Rose Bengal-Amphiphilic Peptide Conjugate for Enhanced Photodynamic Therapy of Malignant Melanoma.

Malignant melanoma is an aggressive skin cancer with poor survival outcomes for patients diagnosed at an advanced stage. While targeted serine/threonine-protein kinase B-Raf (BRAF) and immune checkpoint inhibitors have improved survival outcomes for a proportion of these patients, response rates remain variable. There is a need, therefore, for more effective treatments to bolster the options available for melanoma patients. In this manuscript, we covalently attached Rose Bengal (RB) to the amphipathic peptide (AMP) C(KLAKLAK)2 and determined the effectiveness of the resulting RB-C(KLAKLAK)2 conjugate as a photodynamic therapy (PDT) sensitizer. RB-C(KLAKLAK)2-mediated PDT treatment of subcutaneous B16-F10-Luc2 tumors in C57 mice resulted in lesions that were 479% smaller at the end of the study than animals treated with RB-mediated PDT. The synergistic effect between RB and C(KLAKLAK)2 has been attributed to the AMP sensitizing cells to reactive oxygen species (ROS), making them more susceptible to ROS-induced oxidative stress.

Acute liver failure complication of brucellosis infection: a case report and review of the literature.

BACKGROUND: Brucellosis is one of the most widespread zoonoses worldwide. It can affect any organ system, particularly the gastrointestinal system, but there is no report of acute liver failure as a brucellosis complication. CASE PRESENTATION: We present a case of acute liver failure secondary to brucellosis infection. A 75-year-old Hispanic man presented to a University Hospital in Chía, Colombia, with a complaint of 15 days of fatigue, weakness, decreased appetite, epigastric abdominal pain, jaundice, and 10 kg weight loss. On examination in an emergency room, abdomen palpation was normal with hepatosplenomegaly and the results of a liver function test were elevated. The diagnosis of brucellosis was confirmed by epidemiological contact and positive Rose Bengal agglutination with negative enzyme-linked immunosorbent assay immunoglobulin M for Brucella. He was then treated with doxycycline plus trimethoprim/sulfamethoxazole, with a favorable clinical outcome. CONCLUSIONS: The clinical presentation of brucellosis can be very imprecise because it can affect any organ system; however, there is no report of acute liver failure as a brucellosis complication. This is the first reported case in the Colombian literature of acute liver failure due to brucellosis. We found this case to be of interest because it could be taken into account for diagnosis in future appearances and we described adequate treatment and actions to be taken at presentation.

Enzyme-mediated photoinactivation of Enterococcus faecalis using Rose Bengal-acetate.

Rose Bengal-acetate (RB-Ac) is a pro-photosensitizer claimed to diffuse into target cells, where the acetate groups are hydrolyzed and the photosensitizing properties of Rose Bengal (RB) are restored. Despite promising results on tumor cells, the interaction of RB-Ac with bacteria has never been investigated. This study aimed to assess the interaction of RB-Ac with Enterococcus faecalis and to evaluate its potential use in antimicrobial photodynamic therapy (aPDT). Spectrofluorometry was used to assess the ability of E. faecalis to hydrolyze the RB-Ac compound. Fluorescence microscopy was employed to observe the distribution and to evaluate the cellular uptake of the RB produced. The antibacterial efficiency of RB-Ac-mediated aPDT was assessed by flow cytometry in combination with the LIVE/DEAD® staining. Results showed that RB-Ac was successfully hydrolyzed in the presence of E. faecalis cells. The RB produced appeared to incorporate the membrane of bacteria. Higher concentrations of RB-Ac resulted in higher incorporation of RB. The blue-light irradiation of RB-Ac-treated samples significantly reduced bacterial viability. Less than 0.01% of E. faecalis survived after incubation with 200 µM RB-Ac during 900 min and blue-light activation. The current report indicates that E. faecalis cells can hydrolyze the RB-Ac compound to produce active RB. The use of RB-Ac did not appear to allow cytoplasmic internalization of the RB produced, which rather incorporated the membrane bilayers of E. faecalis. The use of RB-Ac did not provide additional advantages over RB in terms of PS localization. Nonetheless, sufficient RB was produced and incorporated into the membranes of bacteria to elicit effective aPDT.

