Photodynamic Therapy (PDT) Research Group Norwegian University of Science and Technology (NTNU) Projects and Abstracts

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Ruthenium porphyrin-induced photodamage in bladder cancer cells

 Vanya Bogoevaa, , ,

 Monica Siksjøb,

 Kristin G. Sæterbøb,

 Thor Bernt Meløb,

 Astrid Bjørkøyb,

 Mikael Lindgrenb, c,

 Odrun A. Gederaasd

 a Department Molecular Biology of Cell Cycle, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. “G. Bonchev” Str., Bl. 21, Sofia 1113, Bulgaria

 b Department of Physics, Norwegian University of Science and Technology, N-7491 Trondheim, Norway

 c Department of Physics, Chemistry and Biology, Linköping University, SE- 58183 Linköping, Sweden

 d Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7491 Trondheim, Norway Received 19 August 2015, Revised 27 January 2016, Accepted 29 January 2016, Available online 1 February 2016 Show less

doi:10.1016/j.pdpdt.2016.01.012 Get rights and content

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Highlights • Photophysical properties of ruthenium (II) porphyrin, verified that the photosensitizer is capable of activating singlet oxygen. • In vitro experiments on bladder cancer cells, indicated that RuP, irradiated with blue light, induced cellular phototoxicity of 93% • The present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis.

Abstract Photodynamic therapy (PDT) is a noninvasive treatment for solid malignant and flat tumors. Light activated sensitizers catalyze photochemical reactions that produce reactive oxygen species which can cause cancer cell death. In this work we investigated the photophysical properties of the photosensitizer ruthenium (II) porphyrin (RuP), along with its PDT efficiency onto rat bladder cancer cells (AY27). Optical spectroscopy verified that RuP is capable to activate singlet oxygen via blue and red absorption bands and inter system crossing (ISC) to the triplet state. In vitro experiments on AY27 indicated increased photo-toxicity of RuP (20 μM, 18 h incubation) after cell illumination (at 435 nm), as a function of blue light exposure. Cell survival fraction was significantly reduced to 14% after illumination of 20 μM RuP with 15.6 J/cm2, whereas the “dark toxicity” of 20 μM RuP was 17%. Structural and morphological changes of cells were observed, due to RuP accumulation, as well as light-dependent cell death was recorded by confocal microscopy. Flow cytometry verified that PDT-RuP (50 μM) triggered significant photo-induced cellular destruction with a photoxicity of (93% ± 0.9%). Interestingly, the present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis. RuP “dark toxicity” compared to the conventional chemotherapeutic drug cisplatin was higher, both evaluated by the MTT assay (24 h). In conclusion, the present investigation shows that RuP with or without photoactivation induces cell death of bladder cancer cells.

Keywords

 Photodynamic therapy;

 PDT;

 Ruthenium porphyrin;

 photophysical characterization;

 singlet oxygen;

 bladder cancer Download full text in PDF Opens in a new window. Article suggestions will be shown in a dialog on return to ScienceDirect.

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Photochemical internalization of bleomycine and temozolomide – in vitro studies on glioma cell line F98

Odrun A. Gederaas1, Anette Hauge1,2, Pål Ellingsen2,3, Kristian Berg4, Dag Altin5, Tora Bardal6, Anders Høgset7 and Mikael Lindgren2

1) Department of Cancer Research and Molecular Medicine, University of Science and Technology, N-7489 Trondheim, Norway 2) Department of Physics, N-7491 Trondheim, Norway. 3) Department of Artic Geophysics, The university Centre in Svalbard, N-9171 Longyearbyen, Norway 4) Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway 5) Altin BioTrix, Finn Bergs Veg 3, N-7022 Trondheim, Norway 5) Department of Biology, N-7491 Trondheim, Norway 6) PCI Biotech ASA, Strandvegen 55, 1366 Lysaker, Oslo, Norway

