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Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

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This work explores the photochemical degradation of cationic species of 7-hydroxy-1-methyl-2H-pyrido[3,4-b]indole or harmol (1C) and the corresponding partially hydrogenated derivative 7-hydroxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole or harmalol (2C) in aqueous solution. UV-visible absorption and fluorescence emission spectroscopy coupled with multivariate data analysis (MCR-ALS and PARAFAC), HPLC and HRESI-MS techniques were used for both quantitative and qualitative analysis. The formation of hydrogen peroxide reactive oxygen species (ROS) was quantified, and the influence of pH, oxygen partial pressure and photoexcitation source on the photochemical degradation of both compounds was assessed. The potential implications on the biosynthesis of ßCs and their biological role in living systems are discussed.

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This study reports valuable information regarding the presence and concentration of a series of photoactive ß-carboline (ßCs) alkaloids (norharmane, harmane, harmine, harmol, harmaline, and harmalol) and their distribution across the floral age and organs of Passiflora caerulea. UHPLC-MS/MS data reported herein reveal that the ßCs' content ranged from 1 to 110 µg kg-1 , depending on the floral organ and age. In certain physiologically relevant organs, such as anthers, ßCs' content was one order of magnitude higher than in other organs, suggesting a special role for ßCs in this specific organ. ßCs' content also varied in a structure-dependent manner. Alkaloids bearing a hydroxyl group at position C(7) of the main ßC ring were present at concentrations one order of magnitude higher than other ßC derivatives investigated. UV-visible and fluorescence spectroscopy of the flower extracts provided complementary information regarding other biologically relevant groups of chromophores (phenolic/indolic derivatives, flavonoids/carotenes, and chlorophylls). Since flowers are constantly exposed to solar radiation, the presence of photoactive ßCs in floral organs may have several (photo)biological implications that are further discussed.

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Harmaline (1) and harmalol (2) represent two 3,4-dihydro-ß-carboline (DHßCs) most frequently reported in a vast number of living systems. Fundamental aspects including the photosensitizing properties, cellular uptake, as well as the cyto- and phototoxicity of 1 and 2 were investigated herein. The molecular basis underlying the investigated processes are elucidated. Data reveal that both alkaloids show a distinctive pattern of extracellular DNA photodamage. Compound 1 induces a DNA photodamage profile dominated by oxidised purines and sites of base loss (AP sites), whereas 2 mostly induces single-strand breaks (SSBs) in addition to a small extent of purine oxidative damage. In both cases, DNA oxidative damage would occur through type I mechanism. In addition, a concerted hydrolytic attack is suggested as an extra mechanism accounting for the SSBs formation photoinduced by 2. Subcellular internalisation, cyto- and phototoxicity of 1 and 2 and the corresponding full-aromatic derivatives harmine (3) and harmol (4) also showed quite distinctive patterns in a structure-dependent manner. These results are discussed in the framework of the potential biological, biomedical and/or pharmacological roles reported for these alkaloids. The subtle structural difference (i.e., the exchange of a methoxy group for a hydroxyl substituent at C(7)) between harmaline and harmalol, gives rise to distinctive photosensitizing and subcellular localisation patterns.

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Toxoplasmosis is one of the most prevalent and neglected zoonotic global diseases caused by Toxoplasma gondii. The current pharmacological treatments show clinical limitations, and therefore, the search for new drugs is an urgent need in order to eradicate this infection. Due to their intrinsic biological activities, ß-carboline (ßC) alkaloids might represent a good alternative that deserves further investigations. In this context, the in vitro anti-T. gondii activity of three ßCs, harmine (1), 2-methyl-harminium (2), and 9-methyl-harmine (3), was evaluated herein. Briefly, the three alkaloids exerted direct effects on the parasite invasion and/or replication capability. Replication rates of intracellular treated tachyzoites were also affected in a dose-dependent manner, at noncytotoxic concentrations for host cells. Additionally, cell cycle analysis revealed that both methyl-derivatives 2 and 3 induce parasite arrest in S/M phases. Compound 3 showed the highest irreversible parasite growth inhibition, with a half maximal inhibitory concentration (IC50) value of 1.8 ± 0.2 µM and a selectivity index (SI) of 17.2 at 4 days post infection. Due to high replication rates, tachyzoites are frequently subjected to DNA double-strand breaks (DSBs). This highly toxic lesion triggers a series of DNA damage response reactions, starting with a kinase cascade that phosphorylates a large number of substrates, including the histone H2A.X to lead the early DSB marker γH2A.X. Western blot studies showed that basal expression of γH2A.X was reduced in the presence of 3. Interestingly, the typical increase in γH2A.X levels produced by camptothecin (CPT), a drug that generates DSB, was not observed when CPT was co-administered with 3. These findings suggest that 3 might disrupt Toxoplasma DNA damage response.

