Pitavastatin, a new HMG-CoA reductase inhibitor, induces phototoxicity in human keratinocytes NCTC-2544 through the formation of benzophenanthridine-like photoproducts.

This study reports the results of an investigation of the phototoxicity mechanism induced by pitavastatin and its photoproducts, namely 6-cyclopropyl-10-fluoro-7,8-dihydrobenzo[k]phenanthridine (PP3) and 6-cyclopropyl-10-fluorobenzo[k]phenanthridine (PP4). The phototoxicity was tested in human keratinocytes cell lines NCTC-2544, and the results proved that under the same conditions, all three compounds exhibited phototoxic effects in the model tested. The reduction in cell viability was found to be both concentration- and UVA dose-dependent. A point of note is that both the photoproducts produced a dramatic decrease in cell viability with GI(50) values one order of magnitude lower compared to the parent compound. In particular, the fully aromatic derivative (PP4) showed the highest antiproliferative activity. Flow cytometric analysis indicated that pitavastatin and the photoproduct PP4 principally induced necrosis, as revealed by the large appearance of propidium iodide-positive cells and also confirmed by the rapid drop in cellular ATP levels. Further studies committed to better understanding of photoinduced cell death mechanism(s) revealed that neither pitavastatin nor PP4 induced mitochondrial depolarization or lysosomal damage, but, interestingly, extensive cell lipid membrane peroxidation along with a significant oxidation of model proteins occurred, suggesting that pitavastatin and PP4 exert their phototoxic effect mainly in the cellular membranes. The present results suggest that the phototoxicity of pitavastatin may be mediated by the formation of benzophenanthridine-like photoproducts that appear to have high potential as photosensitizers.

Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function.

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC(50) of 10 µM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4'-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.

Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents.

Pyrrolo[3,4-h]quinolin-2-ones were synthesized as nitrogen isosters of the angular furocoumarin angelicin, with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects. A versatile synthetic pathway was approached to allow the isolation of derivatives of the new ring system with a good substitution pattern on the pyrrole moiety. Photobiological screenings of the new compounds revealed a potent phototoxic effect and a great UVA dose dependence, reaching IC(50) values at submicromolar level. The induced cellular photocytotoxicity was related to apoptosis with the involvement of mitochondria and lysosomes, alteration of cell cycle profile and membrane lipid peroxidation.

Virtual screening against nuclear factor κB (NF-κB) of a focus library: Identification of bioactive furocoumarin derivatives inhibiting NF-κB dependent biological functions involved in cystic fibrosis.

In the present study, a structured-based virtual screening (VS) of differently substituted furocoumarins and analogues has been carried out against nuclear factor kappa B (NF-κB), with the objective of selecting molecules able to inhibit the binding of this transcription factor to the DNA. The focus library was developed starting from chemical structures obtained from the literature, as well as retrieving compounds from available commercial databases. A two dimensional substructure searching method based on four different chemical scaffolds was used for this purpose. Among the 10 highest-scored ligands selected from the docking studies, five commercially available molecules were investigated in biological assays. Four furocoumarin derivatives showed IC(50) values in the range of 40-100 µM in inhibiting NF-κB/DNA interactions studied by electrophoretic mobility shift assay (EMSA). Three compounds significantly inhibited NF-κB dependent biological functions (expression of IL-8) in cellular analysis based on Pseudomonas aeruginosa infection of cystic fibrosis IB3-1 cells. These findings validated the virtual screening approach here presented and reinforce the successful results of our previously computational studies aimed at the identification of molecules targeting NF-κB. The discovered novel compounds could be of relevance to identify more potent inhibitors of NF-κB dependent biological functions beneficial to control lung inflammation occurring in patients affected by cystic fibrosis.

Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage.

In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrolo[3,2-h]quinoline--bioisosters of the angular furocoumarin angelicin--were synthesized through a four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A dependence, reaching IC50 values at submicromolar level. The mode of cellular death photoinduced by pyrrolo[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were performed to clarify their mechanism of action.

Pyrano[2,3-e]isoindol-2-ones, new angelicin heteroanalogues.

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC(50) in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.

Isoindolo[2,1-a]quinoxaline derivatives, novel potent antitumor agents with dual inhibition of tubulin polymerization and topoisomerase I.

Isoindoloquinoxalines 4 and 5 were obtained by refluxing 2-(2'-aminoaryl)-1-cyanoisoindoles 3a- e in acetic or formic acid. All derivatives were screened by the National Cancer Institute (Bethesda, MD) for the in vitro one dose primary anticancer assay against a 3-cell line panel. Compounds 4a- e, screened against a panel of about 60 human tumor cell lines, showed remarkable antineoplastic activity; they had GI 50 values in the low micromolar or submicromolar range and reached, in the case of 4c, nanomolar concentrations on 88% of the 59 tested cell lines. Flow cytometric analysis of cell cycle after treatment with 4c demonstrated an arrest of the cell cycle in G2/M phase. This effect was accompanied with apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3 and caspase-9. Moreover, 4c induced a clear increase in the mitotic index, inhibited microtubule assembly in vitro, and interestingly also acted as a topoisomerase I inhibitor.

