Exploring Structural-Photophysical Property Relationships in Mitochondria-Targeted Deep-Red/NIR-Emitting Coumarins.

Organic fluorophores operating in the optical window of biological tissues, namely in the deep-red and near-infrared (NIR) region of the electromagnetic spectrum, offer several advantages for fluorescence bioimaging applications owing to the appealing features of long-wavelength light, such as deep tissue penetration, lack of toxicity, low scattering, and reduced interference with cellular autofluorescence. Among these, COUPY dyes based on non-conventional coumarin scaffolds display suitable photophysical properties and efficient cellular uptake, with a tendency to accumulate primarily in mitochondria, which renders them suitable probes for bioimaging purposes. In this study, we have explored how the photophysical properties and subcellular localization of COUPY fluorophores can be modulated through the modification of the coumarin backbone. While the introduction of a strong electron-withdrawing group, such as the trifluoromethyl group, at position 4 resulted in an exceptional photostability and a remarkable redshift in the absorption and emission maxima when combined with a julolidine ring replacing the N,N-dialkylaminobenzene moiety, the incorporation of a cyano group at position 3 dramatically reduced the brightness of the resulting fluorophore. Interestingly, confocal microscopy studies in living HeLa cells revealed that the 1,1,7,7-tetramethyl julolidine-containing derivatives accumulated in the mitochondria with much higher specificity. Overall, our results provide valuable insights for the design and optimization of new COUPY dyes operating in the deep-red/NIR region.

Exploring the HIV-1 Rev Recognition Element (RRE)-Rev Inhibitory Capacity and Antiretroviral Action of Benfluron Analogs.

Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE-Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE-Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE-Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.

Ru(II)-Cyanine Complexes as Promising Photodynamic Photosensitizers for the Treatment of Hypoxic Tumours with Highly Penetrating 770†nm Near-Infrared Light.

Light-activated treatments, such as photodynamic therapy (PDT), provide temporal and spatial control over a specific cytotoxic response by exploiting toxicity differences between irradiated and dark conditions. In this work, a novel strategy for developing near infrared (NIR)-activatable Ru(II) polypyridyl-based photosensitizers (PSs) was successfully developed through the incorporation of symmetric heptamethine cyanine dyes in the metal complex via a phenanthrimidazole ligand. Owing to their strong absorption in the NIR region, the PSs could be efficiently photoactivated with highly penetrating NIR light (770 nm), leading to high photocytotoxicities towards several cancer cell lines under both normoxic and hypoxic conditions. Notably, our lead PS (Ru-Cyn-1), which accumulated in the mitochondria, exhibited a good photocytotoxic activity under challenging low-oxygen concentration (2 % O2 ) upon NIR light irradiation conditions (770 nm), owing to a combination of type I and II PDT mechanisms. The fact that the PS Protoporphyrin IX (PpIX), the metabolite of the clinically approved 5-ALA PS, was found inactive under the same challenging conditions positions Ru-Cyn-1 complex as a promising PDT agent for the treatment of deep-seated hypoxic tumours.

A near-infrared light-activatable Ru(ii)-coumarin photosensitizer active under hypoxic conditions.

Photodynamic therapy (PDT) represents a promising approach for cancer treatment. However, the oxygen dependency of PDT to generate reactive oxygen species (ROS) hampers its therapeutic efficacy, especially against hypoxic solid tumors. In addition, some photosensitizers (PSs) have dark toxicity and are only activatable with short wavelengths such as blue or UV-light, which suffer from poor tissue penetration. Herein, we developed a novel hypoxia-active PS with operability in the near-infrared (NIR) region based on the conjugation of a cyclometalated Ru(ii) polypyridyl complex of the type [Ru(C^N)(N^N)2] to a NIR-emitting COUPY dye. The novel Ru(ii)-coumarin conjugate exhibits water-solubility, dark stability in biological media and high photostability along with advantageous luminescent properties that facilitate both bioimaging and phototherapy. Spectroscopic and photobiological studies revealed that this conjugate efficiently generates singlet oxygen and superoxide radical anions, thereby achieving high photoactivity toward cancer cells upon highly-penetrating 740 nm light irradiation even under hypoxic environments (2% O2). The induction of ROS-mediated cancer cell death upon low-energy wavelength irradiation along with the low dark toxicity exerted by this Ru(ii)-coumarin conjugate could circumvent tissue penetration issues while alleviating the hypoxia limitation of PDT. As such, this strategy could pave the way to the development of novel NIR- and hypoxia-active Ru(ii)-based theragnostic PSs fuelled by the conjugation of tunable, low molecular-weight COUPY fluorophores.

Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates.

Photodynamic therapy holds great promise as a non-invasive anticancer tool against drug-resistant cancers. However, highly effective, non-toxic, and reliable photosensitizers with operability under hypoxic conditions remain to be developed. Herein, we took the advantageous properties of COUPY fluorophores and cyclometalated Ir(III) complexes to develop novel PDT agents based on Ir(III)-COUPY conjugates with the aim of exploring structure-activity relationships. The structural modifications carried out within the coumarin scaffold had a strong impact on the photophysical properties and cellular uptake of the conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity under green light irradiation, which was attributed to the photogeneration of ROS, while remaining non-toxic in the dark. Among them, two hit conjugates showed excellent phototherapeutic indexes in cisplatin-resistant A2780cis cancer cells, both in normoxia and in hypoxia, suggesting that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting COUPY dyes and transition metal complexes could effectively tackle in vitro acquired resistance to cisplatin.

Mitochondria-Targeted COUPY Photocages: Synthesis and Visible-Light Photoactivation in Living Cells.

Releasing bioactive molecules in specific subcellular locations from the corresponding caged precursors offers great potential in photopharmacology, especially when using biologically compatible visible light. By taking advantage of the intrinsic preference of COUPY coumarins for mitochondria and their long wavelength absorption in the visible region, we have synthesized and fully characterized a series of COUPY-caged model compounds to investigate how the structure of the coumarin caging group affects the rate and efficiency of the photolysis process. Uncaging studies using yellow (560 nm) and red light (620 nm) in phosphate-buffered saline medium have demonstrated that the incorporation of a methyl group in a position adjacent to the photocleavable bond is particularly important to fine-tune the photochemical properties of the caging group. Additionally, the use of a COUPY-caged version of the protonophore 2,4-dinitrophenol allowed us to confirm by confocal microscopy that photoactivation can occur within mitochondria of living HeLa cells upon irradiation with low doses of yellow light. The new photolabile protecting groups presented here complement the photochemical toolbox in therapeutic applications since they will facilitate the delivery of photocages of biologically active compounds into mitochondria.

SARS-CoV-2 Inhibitors Identified by Phenotypic Analysis of a Collection of Viral RNA-Binding Molecules.

Antiviral agents are needed for the treatment of SARS-CoV-2 infections and to control other coronavirus outbreaks that may occur in the future. Here we report the identification and characterization of RNA-binding compounds that inhibit SARS-CoV-2 replication. The compounds were detected by screening a small library of antiviral compounds previously shown to bind HIV-1 or HCV RNA elements with a live-virus cellular assay detecting inhibition of SARS-CoV-2 replication. These experiments allowed detection of eight compounds with promising anti-SARS-CoV-2 activity in the sub-micromolar to micromolar range and wide selectivity indexes. Examination of the mechanism of action of three selected hit compounds excluded action on the entry or egress stages of the virus replication cycle and confirmed recognition by two of the molecules of conserved RNA elements of the SARS-CoV-2 genome, including the highly conserved S2m hairpin located in the 3'-untranslated region of the virus. While further studies are needed to clarify the mechanism of action responsible for antiviral activity, these results facilitate the discovery of RNA-targeted antivirals and provide new chemical scaffolds for developing therapeutic agents against coronaviruses.

Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies.

Ceramides (Cer) are bioactive sphingolipids that have been proposed as potential disease biomarkers since they are involved in several cellular stress responses, including apoptosis and senescence. 1-Deoxyceramides (1-deoxyCer), a particular subtype of noncanonical sphingolipids, have been linked to the pathogenesis of type II diabetes. To investigate the metabolism of these bioactive lipids, as well as to have a better understanding of the signaling processes where they participate, it is essential to expand the toolbox of fluorescent sphingolipid probes exhibiting complementary subcellular localization. Herein, we describe a series of new sphingolipid probes tagged with two different organic fluorophores, a far-red/NIR-emitting coumarin derivative (COUPY) and a green-emitting BODIPY. The assembly of the probes involved a combination of olefin cross metathesis and click chemistry reactions as key steps, and these fluorescent ceramide analogues exhibited excellent emission quantum yields, being the Stokes' shifts of the COUPY derivatives much higher than those of the BODIPY counterparts. Confocal microscopy studies in HeLa cells confirmed an excellent cellular permeability for these sphingolipid probes and revealed that most of the vesicles stained by COUPY probes were either lysosomes or endosomes, whereas BODIPY probes accumulated either in Golgi apparatus or in nonlysosomal intracellular vesicles. The fact that the two sets of fluorescent Cer probes have such different staining patterns indicates that their subcellular distribution is not entirely defined by the sphingolipid moiety but rather influenced by the fluorophore.

