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Transactive DNA Response Element Binding Protein 43 (TDP-43) is a RNA binding protein participating in gene expression on a transcriptional level. It is localized in the cell nucleus. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. In most ALS patients TDP-43 becomes localized into the cytoplasm of neurons and glia cells. The TDP-43 rat ALS model provide insight in ALS disease progression and molecular mechanisms. This animal model has been characterized previously in the literature. Cerebral Dopamine Growth Factor (CDNF) is a neuroprotective and restorative protein in rat animal model of Parkinson's disease. CDNF may have an impact on disease progression in ALS. One of the goals in this work was to recharacterize the TDP-43 rat ALS model and to try repeat published data. The other aim of this work was to treat TDP-43 rats with intraventricular chronic infusion of CDNF, and to compare symptom progression with TDP-43 rats treated with phosphate buffered saline. Behavioral assays were done trice a week and when rats reached endpoint, spinal cords were removed. Motor neuron counting and detection of stress granule formation were investigated in spinal cords with immunohistochemistry. Also, the volume of CDNF diffusion in rat brain after chronic intraventricular CDNF infusion was investigated with immunohistochemistry. In the characterization part, symptom progression was repeated in a similar manner as it has been reported previously. CDNF treatment could not stop the symptom progression nor slow down the progression of symptoms in TDP-43 rats. Motor neuron counting revealed a heavy loss of motor neurons in the lumbal part of the spinal cord in both treatment groups. Diffusion of CDNF was very poor in the rat brain. Higher doses of CDNF and proper administration depth in the brain or route of administration should be reconsidered in the future.

UDP-glucuronosyltransferases (UGTs) catalyse glucuronidation reactions between glucuronic acid and drug molecules, which contain nucleophilic groups, mostly hydroxyls, amines or carboxylic acids. Glucuronidation is the most important reaction in the conjugative drug metabolism. Because these conjugates are not usually able to cross cell membranes passively, they need active efflux transport. Efflux transporters mostly belong to superfamily of ATP-binding cassette transporters (ABC). Subfamily C of ABC transporters (ABCC) are known to be involved in efflux transport of glucuronides. Especially MRP2 (ABCC2) and MRP3 (ABCC3) play key roles in the elimination of glucuronide conjugates of drugs. MRP2 is localized in the apical membranes of hepatocytes and enterocytes, whereas MRP3 is localized in the basolateral membranes of the respective cells. On the other hand, UGT1A1 and UGT2B7 are highly expressed in liver and small intestine and are the most important UGTs in drug metabolism. It is known, that UGTs and efflux transporters work together forming interplay to eliminate drugs. Therefore, studying both of them in the same in vitro system is in important focus of drug metabolism studies. The Madin Darby canine kidney cell line (MDCK) is one of the standard in vitro tools in drug metabolism studies. In this study, MDCK was chosen for a cell line to co-express UGTs (UGT1A1 or UGT2B7) and efflux transporters (MRP2 and MRP3 simultaneously. Therefore, cloning of the UGT2B7 cDNA and the ABCC3 cDNA encoding MRP3 was aimed in this study. On the other hand, the UGT1A1 cDNA was already cloned in-house and MRP2 expressing MDCK cells were established earlier. Cloning of the UGT2B7 cDNA was not successful in this study despite of several different strategies such as PCR-amplification of the cDNA fragment using kidney or liver sscDNA as template. Cloning of the ABCC3 cDNA encoding MRP3 was achieved and a mammalian expression vector containing this cDNA was constructed. In addition, the mammalian expression vector containing the UGT1A1 cDNA was used to establish MDCK-UGT1A1 cells and this cell line was characterized regarding the expression of UGT1A1 mRNA and UGT1A1 protein amount. Furthermore, establishment of MDCK-UGT1A1-MRP2 cell line was attempted in this study without success. The mammalian expression vector containing the ABCC3 cDNA encoding MRP3 could be used for future experiments to achieve novel cell lines such as MDCK-UGT1A1-MRP3 and MDCK-UGT1A1-MRP2-MRP3 for drug metabolism studies. In addition, the novel cell line MDCK-UGT1A1 could be used for drug metabolism studies in further experiments, but also as a cell line for further establishment of above cell lines. On the other hand, the cloning of the UGT2B7 cDNA needs optimization and several different strategies should be used to achieve the mammalian expression vector containing this cDNA.