Biosorption of Basic Blue 7 by fungal cells immobilized on the green-type biomatrix of Phragmites australis spongy tissue.

Biosorption is an effective alternative method for the control of water pollution caused by different pollutants such as synthetic dyes and metals. A new and efficient biomass system was developed from the passively immobilized fungal cells. The spongy tissue of Phragmites australis was considered as the carrier for the immobilization of Neurospora sitophila cells employed for the biosorption of Basic Blue 7. This plant tissue was used for the first time as a carrier for fungal cells. The biosorption was examined through batch- and continuous-mode operations. The biosorption process conformed well to the Langmuir model. Maximum monolayer biosorption capacity of the biosorbent was recorded as 154.756 mg g-1. Kinetic findings showed a very good compliance with the pseudo-second-order model. The negative values of ΔG° indicated a spontaneous nature of the biosorption process and a positive value of ΔH° (14.69 kJ mol-1) concluded favorable decolorization at high temperature. The scanning electron microscopy analysis showed that a porous, rippled, and rough surface of biomass system was covered with BB7 molecular cloud. IR results revealed that functional groups like -OH, -NH, and CË­O participated to the decolorization. Breakthrough and exhausted points were found as 360 and 570 minutes, respectively. The biomass system was successfully applied to the treatment of real wastewater.

Tumor ablation using low-intensity ultrasound and sound excitable drug.

The cell membrane is a semi-fluid container that defines the boundary of cells, and provides an enclosed environment for vital biological processes. A sound excitable drug (SED) that is non-cytotoxic to cells is developed to disrupt the plasma membrane under gentle ultrasound insonation, 1MHz, 1W/cm2. The frequency and power density of insonation are within the physical therapy and medical imaging windows; thus the applied ultrasound is safe and not harmful to tissues. The insertion of SEDs into the plasma membrane is not toxic to cells; however, the intruding SEDs weaken the membrane's integrity. Under insonation, the ultrasound energy destabilized the SED disrupted membranes, resulting in membrane rupture and eventual cell death. In a xenograft breast tumor model, the SED alone or the ultrasound alone caused little adverse effects to tumor tissue, while the combined treatment triggered necrosis with a brief local insonation of 3min. The described sono-membrane rupture therapy could be a safe alternative to the currently used high-energy tissue ablation technology, which uses X-rays, gamma rays, electron beams, protons, or high-intensity focused ultrasound.

Magnetic and pH dual-responsive mesoporous silica nanocomposites for effective and low-toxic photodynamic therapy.

Nonspecific targeting, large doses and phototoxicity severely hamper the clinical effect of photodynamic therapy (PDT). In this work, superparamagnetic Fe3O4 mesoporous silica nanoparticles grafted by pH-responsive block polymer polyethylene glycol-b-poly(aspartic acid) (PEG-b-PAsp) were fabricated to load the model photosensitizer rose bengal (RB) in the aim of enhancing the efficiency of PDT. Compared to free RB, the nanocomposites (polyethylene glycol-b-polyaspartate-modified rose bengal-loaded magnetic mesoporous silica [RB-MMSNs]) could greatly enhance the cellular uptake due to their effective endocytosis by mouse melanoma B16 cell and exhibited higher induced apoptosis although with little dark toxicity. RB-MMSNs had little dark toxicity and even much could be facilitated by magnetic field in vitro. RB-MMSNs demonstrated 10 times induced apoptosis efficiency than that of free RB at the same RB concentration, both by cell counting kit-8 (CCK-8) result and apoptosis detection. Furthermore, RB-MMSNs-mediated PDT in vivo on tumor-bearing mice showed steady physical targeting of RB-MMSNs to the tumor site; tumor volumes were significantly reduced in the magnetic field with green light irradiation. More importantly, the survival time of tumor-bearing mice treated with RB-MMSNs was much prolonged. Henceforth, polyethylene glycol-b-polyaspartate-modified magnetic mesoporous silica (MMSNs) probably have great potential in clinical cancer photodynamic treatment because of their effective and low-toxic performance as photosensitizers' vesicles.