Improving Photodynamic Therapy Anticancer Activity of a Mitochondria-Targeted Coumarin Photosensitizer Using a Polyurethane-Polyurea Hybrid Nanocarrier.

Integration of photosensitizers (PSs) within nanoscale delivery systems offers great potential for overcoming some of the "Achiles' heels" of photodynamic therapy (PDT). Herein, we have encapsulated a mitochondria-targeted coumarin PS into amphoteric polyurethane-polyurea hybrid nanocapsules (NCs) with the aim of developing novel nanoPDT agents. The synthesis of coumarin-loaded NCs involved the nanoemulsification of a suitable prepolymer in the presence of a PS without needing external surfactants, and the resulting small nanoparticles showed improved photostability compared with the free compound. Nanoencapsulation reduced dark cytotoxicity of the coumarin PS and significantly improved in vitro photoactivity with red light toward cancer cells, which resulted in higher phototherapeutic indexes compared to free PS. Importantly, this nanoformulation impaired tumoral growth of clinically relevant three-dimensional multicellular tumor spheroids. Mitochondrial photodamage along with reactive oxygen species (ROS) photogeneration was found to trigger autophagy and apoptotic cell death of cancer cells.

Novel Tumor-Targeted Self-Nanostructured and Compartmentalized Water-in-Oil-in-Water Polyurethane-Polyurea Nanocapsules for Cancer Theragnosis.

Encapsulation of water-soluble bioactive compounds for enabling specific accumulation in tumor locations, while avoiding premature clearance and/or degradation in the bloodstream, is one of the main hallmarks in nanomedicine, especially that of NIR fluorescent probes for cancer theragnosis. The herein reported technology furnishes water-dispersible double-walled polyurethane-polyurea hybrid nanocapsules (NCs) loaded with indocyanine green (ICG-NCs), using a versatile and highly efficient one-pot and industrially scalable synthetic process based on the use of two different prepolymers to set up the NCs walls. Flow cytometry and confocal microscopy confirmed that both ICG-loaded NCs internalized in monocyte-derived dendritic cells (moDCs). The in vivo analysis of xenograft A375 mouse melanoma model revealed that amphoteric functionalization of NCs' surface promotes the selective accumulation of ICG-NCs in tumor tissues, making them promising agents for a less-invasive theragnosis of cancer.

COUPY Coumarins as Novel Mitochondria-Targeted Photodynamic Therapy Anticancer Agents.

Photodynamic therapy (PDT) for cancer treatment has drawn increased attention over the last decades. Herein, we introduce a novel family of low-molecular-weight coumarins as potential PDT anticancer tools. Through a systematic study with a library of 15 compounds, we have established a detailed structure-activity relationship rationale, which allowed the selection of three lead compounds exhibiting effective in vitro anticancer activities upon visible-light irradiation in both normoxia and hypoxia (phototherapeutic indexes up to 71) and minimal toxicity toward normal cells. Acting as excellent theranostic agents targeting mitochondria, the mechanism of action of the photosensitizers has been investigated in detail in HeLa cells. The generation of cytotoxic reactive oxygen species, which has been found to be a major contributor of the coumarins' phototoxicity, and the induction of apoptosis and/or autophagy have been identified as the cell death modes triggered after irradiation with low doses of visible light.

A Cyclometalated IrIII Complex Conjugated to a Coumarin Derivative Is a Potent Photodynamic Agent against Prostate Differentiated and Tumorigenic Cancer Stem Cells.