Lactose is probably the most used tablet excipient in the field of pharmacy. Although lactose is thoroughly characterized and available in many different forms there is a need to find a replacer for lactose as a filler/binder in tablet formulations because it has some downsides. Melibiose is a relatively unknown disaccharide that has not been thoroughly characterized and not previously used as an excipient in tablets. Structurally melibiose is close to lactose as it is also formed from the same two monosaccharides, glucose and galactose. Aim of this research is to characterize and to study physicochemical properties of melibiose. Also the potential of melibiose to be used as pharmaceutical tablet excipient, even as a substitute for lactose is evaluated. Current knowledge about fundamentals of tableting and methods for determinating of deformation behavior and tabletability are reviewed. In this research Raman spectroscopy, X-ray powder diffraction (XRPD), near-infrared spectroscopy (NIR) and Fourier-transform infrared spectroscopy (FT-IR) were used to study differences between two melibiose batches purchased from two suppliers. In NIR and FT-IR measurements no difference between materials could be observed. XPRD and Raman however found differences between the two melibiose batches. Also the effects of moisture content and heating to material properties were studied and moisture content of materials seems to cause some differences. Thermal analytical methods, differential scanning calorimetry (DSC) and thermogravimetry (TG) were used to study thermal behaviour of melibiose and difference between materials was found. Other melibiose batch contains residual water which evaporates at higher temperatures causing the differences in thermal behaviour. Scanning electron microscopy images were used to evaluate particle size, particle shape and morphology. Bulk, tapped and true densities and flow properties of melibiose was measured. Particle size of the melibiose batches are quite different resulting causing differences in the flowability. Instrumented tableting machine and compression simulator were used to evaluate tableting properties of melbiose compared to α-lactose monohydrate. Heckel analysis and strain-rate sensitivity index were used to determine deformation mechanism of melibiose monohydrate in relation to α-lactose monohydrate during compaction. Melibiose seems to have similar deformation behaviour than α-lactose monohydrate. Melibiose is most likely fragmenting material. Melibiose has better compactibility than α - lactose monohydrate as it produces tablets with higher tensile strength with similar compression pressures. More compression studies are however needed to confirm these results because limitations of this study.

The human immune system can provide a powerful tool in developing therapies against various cancers. Even though the idea of an immune system actively searching for and disposing of potential mutated tumor cells is over a century old, only recent developments in various fields such as mass spectrometry, immuno-checkpoint blockade strategies and in silico modelling have enabled the realization of the full potential of recruiting immune system to fight cancer and the possibilities of personalized therapies. These therapeutic methods, including but not limited to oncolytic virus therapies, T-cell therapies and cancer vaccines, are based on the body’s ability to recognize mutated antigen peptides presented on the cell surface by MCH-receptors (also known as HLA-receptors in humans) and the disposal of the malignant cells by cytotoxic T-cells. Thus, the capability to map the individual HLA-presented peptidome and differentiate the immunogenic peptides is a foundation for this plethora of therapies and is in focus of ongoing research. This master thesis is a part of a project aiming to set up immunoaffinity-purification/MS based method in order to analyse the ligandome and determine T-cell recognized cancer associated antigens from tumor cells. Objectives of the work: 1. Characterizing tumor cell lines. 2. Immunological assay set up. 3. Collecting cell culture material for the ligandome affinity purification. 4. In silico prediction if the immunogenicity of selected peptides and assessing their source proteins. Methods used: 1. Cell culture. 2. FACS-analysis. 3. MTS-viability assay. 4. Immunological assays (ELISA, ELISPOT). 5. Immunological bioinformatics analysis tools (IEDB) and database search (UniPROT). Results: 1. Flow cytometric analysis provided essential information of the cell line HLA-1 expression. Additional information of PD-L1 expression can be used to evaluate cell line’s immune-evasion abilities. Preliminary MTS assay is used to determine linear range and optimal time frame for the PBMC/cancer cell co-culture killing assay. 2. Interferon γ cytokine secretion was determined by ELISPOT to assess PBMC response against known antigens in a preliminary experiment to approximate usable range for the following antigen specific PBMC assays. ELISA is used to confirm the presence of HLA-I receptors in the ligandome affinity purification eluates and to estimate the efficacy of purification. 3. Feasibility of in silico methods in the prediction of immunogenic peptides was explored. The experiments provided information that can be applied to the further development of the immune ligandome discovery project. In silico methods were successfully used to characterize previously identified HLA-restricted peptides and one previously identified immunogenic T-cell epitope. Even if the data acquired in silico can be considered only nominally verified at this stage, the results are encouraging.

