Synthesis and Tau RNA Binding Evaluation of Ametantrone-Containing Ligands

We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules

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 physiologicallike 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

A green light-triggerable RGD peptide for photocontrolled targeted drug delivery: synthesis and photolysis studies

We describe for the first time the synthesis and photochemical properties of a coumarin-caged cyclic RGD peptide and demonstrate that uncaging can be efficiently performed with biologically compatible green light. This was accomplished by using a new dicyanocoumarin derivative (DEAdcCE) for the protection of the carboxyl function at the side chain of the aspartic acid residue, which was selected on the basis of Fmoc-tBu SPPS compatibility and photolysis efficiency. The shielding effect of a methyl group incorporated in the coumarin derivative near the ester bond linking both moieties in combination with the use of acidic additives such as HOBt or Oxyma during the basic Fmoc-removal treatment were found to be very effective for minimizing aspartimide-related side reactions. In addition, a conjugate between the dicyanocoumarin-caged cyclic RGD peptide and ruthenocene, which was selected as a metallodrug model cargo, has been synthesized and characterized. The fact that green-light triggered photoactivation can be efficiently performed both with the caged peptide and with its ruthenocenoyl bioconjugate reveals great potential for DEAdcCE-caged peptide sequences as selective drug carriers in the context of photocontrolled targeted anticancer strategies

Somatostatin receptor-targeted organometallic iridium(III) complexes as novel theranostic agents

A novel somatostatin receptor-targeted anticancer agent based on the conjugation of a highly cytotoxic and luminiscent cyclometalated iridium(III) complex to tumor-targeting vectors based on octreotide peptide has been described, and its potential for targeted theranostic applications has been demonstrated

Ametantrone-based compounds as potential regulators of Tau pre-mRNA alternative splicing

We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules

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

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 physicological-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 delivering caged analogues of biologically active compounds into this organell

A cyclometallated 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 tumorspheres, 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 CSC

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

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 hipoxia (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