A cyclometalated IrIII complex conjugated to a far-red-emitting coumarin, IrIII -COUPY (3), was recently shown as a very promising photosensitizer suitable for photodynamic therapy of cancer. Therefore, the primary goal of this work was to deepen knowledge on the mechanism of its photoactivated antitumor action so that this information could be used to propose a new class of compounds as drug candidates for curing very hardly treatable human tumors, such as androgen resistant prostatic tumors of metastatic origin. Conventional anticancer chemotherapies exhibit several disadvantages, such as limited efficiency to target cancer stem cells (CSCs), which are considered the main reason for chemotherapy resistance, relapse, and metastasis. Herein, we show, using DU145 tumor cells, taken as the model of hormone-refractory and aggressive prostate cancer cells resistant to conventional antineoplastic drugs, that the photoactivated conjugate 3 very efficiently eliminates both prostate bulk (differentiated) and prostate hardly treatable CSCs simultaneously and with a similar efficiency. Notably, the very low toxicity of IrIII -COUPY conjugate in the prostate DU145 cells in the dark and its pronounced selectivity for tumor cells compared with noncancerous cells could result in low side effects and reduced damage of healthy cells during the photoactivated therapy by this agent. Moreover, the experiments performed with the 3D spheroids formed from DU145 CSCs showed that conjugate 3 can penetrate the inner layers of tumor spheres, which might markedly increase its therapeutic effect. Also interestingly, this conjugate induces apoptotic cell death in prostate cancer DU145 cells associated with calcium signaling flux in these cells and autophagy. To the best of our knowledge, this is the first study demonstrating that a photoactivatable metal-based compound is an efficient agent capable of killing even hardly treatable CSCs.

A versatile click chemistry-based approach for functionalizing biomaterials of diverse nature with bioactive peptides.

A novel and versatile toolkit approach for the functionalization of biomaterials of different nature is described. This methodology is based on the solid-phase conjugation of specific anchoring units onto a resin-bound azido-functionalized peptide by using click chemistry. A synergistic multifunctional peptidic scaffold with cell adhesive properties was used as a model compound to showcase the versatility of this new approach. Titanium, gold and polylactic acid surfaces were biofunctionalized by this method, as validated by physicochemical surface characterization with XPS. In vitro assays using mesenchymal stem cells showed enhanced cell adhesion on the functionalized samples, proving the capacity of this strategy to efficiently bioactivate different types of biomaterials.

Unexpected photoactivation pathways in a folate-receptor-targeted trans-diazido Pt(IV) anticancer pro-drug.

A conjugate between a photoactive trans-diazido Pt(iv) pro-drug, trans,trans,trans-[Pt(N3)2(OH)2(py)2], and folic acid has been synthesized and fully characterized by high resolution ESI-MS, NMR and UV-vis spectroscopy. Photoactivation of the Pt-folate conjugate with visible light confirmed the generation of cytotoxic Pt(ii) species capable of binding to guanine nucleobases. Importantly, photoreduction of the Pt(iv) complex triggered the photodecomposition of the folate vector into a p-aminobenzoate-containing fragment and several pterin derivatives, including 6-formylpterin. Besides exhibiting high dark stability in physiological-like conditions, the Pt-folate conjugate was ca. 2× more photocytotoxic towards MCF-7 breast cancer cell line than its parent Pt(iv) complex with a high photoselectivity index (PI > 6.9). The higher photocytotoxicity of the conjugate may be a consequence of its higher cellular accumulation and of the generation of a set of different cytotoxic species, including Pt(ii) photoproducts and several pterin derivatives, which are known to generate ROS.

Transformation of COUPY Fluorophores into a Novel Class of Visible-Light-Cleavable Photolabile Protecting Groups.

Although photolabile protecting groups (PPGs) have found widespread applications in several fields of chemistry, biology and materials science, there is a growing interest in expanding the photochemical toolbox to overcome some of the limitations of classical caging groups. In this work, the synthesis of a new class of visible-light-sensitive PPGs based on low-molecular weight COUPY fluorophores with several attractive properties, including long-wavelength absorption, is reported. Besides being stable to spontaneous hydrolysis in the dark, COUPY-based PPGs can be efficiently photoactivated with yellow (560 nm) and red light (620 nm) under physiological-like conditions, thereby offering the possibility of unmasking functional groups from COUPY photocages under irradiation conditions in which other PPGs remain stable. Additionally, COUPY photocages exhibit excellent cellular uptake and accumulate selectively in mitochondria, opening the door to the delivery of caged analogues of biologically active compounds into these organelles.

Modulating Photostability and Mitochondria Selectivity in Far-Red/NIR Emitting Coumarin Fluorophores through Replacement of Pyridinium by Pyrimidinium.

Mitochondrial dysfunction has been associated with several human pathological conditions, including cancer, aging, and neurodegenerative diseases. Thus, the availability of selective fluorescent probes for mitochondria could play an important role in the future for monitoring cellular functions and disease progression. In this work, we have studied how the photophysical properties and subcellular accumulation of nonconventional coumarin-based COUPY fluorophores can be fine-tuned through replacement of the para-pyridinium moiety with several heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties for bioimaging applications, including emission in the far-red to NIR region, large Stokes' shifts, and high photostability. Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria compared with the parent COUPY fluorophores. Overall, these results provided useful insights into the development of novel mitochondria-targeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-pyrimidinium counterparts.