Photodynamic therapy (PDT) is an effective treatment for malignant1 and non- malignant diseases, and testing of novel photosensitizers is important prior to in vivo experiments and clinical trials. During PDT experiments the activated photosensitizer 1 transfers energy to nearby oxygen molecules, generating singlet oxygen ( O2) resulting in oxidative stress in cells, which further elicit cell death by necrosis and apoptosis. In the present study we review the use of the photosensitizer meso- tetraphenyl chlorin disulphonate (TPCS2a - Amphinex®) in rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique2. The tested anticancer drugs were Bleomycin (BLM) and Temozolomide (TMZ). Glioma o cells were incubated with TPCS2a (0.2ug/ml, 18h, 37 C) before BLM or TMZ stimulation (4 h) prior to red light illumination (Quanta System, 652 nm, 50 mW/cm2). The demonstration of structural and morphological changes within glioma cells upon the PCI technique, shown that BLM-PCI is an effective method for killing of F98 glioma cells, but smaller effects were observed using TMZ. The cell survival after BLM-PCI, quantified by the MTT assay, was reduced about 25% after 24 h relative to controls, and 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1uM), indicated a long–term cytotoxic effect. The surviving fraction of clonogenic cells was reduced to 5% after light exposure (80s) with PCI compared to 70% in the case of PDT. The present study demonstrates that PCI of BLM is an effective method for killing of F98 glioma cells, but smaller effects was observed using TMZ following the “light after” strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.

*** Studies of the photosensitizer disulfonated meso-tetraphenyl chlorin in an orthotopic rat bladder tumor model.

Baglo Y, Peng Q, Hagen L, Berg K, Høgset A, Drabløs F, Gederaas OA.

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Photodiagnosis Photodyn Ther. 2015 Mar; 12(1):58-66. doi: 10.1016/j.pdpdt.2014.12.005. Epub 2015 Jan 6.

Abstract

BACKGROUND:

Photochemical internalization (PCI) is a novel technology for the release of a therapeutic molecule from endocytic vesicles into the cytosol of a cell. The release of molecules occurs after activation of an endocytic membrane-embedded photosensitizer by light. In this study uptake and localization of the photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) were explored to optimize a PCI protocol in an orthotopic rat bladder tumor model.

METHODS:

Female Fischer F344 rats were intravesically instilled with 0.4×10(6) AY-27 transitional carcinoma cells before allowing tumor growth for 14 days. The photosensitizer TPCS2a was intravesically instilled at different concentrations, and bladders were excised after different time intervals. The retention, penetration, and localization of intratumoral TPCS2a were explored ex vivo using fluorescence spectroscopy and fluorescence microscopy to determine an optimal PCI protocol. These results were compared to histological analysis of necrotic areas after activation of intratumoral TPCS2a by red light (652nm, 0.5J/cm(2)).

RESULTS:

A superficial distribution pattern of the photosensitizer TPCS2a was seen in bladder tumor tissue, and TPCS2a was almost cleared from the tumors after 72h. The highest retention of TPCS2a was found at 24h after instillation when using a concentration of 3mg/ml.

CONCLUSION:

An optimal PCI protocol was defined for the tumor model, including a 24-h TPCS2a- to-light interval and a dose of 3mg/ml TPCS2a. This protocol will be utilized for the study of PCI-enhanced therapeutic effects on non-muscle invasive bladder cancer, using a potent chemotherapeutic under an optimal light dose.

Copyright © 2014 Elsevier B.V. All rights reserved.

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Figure 2: Fluorescence emission spectra of TPCS2a in bladder tumor tissues. Data were obtained from

JETI PDT fluorometer at calibrated integration time of 200 ms. Fluorescence of TPCS2a was excited at

405 nm by JETI equipped laser. TPCS2a fluorescence was detected ex vivo only from TPCS2a-treated bladder tumor tissue, not the controls.