In vitro and in vivo clearance of Rose Bengal Acetate-PhotoDynamic Therapy-induced autophagic and apoptotic cells.

This study focuses on the clearance of Rose Bengal Acetate (RBAc)-PhotoDynamic Therapy (PDT)-generated apoptotic and autophagic HeLa cells by murine and human macrophages. Indeed, phagocytosis of dead cells drives the therapeutic efficacy of PDT through both efficient removal of dead/dying cells and macrophages response evoked during engulfment and, up to now, clearance of dying photosensitized cells has been less investigated than PDT mechanisms of cell death induction. RBAc-PDT ensures a long onset of cytotoxicity and a time-related cell death of HeLa cells by signals originating from or converging on almost all intracellular organelles. On this basis, to clarify whether the efficacious cell death commitment is followed by an efficient clearance mechanism, we primarily focused on the analysis of 'eat me' signals exposure and 'find me' signals release, and then investigated the migration, recognition, engulfment and response of murine Raw 264.7 and human blood isolated macrophages. Dead cells secreted 'find me' signals, i.e. fractalkine and Heat Shock Protein 70 (HSP 70), to recruit macrophages and promote their fast phagocytosis. Macrophages phagocytosed apoptotic and autophagic PDT-treated cells more efficiently than the respective positive controls, i.e. puromycin-induced apoptotic and Earle's balanced salt solution-starved autophagic cells. Phagocytosis depends on the glycans exposed on dead cells. The macrophages internalization of photokilled cells elicits the production of Interleukin-10, Transforming Growth Factor-ß and Tumour Necrosis Factor-α by macrophages. TNFα production, along with HSP70 release and plasma membrane translocation on dead cells, suggest an immunogenic impact of RBAc-PDT. In fact, macrophages, activated fibroblasts and endothelial cells colonized the inoculum site of photosensitized cells in rat calf muscles, endorsing the hypothesis of immunogenic elicitation of RBAc-PDT.

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine.

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound.

Pulsed high intensity focused ultrasound (HIFU) has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure >7 MPa (~27 W acoustic power at 1.4 MHz) destroys >95% of breast cancer cells MDA-MB-231 in suspension with >10 µM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (N-acetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected i.v. yielded a substantial synergistic improvement.

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy.

Rose Bengal acetate photodynamic therapy (RBAc-PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.

Rose bengal acetate photodynamic therapy-induced autophagy.

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.

Rose Bengal analogs and vesicular glutamate transporters (VGLUTs).

Vesicular glutamate transporters (VGLUTs) allow the loading of presynaptic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. Rose Bengal (RB) is the most potent known VGLUT inhibitor (Ki 25 nM); therefore we designed, synthesized and tested in brain preparations, a series of analogs based on this scaffold. We showed that among the two tautomers of RB, the carboxylic and not the lactonic form is active against VGLUTs and generated a pharmacophore model to determine the minimal structure requirements. We also tested RB specificity in other neurotransmitter uptake systems. RB proved to potently inhibit VMAT (Ki 64 nM) but weakly VACHT (Ki>9.7 microM) and may be a useful tool in glutamate/acetylcholine co-transmission studies.

Photodynamic therapy-induced apoptosis of HeLa cells.