A simple method for the synthesis of N-difluoromethylated pyridines and 4-pyridones/quinolones by using BrCF2COOEt as the difluoromethylation reagent.

We describe a novel transition metal-free method for the synthesis of N-difluoromethylated pyridines and 4-pyridones/quinolones by using readily available ethyl bromodifluoroacetate as a fluorine source. The formation of N-difluoromethylated pyridines involves a two-step process in which N-alkylation by ethyl bromodifluoroacetate is followed by in situ hydrolysis of the ester and decarboxylation. Besides optimizing the N-difluoromethylation conditions and assessing the influence of steric and electronic effects on the outcome of the reaction, we have synthesized the N-difluoromethylated analogues of two fluorophores and demonstrated that their spectroscopic properties can be improved through replacement of N-CH3 group by N-CF2H.

Towards Novel Photodynamic Anticancer Agents Generating Superoxide Anion Radicals: A Cyclometalated IrIII Complex Conjugated to a Far-Red Emitting Coumarin.

Although cyclometalated IrIII complexes have emerged as promising photosensitizers for photodynamic therapy, some key drawbacks still hamper clinical translation, such as operability in the phototherapeutic window and reactive oxygen species (ROS) production efficiency and selectivity. In this work, a cyclometalated IrIII complex conjugated to a far-red-emitting coumarin, IrIII -COUPY, is reported with highly favourable properties for cancer phototherapy. IrIII -COUPY was efficiently taken up by HeLa cells and showed no dark cytotoxicity and impressive photocytotoxicity indexes after irradiation with green and blue light, even under hypoxia. Importantly, a clear correlation between cell death and intracellular generation of superoxide anion radicals after visible light irradiation was demonstrated. This strategy opens the door to novel fluorescent photodynamic therapy agents with promising applications in theragnosis.

Solid-Phase Approaches for Labeling Targeting Peptides with Far-Red Emitting Coumarin Fluorophores.

Fluorophores based on organic molecules hold great potential for ligand-targeted imaging applications, particularly those operating in the optical window in biological tissues. In this work, we have developed three straightforward solid-phase approaches based on amide-bond formation or a Cu(I)-catalyzed azide-alkyne click (CuAAC) reaction for labeling an octreotide peptide with far-red emitting coumarin-based COUPY dyes. First, the conjugatable versions of COUPY fluorophores incorporating the required functional groups (e.g., carboxylic acid, azide, or alkyne) were synthesized and characterized. All of them were found fully compatible with Fmoc/ tBu solid-phase peptide synthesis, which allowed for the labeling of octreotide either through amide-bond formation or by CuAAC reaction. A near quantitative conversion was obtained after only 1 h of reaction at RT when using CuSO4 and sodium ascorbate independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). COUPY-octreotide conjugates were found stable in cell culture medium as well as noncytotoxic in HeLa cells, and their spectroscopic and photophysical properties were found similar to those of their parent coumarin dyes. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy through the visualization of living HeLa cells overexpressing the somatostatin subtype-2 receptor.

High Photostability in Nonconventional Coumarins with Far-Red/NIR Emission through Azetidinyl Substitution.

Replacement of electron-donating N,N-dialkyl groups with three- or four-membered cyclic amines (e.g., aziridine and azetidine, respectively) has been described as a promising approach to improve some of the drawbacks of conventional fluorophores, including low fluorescent quantum yields (ΦF) in polar solvents. In this work, we have explored the influence of azetidinyl substitution on nonconventional coumarin-based COUPY dyes. Two azetidine-containing scaffolds were first synthesized in four linear synthetic steps and easily transformed into far-red/NIR-emitting fluorophores through N-alkylation of the pyridine moiety. Azetidine introduction in COUPY dyes resulted in enlarged Stokes' shifts with respect to the N,N-dialkylamino-containing parent dyes, but the ΦF were not significantly modified in aqueous media, which is in contrast with previously reported observations in other fluorophores. However, azetidinyl substitution led to an unprecedented improvement in the photostability of COUPY dyes, and high cell permeability was retained since the fluorophores accumulated selectively in mitochondria and nucleoli of HeLa cells. Overall, our results provide valuable insights for the design and optimization of novel fluorophores operating in the far-red/NIR region, since we have demonstrated that three important parameters (Stokes' shifts, ΦF, and photostability) cannot be always simultaneously addressed by simply replacing a N,N-dialkylamino group with azetidine, at least in nonconventional coumarin-based fluorophores.