Oncolytic adenoviruses are a new cancer treatment platform which aims to eliminate cancer through direct lysis of cancer cells by viral replication and the activation of the immune system by the release of tumor antigens upon oncolysis. In the PeptiCRAd technology, the activation of an anti-cancer immune response is enhanced by the addition of poly-lysine modified cancer peptides, where the antigen presentation to the immune system is improved in comparison to plain oncolytic viruses. PeptiCRAd complexes have been assumed to form solely by electrostatic interactions, but the thermodynamic profiles and mechanisms involved in the complexation have not been previously addressed. Thus, by adding isothermal titration calorimetry as part of the analysis repertoire provides valuable information of the characteristics of PeptiCRAd complexes. In this study, the applicability of isothermal titration calorimetry in PeptiCRAd complexation analyses was evaluated based on initial peptide-to-virus and virus-to-peptide titrations, and a method of analysis was created for the thermodynamics of the interactions of the complex. Optimization of the experimental method (i.e., titration protocol) and the data analysis (i.e., calculation models) remains inconclusive for quantitative analysis as data obtained from the measurements was mainly of bad quality, thus requiring further optimization to obtain reliable data. However, using surface plasmon resonance as an already established method for poly-lysine peptide-virus interaction studies gave robust data and can be used as a base or guideline to further develop isothermal titration calorimetry analyses for characterizing PeptiCRAd complexes. Although isothermal titration calorimetry measurements were unsuccessful for quantification purposes, it was possible to qualitate the mechanisms of PeptiCRAd complexation for four different peptides with fair confidence. The peptides showed low heats of binding, and positive and negative cooperative binding in ionic and non-ionic solutions, respectively. Based on this, the binding of peptides in PeptiCRAd complexes was determined to be driven by hydrophobic inter-peptide interactions on the virus surface, although an electrostatic attraction is indeed present at the virus-peptide interface, initiating the binding event. Also, improvements to the titration protocol for PeptiCRAd analyses with isothermal titration calorimetry are suggested for further optimizations in the future to conclusively determine the applicability of the isothermal titration calorimetry technique for characterizing peptide-virus interactions of PeptiCRAd complexes.