Thiopyrano[2,3-e]indol-2-ones: angelicin heteroanalogues with potent photoantiproliferative activity.

A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular 7,8-dimethyl-thiopyranoindolone 6c s showed a remarkable phototoxicity and a great dose UVA dependence reaching IC(50) values at submicromolar level. This latter photoinduced a massive apoptosis and a remarkable photodamage to lipids and proteins. Although it did not intercalate DNA, it was able to cause photooxidation of DNA bases.

Furocoumarins photolysis products induce differentiation of human erythroid cells.

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.

Photophysical properties and photobiological behavior of amodiaquine, primaquine and chloroquine.

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.

DNA cleavage induced by photoexcited antimalarial drugs: a photophysical and photobiological study.

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants Ka approximately 10(5) M(-1)). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug]/ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and/or radical cation production. These findings are further supported by the determination of two photoproducts of 2'-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-beta-D-erythro-pentofuranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2'-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.

Intercalation of organic dye molecules into double-stranded DNA. Part 2: the annelated quinolizinium ion as a structural motif in DNA intercalators.

DNA intercalators represent an important class of compounds with a high potential as DNA-targeting drugs. In this review it is demonstrated that annelated quinolizinium derivatives such as coralyne and derivatives thereof intercalate into DNA and that this structural motif allows several variations of the substitution pattern without loss of intercalating properties. The commonly applied methods for the evaluation of the DNA association, mainly spectroscopic studies, are pointed out. In addition, studies on the biological activities of annelated quinolizinium derivatives, such as topoisomerase poisoning or cell toxicity, are highlighted.

PUVA-induced apoptosis involves mitochondrial dysfunction caused by the opening of the permeability transition pore.

The mechanism of cell death was investigated in Jurkat cells exposed to the combination of psoralen and UVA irradiation (PUVA). Apoptosis was by far prevailing over necrosis and involved mitochondrial dysfunction. The collapse of mitochondrial membrane potential, appears to be caused by the opening of the mitochondrial permeability transition pore since its inhibitor, cyclosporin A, prevented mitochondrial dysfunction and largely attenuated apoptosis. Apoptosis also occurred in cells treated with the photoproducts generated by irradiating psoralen in vitro with an oxygen-dependent process. Thus, the involvement of reactive oxygen species in the onset of PUVA-induced apoptosis appears mostly related to psoralen photooxidation.

A novel concept to activate enediynes for DNA cleavage.

A novel concept is presented to activate enediynes via a biscumulenic intermediate using photoinduced electron transfer (PET).

Photophysical and photobiological behavior of antimalarial drugs in aqueous solutions.

This article describes the results of a combined photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs quinine (Q), quinacrine (QC) and mefloquine (MQ). Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed on irradiation. The results obtained showed that fluorescence and intersystem crossing account for all the adsorbed quanta for Q and MQ (quantum yield of about 0.1 and 0.9, respectively) and only for 24% in the case of QC, which has a negligible fluorescence quantum yield (0.001). Laser flash photolysis experiments evidenced, for QC and MQ, the occurrence of photoionization processes leading to the formation of the radical cations of the drugs. The effects of tryptophan and histidine on the excited states and transient species of the three drugs were also investigated. In parallel, the photoactivity of the antimalarial drugs was investigated under UV irradiation on various biological targets through a series of in vitro assays in the presence and in the absence of oxygen. Phototoxicity on 3T3 cultured fibroblasts and lipid photoperoxidation were observed for all the drugs. The photodamage produced by the drugs was also evaluated on proteins by measuring the photosensitized cross-linking of spectrin. The combined approaches were proven to be useful for understanding the mechanism of phototoxicity induced by the antimalarial drugs.

Excited-state properties and in vitro phototoxicity studies of three phenothiazine derivatives.

This work concerns a combined photophysical, photochemical and photobiological study of three drugs (psychotherapeutic agents) of the phenothiazine series: perphenazine, fluphenazine hydrochloride and thioridazine hydrochloride. The excited-state properties were first investigated by stationary and time-resolved fluorimetry and by laser flash photolysis. The spectral description was assisted by quantum-mechanical calculations with the INDO/1-CI method. In organic media the lowest excited singlet state was found to decay by fluorescence (small quantum yield) and mainly by intersystem crossing to the lowest triplet state, which is responsible for oxygen photosensitization (high yields of singlet oxygen production) and photodegradation. A further decay pathway in aqueous solutions was the photoionization process, which led to the formation of the phenothiazine radical cations and the solvated electron. After the preliminary study of the photobehavior in organic solvents and in water, the phototoxicity of the three drugs was investigated on various biological substrates through a series of in vitro assays under UVA irradiation. Photohemolysis of mouse erythrocytes and phototoxicity on cultured murine fibroblasts were observed for all three compounds. Lipid photoperoxidation was then investigated using linoleic acid as the unsaturated lipid model and isolated red blood cell membranes. The drug-induced photodamage was also evaluated on proteins by measuring the photosensitizing cross-linking in erythrocyte ghosts. The combined approach proved to be useful in understanding the mechanism by which these phenothiazine derivatives induce skin photosensitization. In particular, the photophysical properties of the compounds under investigation and the results of the study on their phototoxicity are in agreement with a mechanism that involves the radical cation of the drugs as a main intermediate.

