ursolic acid.

A series of novel indolequinone derivatives of ursolic acid bearing ester, hydrazide, or amide moieties were designed, synthesized, and screened for their in vitro antiproliferative activities against three cancer cell lines (MCF-7, HeLa, and HepG2) and a normal gastric mucosal cell line (Ges-1). A number of compounds showed significant activity against tested cancer cell lines. Among them, compound 6t exhibited the most potent activity against three cancer cell lines with IC50 values of 1.66 ± 0.21, 3.16 ± 0.24, and 10.35 ± 1.63 µM, respectively, and considerably lower cytotoxicity to Ges-1 cells. Especially, compound 6t could arrest cell cycle at S phase, suppress the migration of MCF-7 cells, elevate intracellular reactive oxygen species (ROS) level, and decrease mitochondrial membrane potential. Western blot analysis showed that compound 6t upregulated Bax, cleaved caspase-3/9, cleaved PARP levels and downregulated Bcl-2 level of MCF-7 cells. All these results indicated that compound 6t could significantly induce the apoptosis of MCF-7 cells. Meanwhile, compound 6t markedly decreased p-AKT and p-mTOR expression, which revealed that compound 6t probably exerted its cytotoxicity through targeting PI3K/AKT/mTOR signaling pathway. Therefore, compound 6t could be a promising lead for the discovery of novel anticancer agents.

Probing Molecular-Scale Oxidative Generation of Quinone Methides and Their Transformation Using Tip-Enhanced Raman Spectroscopy.

Quinone methides (QMs) are very important intermediates in chemistry. These species are most often generated in situ with metal oxidants and transition metal complexes. Here, tip-enhanced Raman spectroscopy (TERS) has been implemented to investigate the in situ oxidative generation of a QM species from alkylphenols facilitated by a transition metal complex. Using TERS, the metal oxidant-mediated transformation of a phenol species has been observed. The subsequent oxidative addition reaction of QM has also been identified based on distinct vibrational features, which have been assigned based on density functional theory (DFT). This study may establish TERS as a chemical detection tool for various QM-mediated reactions.

The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins.

Tryptophan (Trp) holds a unique place in biology for a multitude of reasons. It is the largest of all twenty amino acids in the translational toolbox. Its side chain is indole, which is aromatic with a binuclear ring structure, whereas those of Phe, Tyr, and His are single-ring aromatics. In part due to these elaborate structural features, the biosynthetic pathway of Trp is the most complex and the most energy-consuming among all amino acids. Essential in the animal diet, Trp is also the least abundant amino acid in the cell, and one of the rarest in the proteome. In most eukaryotes, Trp is the only amino acid besides Met, which is coded for by a single codon, namely UGG. Due to the large and hydrophobic π-electron surface area, its aromatic side chain interacts with multiple other side chains in the protein, befitting its strategic locations in the protein structure. Finally, several Trp derivatives, namely tryptophylquinone, oxitriptan, serotonin, melatonin, and tryptophol, have specialized functions. Overall, Trp is a scarce and precious amino acid in the cell, such that nature uses it parsimoniously, for multiple but selective functions. Here, the various aspects of the uniqueness of Trp are presented in molecular terms.

Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5'- or 3'-terminus to mimic a quadruplex-duplex (Q-D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q-D hybrids with a very high affinity in the order Ka ≈107  m-1 irrespective of the duplex location at the quadruplex 3'- or 5'-end. NMR chemical shift perturbations identified the tetrad face of the Q-D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3'- or 5'-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3'-Q-D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5'-Q-D hybrid.

ß-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens.

The ability to detect cell surface proteins using fluorescent-dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter-wavelength region. Herein we report on a new method, quinone methide-based catalyzed labeling for signal amplification (CLAMP), in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used ß-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, that contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by ß-galactosidase labeling of the target cell surface proteins, the resulting product containing the quinone methide group was found to be both cell-membrane-permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins, including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent-dye-labeled antibodies.

Aza-ortho-Quinone Methide and Its Conjugated Acid: Reactivity, Stability and Acidity.

Aza-o-quinone methides and their conjugated acids are reactive drug metabolites that might react with nucleophilic sites of DNAs and proteins to cause cancer or immune responses, and their reactivity with water is the key information to judge if these metabolites are harmful in living systems. For the first time, aza-o-quinone methide (1) and its conjugated acid (2) are observed by laser flash photolysis, and their reactivity, stability and acidity in water are determined.

Signal Amplification for Imaging Mass Cytometry.

An enzyme-catalyzed reporter deposition stain has been developed for Imaging Mass Cytometry (IMC). The reagent consists of an alkaline phosphatase substrate tethered to a tellurophene which serves as reporter group for mass cytometry. Upon phosphate hydrolysis, a quinone methide is released which covalently labels local nucleophiles. This strategy is a useful complement to heavy isotope antibody conjugates as it facilitates signal amplification for low-abundance biomarker detection. The workflow is conveniently integrated with standard IMC antibody staining to allow multiparametric antigen detection.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2 Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Atypical Lone Pair-π Interaction with Quinone Methides in a Series of Imido-Ferrociphenol Anticancer Drug Candidates.

Ferrociphenols, especially those possessing a heterocycle at the terminus of an aliphatic chain, display strong anticancer activity through a novel redox mechanism that generates active metabolites such as quinone methides (QMs). X-ray crystallography and UV/Vis spectroscopy reveal that the specific lone pair (lp)-π interaction between a carbonyl group of the imide and the quinone motif of the QM plays an important role in the exceptional cytotoxic behaviour of their imido-ferrociphenol precursors. This intramolecular lp-π interaction markedly enhanced the stability of the QMs and lowered the pKa values of the corresponding phenol/phenolate couples. As the first example of such a non-covalent interaction that stabilizes QMs remotely, it not only expands the scope of the lp-π interaction in supramolecular chemistry, but also represents a new mode of stabilization of a QM. This unprecedented application of lp-π interactions in imido-ferrociphenol anticancer drug candidates may also have great potential in drug discovery and organocatalyst design.

The Redox Properties of a Cysteine Tryptophylquinone-Dependent Glycine Oxidase Are Distinct from Those of Tryptophylquinone-Dependent Dehydrogenases.

GoxA is a cysteine tryptophylquinone (CTQ)-dependent glycine oxidase that is a member of a family of LodA-like proteins. The electrochemical midpoint potential ( Em) values for the quinone/semiquinone couple and the semiquinone/quinol couple were determined to be 111 and 21, respectively. The Em value for the overall two-electron quinone/quinol couple was similar to those of CTQ- and tryptophan tryptophylquinone (TTQ)-bearing dehydrogenases. However, for the well-studied TTQ-dependent methylamine dehydrogenase, the quinone/semiquinone couple is more negative than the semiquinone/quinol couple, the opposite of what was determined for GoxA. The change in Em value for the two-electron quinone/quinol couple of CTQ in GoxA with pH indicates that the overall two-electron transfer process is associated with the transfer of one proton. Thus, the quinol is anionic. The data reported herein further suggest that in GoxA the CTQ semiquinone is neutral, in contrast to the TTQ-dependent dehydrogenases, in which it is an anionic TTQ semiquinone. These results are discussed in the context of the structure and function of this glycine oxidase, compared to that of the tryptophylquinone-dependent dehydrogenases.