Photodynamic therapy (PDT), which is a treatment for cancer and certain noncancerous conditions, requires exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength. By using Rose Bengal Acetate (RBAc) as the photosensitizer and an innovative green light-emitting diode, we investigated the efficiency with which apoptosis is induced in HeLa cells, focusing our study on mitochondria alteration and cytochrome c release. Indeed, RBAc is a very efficient fluorogenic substrate and easily enters the cells where the original photoactive molecule is restored by specific esterases. HeLa cells after PDT underwent a consistent rate of apoptosis (peaked at 12 h of recovery post-PDT). Necrosis was observed at the longest times of recovery as a result of secondary necrosis. PDT gave rise to a series of shape modifications, mainly referable to apoptotic-related changes (i.e., extensive blebs formation) involving both F-actin and tubulin networks. Soon after PDT, mitochondria lose their potential membranes and release large quantities of cytochrome c.

Multivariate standard addition method solved by net analyte signal calculation and rank annihilation factor analysis.

This article describes the use of the net analyte signal (NAS) concept and rank annihilation factor analysis (RAFA) for building two different multivariate standard addition models called "SANAS" and "SARAF." In the former, by the definition of a new subspace, the NAS vector of the analyte of interest in an unknown sample as well as the NAS vectors of samples spiked with various amounts of the standard solutions are calculated and then their Euclidean norms are plotted against the concentration of added standard. In this way, a simple linear standard addition graph similar to that in univariate calibration is obtained, from which the concentration of the analyte in the unknown sample and the analytical figures of merit are readily calculated. In the SARAF method, the concentration of the analyte in the unknown sample is varied iteratively until the contribution of the analyte in the response data matrix is completely annihilated. The proposed methods were evaluated by analyzing simulated absorbance data as well as by the analysis of two indicators in synthetic matrices as experimental data. The resultant predicted concentrations of unknown samples showed that the SANAS and SARAF methods both produced accurate results with relative errors of prediction lower than 5% in most cases.

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells.

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage.

Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group-i.e. acetate-to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.

Synthesis of amphiphilic derivatives of rose bengal and their tumor accumulation.

It is known that the combination of laser light and its sensitizer is effective for noninvasive tumor treatment, referred to as photodynamic therapy. Using the combination of ultrasound and its sensitizer has also been suggested for a similar kind of tumor treatment, referred to as sonodynamic therapy. The purpose of this paper is to obtain such sensitizers accumulating selectively in tumors. Amphiphilic derivatives of rose bengal (RB) were synthesized to add a tumor-accumulating property to RB. One type of the synthesized RB derivatives (RBD3), having an alkyl chain with a branching carboxyl group, was found to be superior in amphiphilicity to the other types. Tumor tissue distribution of the synthesized derivatives in mice bearing colon 26 carcinoma was evaluated. It was found that RBD3s with carbon chain lengths of 12, 14, and 16 had higher concentrations in the tumor tissue than RB by more than 1 order of magnitude, several hours after administration. The concentrations correlated well with their water/1-octanol partition coefficients. Since RB is known to induce in vitro cell damage in combination with either laser light or ultrasound, the newly synthesized amphiphilic RB derivatives may be potentially useful as a tumor-selective sensitizer for both light and ultrasound.

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells.

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10(-5) M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm2). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24-72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.

Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate.

The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.

Inhibition of vesicular glutamate uptake by Rose Bengal-related compounds: structure-activity relationship.

Synaptic vesicular accumulation of glutamate is a vital initial step in glutamate transmission. We have previously shown that Rose Bengal, a polyhalogenated fluorescein analog, is a potent inhibitor of glutamate uptake into synaptic vesicles. Here, we report the structural features of Rose Bengal required for this inhibition. Various Rose Bengal-related compounds, with systematic structural variations, were tested. Results indicate that the four iodo groups and the phenyl group attached to the xanthene moiety are critical for potent inhibitory activity. Replacement of these groups with two iodo groups and an alkyl group, respectively, results in substantial reduction in potency. Of further interest in creating high potency is the critical nature of the oxygen atom which links the two benzene rings of xanthene. Thus, the phenyl group and multiple iodo groups, as well as the bridging oxygen of xanthene, are crucial elements of Rose Bengal required for its potent inhibitory action.