Photophysical and phototoxic properties of the antibacterial fluoroquinolones levofloxacin and moxifloxacin.

Two antibacterial fluoroquinolones, levofloxacin and moxifloxacin, were investigated to evaluate their photophysical properties and to explore the mechanism of their phototoxicity. Photophysical experiments were carried out in aqueous solution by stationary and time-resolved fluorimetry, and by laser flash photolysis, to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. The results obtained show that levofloxacin is able to photosensitize red blood cell lysis in an oxygen-independent way and induce a high decrease in cell viability after UVA irradiation, although to a lesser degree than the racemic mixture ofloxacin. Moxifloxacin, which is an 8-MeO-substituted fluoroquinolone, is less phototoxic than the other compounds. Cellular phototoxicity was inhibited by the addition of superoxide dismutase, catalase, and free radical and hydroxyl radical scavengers (BHA, GSH, mannitol, and DMTU), indicating the involvement of superoxide anion and/or a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation, protein photodamage, and cellular phototoxicity, indicating that test compounds exert their toxic effects mainly in the cellular membrane. Experiments carried out on pBR322 DNA show that these derivatives do not significantly photocleave DNA directly, but single-strand breaks were evidenced after treatment of photosensitized DNA by two base-excision-repair enzymes, and Endo III.

Synthesis, cytotoxicity, and apoptosis induction in human tumor cells by geiparvarin analogues.

A series of geiparvarin analogues modified on the unsaturated alkenyloxy bridge, where a H-atom replaced the 3'-Me group, were synthesized and evaluated against a panel of human tumor cell lines in vitro. These compounds demonstrated a stronger increase in growth inhibitory activity when compared to the parent compound geiparvarin (8). In particular, the activity increased even further in the series of demethylated compounds when a Me substituent in the coumarin moiety is introduced. On the contrary, the same modifications exerted on the parent compound led to an activity reduction. Interestingly, the new derivatives proved to be fully inhibitory to drug-resistant cell lines, thus suggesting that they are not subject to the pump-mediating efflux of antitumor drugs. On the basis of their cytotoxic profiles, the most-active compounds were selected for further biological evaluation. The extracellular acidification rate by the new geiparvarin analogues was measured with the Cytosensor microphysiometer. The new derivatives significantly increased the acidification rate during the 24-48 h of incubation in a concentration-dependent manner. Cell-cycle analysis, evaluated by flow cytometry, revealed a strong apoptotic induction by these compounds confirmed by DNA laddering and observation by electron microscopy. Interestingly, the apoptotic pathway did not appear to be mediated by the activation of caspase-3.

1-Thioangelicin: crystal structure, computer-aided studies and photobiological activity.

1-Thioangelicin is a furocoumarin analog synthesized to investigate the role of the substitution of sulfur for oxygen in the parent compound angelicin. The compound was examined by X-ray diffraction, and its interaction with DNA, both in the dark and by UVA irradiation, studied by means of linear flow dichroism, chromatography and (1)H NMR. Further insight into the steric and electronic features of 1-thioangelicin has been reached through theoretical calculations, including molecular mechanics optimization, docking studies and frontier molecular orbital investigations. The experimental data indicate that thioangelicin is able to intercalate in the DNA helix and subsequent irradiation yields a cis-syn adduct, in agreement with theoretical calculations within the lower/higher singly occupied molecular orbital formalism. Antiproliferative activity has been assessed on Balb/c 3T3 cultured cells.

Development of a novel furocoumarin derivative inhibiting NF-κB dependent biological functions: design, synthesis and biological effects.

Nuclear Factor kappaB (NF-κB) plays a very important role in the control of gene expression and is deeply involved in several human pathologies. Accordingly, molecules targeting NF-κB dependent biological functions are considered of great interest. Virtual screening of furocoumarin libraries against NF-κB p50 allowed to rank compounds in respect to their expected ability to bind NF-κB and the identified compound might be considered for the development of analogs to be tested for biological activity on inhibition of NF-κB/DNA complex formation. The data reported in the present paper suggest that, following this approach, the best ranked compounds identified by virtual screening (a) strongly bind in silico to NF-κB and (b) efficiently inhibit the molecular interactions between (32)P-labeled NF-κB double stranded DNA and p50 or p50/p65 complex. These data allowed to develop a novel lead of great interest for inhibiting NF-κB dependent biological functions. This novel molecule (compound 2), bearing a methyl group in the 9 position of the psoralen nucleus, exhibits high efficiency in inhibiting NF-κB/DNA interactions. In addition, we found that compound 2 is a potent inhibitor of IL-8 gene expression in TNF-α treated IB3-1 cystic fibrosis cells. Taken together, our data indicate that compound 2 might find an important place in the set of molecules of interest for the development of pharmaceutical strategies against the inflammatory phenotype of cystic fibrosis.