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Halogenated and nitro ß-carboline (ßCs) alkaloids have garnered increasing interest for their role in a broad range of biological, pharmacological and biotechnological processes. Addressing their spectroscopic and photophysical properties provide tools to further explore the presence of these alkaloids in complex biological matrices. In addition, these studies help to elucidate processes where these alkaloids are involved. The UV-visible and steady-state room temperature fluorescence of bromo- and nitro-harmines in an aqueous environment at different pHs, low-temperature phosphorescence (at 77 K) and quantum yields of singlet oxygen production are reported herein. Singlet (S0 and S1) and triplet (T1) electronic states are further analyzed using density functional theory (DFT) and the results compared with experimental data. Data are discussed in the framework of potential biotechnological applications of these ßC alkaloids.

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3,4-Dihydro-ß-carbolines (DHßCs) are a set of endogenously synthesized alkaloids spread over a great variety of living species (e.g., plants, animals and microorganisms), playing a broad spectrum of biological, biochemical and/or pharmacological roles, in a structure-dependent manner. Addressing unresolved fundamental aspects related to the photophysical properties of DHßCs might help to gain further insights into the molecular basis of the mechanisms of the biological processes where these alkaloids are involved. In this work, the UV-visible spectroscopic features of DHßCs are revisited and they are further analyzed by calculations at the Density Functional Theory (DFT) level. In addition, steady-state and time-resolved fluorescence spectroscopy, as well as quantitative singlet oxygen production analysis is reported. Data obtained herein are discussed in the framework of the potential biological role of these alkaloids.

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N-Methyl-ß-carboline (ßC) alkaloids, including normelinonine F (1b) and melinonine F (2b), have been found in a vast range of living species playing different biological, biomedical and/or pharmacological roles. Despite this, molecular bases of the mechanisms through which these alkaloids would exert their effect still remain unknown. Fundamental aspects including the photosensitizing properties and intracellular internalization of a selected group of N-methyl-ßC alkaloids were investigated herein. Data reveal that methylation of the ßC main ring enhances its photosensitizing properties either by increasing its binding affinity with DNA as a biomolecular target and/or by increasing its oxidation potential, in a structure-dependent manner. As a general rule, N(9)-substituted ßCs showed the highest photosensitizing efficiency. With the exception of 2-methyl-harminium, all the N-methyl-ßCs investigated herein induce a similar DNA photodamage profile, dominated largely by oxidized purines. This fact represents a distinctive behavior when comparing with N-unsubstituted-ßCs. On the other hand, although all the investigated compounds might accumulate mainly into the mitochondria of HeLa cells, methylation provides a distinctive dynamic pattern for mitochondrial uptake. While rapid (passive) diffusion is most probably reponsible for the prompt uptake/release of neutral ßCs, an active transport appears to mediate the (reatively slow) uptake of the quaternary cationic ßCs. This might be a consequence of a distinctive subcellular localization (mitochondrial membrane and/or matrix) or interaction with intracellular components. Biomedical and biotechnological implications are also discussed herein.

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ReI -polypyridyl complexes have interesting and distinctive photochemical and photosensitizing properties. This work describes the capability to induce (or photoinduce) DNA damage of three ReI -complexes with a naturally occurring alkaloid called norharmane (nHo) as ligand: [Re(CO)3 (nHo)(L)]CF3 SO3 where L=2,2'-bipyridine (ReBpy), phenanthroline (RePhen) or dipyrido[3,2-a:2',3'-c]phenazine (ReDppz). The interaction of the complexes with DNA was investigated by steady-state and time-resolved spectroscopy. Data show that the mode and strength of interaction depend on the chemical structure of the bidentate ligand. The complexes show a major static contribution to the overall interaction, giving rise to the formation of noncovalent adducts with DNA, and the particular trend observed was RePhen>ReDppz>ReBpy. Photo-oxidation at the purine bases represents the major DNA damaging mechanism. RePhen also induces single-strand breaks in a yield similar to that of base damage, suggesting an additional photosensitizing pathway. We also performed the Ames test to evaluate the cytotoxic and mutagenic properties of both non-irradiated and photoexcited complexes. RePhen, but not the other complexes, turned out to be both toxic and phototoxic for the bacteria.

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