Figure 3: Uptake and retention of TPCS2a in bladder tumor tissues. (A) Relative florescence intensity

(RFI) of TPCS2a measured 72 h after intravesical instillation at concentration of 1, 2, 3, and 5 mg/ml respectively (protocol A); (B) RFI of TPCS2a with an originally instilled concentration of 3 mg/ml measured at retention time of 4, 24, 48, and 72 h respectively (protocol B). Data were obtained from a JETI PDT fluorometer at calibrated integration time of 200 ms. Mean RFI of each group is shown as a horizontal line. Please observe the difference in vertical scale between A and B.

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Transl Oncol. 2014 Dec;7(6):812-23. doi: 10.1016/j.tranon.2014.10.005.

Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when Combined with a PCNA Targeting Peptide.

Gederaas OA1, Søgaard CD1, Viset T2, Bachke S1, Bruheim P3, Arum CJ4, Otterlei M5.

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Abstract Non-muscle-invasive bladder cancers (NMIBCs) are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell's DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA) is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM), including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs). In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX-101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC), as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC.

Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved

Figure 3:Macroscopic and HES-stained images of recovered normal and cancer-containing rat bladders (AY-27 model). (A) Images of opened and mounted rat bladders recovered from animals with different tumor grades as evaluated by histopathology. Bladder weights are given. (B) Images of HES-stained rat bladders (20 ×).

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J Biomed Opt. 2014 Aug;19(8):088002. doi: 10.1117/1.JBO.19.8.088002.

Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro. Helander L1, Krokan HE1, Johnsson A2, Gederaas OA1, Plaetzer K3.

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Abstract

Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT₆₂₄ induced more apoptosis than HAL-PDT₄₁₀ and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

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Acta Oncol. 2014 Mar;53(3):307-15. doi: 10.3109/0284186X.2013.819996. Epub 2013 Aug 19. Can dogs smell lung cancer? First study using exhaled breath and urine screening in unselected patients with suspected lung cancer. Amundsen T1, Sundstrøm S, Buvik T, Gederaas OA, Haaverstad R.

Abstract

BACKGROUND:

On the basis of our own experience and literature search, we hypothesised that a canine olfactory test may be useful for detecting lung cancer in an unselected population of patients suspected to have lung cancer.

MATERIAL AND METHODS:

We conducted a prospective study of 93 patients consecutively admitted to hospital with suspected lung cancer. Exhaled breath and urine were sampled before the patients underwent bronchoscopy. The canine olfactory test was performed in a double-blinded manner. Sensitivity and specificity were outcome measures.

RESULTS:

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With 99% sensitivity, the olfactory test demonstrated that dogs have the ability to distinguish cancer patients from healthy individuals. With an intensified training procedure, the exhaled breath and urine tests showed sensitivity rates of 56-76% and specificity rates of 8.3-33.3%, respectively, in our heterogeneous study population.

CONCLUSION:

Although the olfactory test appears to be a promising tool for the detection of cancer, the main challenge is to determine whether the test can sufficiently discriminate between patients at risk, patients with benign disease, and patients with malignant disease. We need to gain a deeper understanding of this test and further refine it before applying it as a screening tool for lung cancer in clinical settings.

*** Baglo, Yan. PhD Thesis. Preclinical Studies of Photodynamic Therapy and Photochemical Internalization in Bladder Cancer. Trondheim, Norway:

NTNU-trykk 2014 (ISBN 978-82-326-0026-7) 152 s. ***

BioMed Research International Volume 2014 (2014), Article ID 921296, 10 pages http://dx.doi.org/10.1155/2014/921296

Research Article Enhanced Efficacy of Bleomycin in Bladder Cancer Cells by Photochemical Internalization Yan Baglo,1 Lars Hagen,1 Anders Høgset,2 Finn Drabløs,1 Marit Otterlei,1,3 and Odrun A. Gederaas1

Abstract

Bleomycin is a cytotoxic chemotherapeutic agent widely used in cancer treatment. However, its efficacy in different cancers is low, possibly due to limited cellular internalization. In this study, a novel approach known as photochemical internalization (PCI) was explored to enhance bleomycin delivery in bladder cancer cells (human T24 and rat AY-27), as bladder cancer is a potential indication for use of PCI with bleomycin. The PCI technique was mediated by the amphiphilic photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) and blue light (435 nm). Two additional strategies were explored to further enhance the cytotoxicity of

8 bleomycin; a novel peptide drug ATX-101 which is known to impair DNA damage responses, and the protease inhibitor E-64 which may reduce bleomycin degradation by inhibition of bleomycin hydrolase. Our results demonstrate that the PCI technique enhances the bleomycin effect under appropriate conditions, and importantly we show that PCI-bleomycin treatment leads to increased levels of DNA damage supporting that the observed effect is due to increased bleomycin uptake. Impairing the DNA damage responses by ATX-101 further enhances the efficacy of the PCI-bleomycin treatment, while inhibiting the bleomycin hydrolase does not.

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Photochem Photobiol Sci. 2011 Jun; 10(6):1072-9. doi: 10.1039/c0pp00393j. Epub 2011 Mar 22. Photo induced hexylaminolevulinate destruction of rat bladder cells AY-27. Ekroll IK1, Gederaas OA, Helander L, Hjelde A, Melø TB, Johnsson A.

Abstract Photodynamic therapy (PDT) is of increasing interest as a relevant treatment for human urinary bladder cancer. In the present experiments, the rat bladder transitional carcinoma cell line AY-27 was used as a model to study cell destruction mechanisms induced by PDT. Red LED light (630 nm) PDT with hexylaminolevulinate (HAL) as precursor for the photosensitizer protoporphyrin IX (PpIX) was used in treatment of the cells. Flow cytometry with fluorescent markers annexin V, propidium iodide and YO-PRO-1, as well as MTT assay and confocal microscopy, were used to map cell inactivation after PDT. Dark toxicity of HAL alone was low in these procedures and LD(50) (24 h, MTT assay) was approximately 1.6 J cm(-2) for standard red light (LED) irradiation (36 mW cm(-2)). Measurements done 1 h after HAL-PDT showed a maximum apoptotic level of about 10% at 6 J cm(-2), however the dominating mode of cell death was necrosis. Forward light scattering indicated an increase in cell size at low doses, possibly due to necrosis. Survival curves had a dual-slope shape, a fit to single hit, multi-target approximation gave a parameter estimate of n = 10 and D(0) about 2.6 J cm(-2). Replacing continuous light with fractionated light delivery (45 s light/60 s darkness) did not affect the treatment outcome.

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J Biomed Opt. 2011 Feb;16(2):028001. doi: 10.1117/1.3536536. Tissue responses to hexyl 5-aminolevulinate-induced photodynamic treatment in syngeneic orthotopic rat bladder cancer model: possible pathways of action. Arum CJ1, Gederaas OA, Larsen EL, Randeberg LL, Hjelde A, Krokan HE, Svaasand LO, Chen D, Zhao CM.

Abstract

Orthotopic bladder cancer model in rats mimics human bladder cancer with respect to urothelial tumorigenesis and progression. Utilizing this model at pT1 (superficial stage), we analyze the tissue responses to hexyl 5-aminolevulinate-induced photodynamic therapy (HAL-PDT). In comparison to untreated rats, HAL-PDT causes little change in tumor-free rat bladder but induces inflammatory changes with increased lymphocytes and mononuclear cell infiltration in rat bladders with tumor. Immunohistochemistry reveals that HAL-PDT is without effect on proliferating cell nuclear antigen expression within the tumor and increases caspase-3 expression in both normal urothelium and the tumor. Transmission electron microscopy reveals severe mitochondrial damage, formations of apoptotic bodies, vacuoles, and lipofuscin bodies, but no microvillus-formed niches in HAL-PDT-treated bladder cancer rats. Bioinformatics analysis of the gene expression profile indicates an activation of T-cell receptor signaling pathway in bladder cancer rats without PDT. HAL-PDT increases the expression of CD3 and CD45RA in the tumor (determined by immunohistochemistry). We suggest that pathways of action of HAL-PDT may include, at least, activations of mitochondrial apoptosis and autophagy, breakdown of cancer stem cell niches, and importantly, enhancement of T-cell activation.

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J Biomed Opt. 2008 Jul-Aug;13(4):044031. doi: 10.1117/1.2967909. Monitoring of hexyl 5-aminolevulinate-induced photodynamic therapy in rat bladder cancer by optical spectroscopy. Larsen EL1, Randeberg LL, Gederaas OA, Arum CJ, Hjelde A, Zhao CM, Chen D, Krokan HE, Svaasand LO.

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Abstract

Monitoring of the tissue response to photodynamic therapy (PDT) can provide important information to help optimize treatment variables such as drug and light dose, and possibly predict treatment outcome. A urinary bladder cancer cell line (AY- 27) was used to induce orthotopic transitional cell carcinomas (TCC) in female Fischer rats, and hexyl 5-aminolevulinate (HAL, 8 mM, 1 h)-induced PDT was performed on day 14 after instillation of the cancer cells (20 J/cm(2) fluence at 635 nm). In vivo optical reflectance and fluorescence spectra were recorded from bladders before and after laser treatment with a fiberoptic probe. Calculated fluorescence bleaching and oxygen saturation in the bladder wall were examined and correlated to histology results. Reflectance spectra were analyzed using a three-layer optical photon transport model. Animals with TCC treated with PDT showed a clear treatment response; decreased tissue oxygenation and protoporphyrin IX (PpIX) fluorescence photobleaching were observed. Histology demonstrated that 3 of 6 animals with treatment had no sign of the tumor 7 days after PDT treatment. The other 3 animals had significantly reduced the tumor size. The most treatment-responsive animals had the highest PpIX fluorescence prior to light irradiation. Thus, optical spectroscopy can provide useful information for PDT. The model has proved to be very suitable for bladder cancer studies.

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Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed expression of proteins in cell survival and cell death pathways. (2011) Yan Baglo, Mirta M.L. Sousa, Geir Slupphaug, Lars Hagen, Sissel Håvåg, Linda Helander, Kamila A. Zub, Hans E. Krokan and Odrun A. Gederaas* Published in Photochem. Photobiol. Sci. 2011, 10, 1137

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Photodynamic therapy (PDT) using blue light and the potent precursor for protoporphyrin IX, hexyl aminolevulinate (HAL), has been shown to induce apoptosis and necrosis in cancer cells, but the mechanism remains obscure. In the present study, we examined protein carbonylation, expression levels and post- translational modifications in rat bladder cells (AY-27) after PDT with HAL. Altered levels of expression and/or post-translational modifications induced by PDT were observed for numerous proteins, including proteins required for cell mobility, energy supply, cell survival and cell death pathways, by using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). And, 10 carbonylated proteins associated with cytoskeleton, transport, oxidative stress response, protein biosynthesis and stability, and DNA repair were identified, using immunoprecipitation, two-dimensional gel electrophoresis and MS. Overall, the results indicate that HAL-mediated PDT triggers a complex cellular response involving several biological pathways. Our findings may account for the elucidation of mechanisms modulated by PDT, paving the way to improve clinic PDT-efficacy.

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Homology Modeling of Human γ-Butyric Acid Transporters and the Binding of Pro-Drugs 5-Aminolevulinic Acid and Methyl Aminolevulinic Acid Used in Photodynamic Therapy (2013) Yan Baglo, Mari Gabrielsen, Ingebrigt Sylte, Odrun A. Gederaas Published in PLoS ONE 2013, 8, 6

Photodynamic therapy (PDT) is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA), or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX). Activation of PpIX by light causes the formation of reactive oxygen species (ROS) and toxic responses. Studies have indicated that ALA and its methyl ester (MAL) are taken up into the cells via γ-butyric acid (GABA) transporters (GATs). Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain.

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In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT) as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs) of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations). Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA- induced pain.

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