Making the treatment of these infections even harder is the fact, that Chlamydia pneumoniae can produce persistent forms of itself, which are immune to antibiotic treatment. When the bacteria sense a stress factor, for example the presence of a β-lactam antibiotic or interferon γ, they start producing these persistent forms called aberrant bodies. When the stress factor is removed, the bacteria can switch back to their replicating form and start infecting the tissues again. It is also known, that C. pneumoniae bacteria will trigger persistence when the bacteria migrate from lung epithelia into monocytes. Interestingly the onset of this mode of persistence does not require any other triggers besides the invasion of the monocyte. These persistence mechanisms enable latent, quiet, and recurring infections. This master’s thesis aimed to study the coculture of lung epithelial (HL cells) and monocytes (THP-1 cells), and by utilising the magnetic separation method presented by Kortesoja et al, to find a positive control compound in the prevention of Chlamydia pneumoniae internalisation into the THP-1 cells for said protocol. In these cocultures the inhibitory effect of different compound groups such as lignans present in Schisandra chinensis plant, MAPK-inhibitors, and β2,2-amino acid derivatives in C. pneumoniae migration from HL cells to THP-1 cells was assessed. Statistic relevance was observed in JNK inhibitor SP600125, MAPKAP-kinase-2 inhibitor SB203580, and ERK1/2 inhibitor FR180204 compounds. These compounds inhibited the internalisation of Chlamydia pneumoniae into THP-1 cells in the cell coculture by 61,05 ± 16,63 % (p = 0,0001), 54,06 ± 16,02 % (p = 0,0002), and 36,76 ± 10,33 % (p = 0,009) respectively. SP600125 and SB 203580 compounds also had an inhibitory effect on the internalisation of C. pneumoniae into the THP-1 cells in a cell monoculture (39,98 ± 18,92 %, p = 0,026 and 37,89 ± 19,47 %, p = 0,035 respectively), whereas FR180204 had no statistical significance, even though it inhibited the internalisation of C. pneumoniae into the THP-1 cells in cell monoculture by 27,53 ± 21,17 %. From the compounds used in the experiments, only MAPK inhibitors had an effect in inhibiting the C. pneumoniae internalisation into the THP-1 cells. The most potent compound in said inhibition was the JNK inhibitor SP600125. JNK pathway has been thought to take part in chlamydial infections but only little research has been done. The results of this master’s thesis’ experiments support the thought of JNK enzyme taking part in chlamydial infections but determining how exactly it affects the infection cycle of C. pneumoniae bacteria still needs further investigation.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons in brain and spinal cord. The degeneration of motor neurons leads to muscle atrophy and paralysis. Currently there is no cure for ALS. Available drugs for ALS can lengthen the survival time by a couple of months. Several factors involve the pathophysiology of ALS, such as endoplasmic reticulum stress and neuroinflammation. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein which has shown neuroprotective effects on animal models of Parkinson disease and brain ischemia. C-terminal fragment of MANF can cross the blood-brain barrier, allowing it to be administered subcutaneously instead of injected directly into the brain. The experimental part consists of two parts. The aim of the first part was to study the pharmacokinetic properties of next generation MANF (C-MANF). The aim of the second part was to elucidate the effect of twice a week administered subcutaneous injection of C-MANF in genetic SOD1-G93A mouse model and its neuroprotective effects by assessing protection of lumbar motor neurons. Pharmacokinetic properties of C-MANF were determined in wild type mice after a single subcutaneous injection of C-MANF at different time points by using indirect ELISA assay. The effects of C-MANF in SOD1-G93A mouse model were assessed by subcutaneous injection of either C-MANF or PBS twice a week and by monitoring clinical score and motor behavior of mice from 10 weeks of age to clinical endpoint. Hematoxylin eosin staining was used to study neuroprotective effects of C-MANF. C-MANF administered subcutaneously is absorbed into the blood circulation and the highest serum concentration of C-MANF is after 60 minutes of dosing. Subcutaneously injected C-MANF also crosses the blood-brain barrier and reach the brain in 120 minutes. C-MANF did not preserve motor function or ameliorated ALS symptoms in SOD1-G93A mouse model. In this study C-MANF did not increase the survival of SOD1-G93A mice. C-MANF did not significantly protect motor neurons from degeneration even though there was a slight trend between the groups. No beneficial effects were observed with C-MANF in SOD1-G93A mouse model and therefore the dose and frequency of administration of C-MANF were not optimal. Subcutaneously injected C-MANF provides a safer dosing option for neurodegenerative disorders.

Viral promoters are an essential part of a normally functioning virus. Their main task is to drive the transcription of genes which govern hijacking of cell function and replication of viral particles. In addition to supporting normal function of a virus, they can be used to drive the transcription of transgenes which can be used in different therapies. In oncolytic therapies, transgenes can be used to prime the host system against neoplasms which has been shown to generate long term anti-tumour immunity. Human adenoviruses (Ad) are commonly used as a platform for oncolytic virotherapies. Human Ad’s replicate poorly in mouse tumour cell lines, yet some promoters, which are included in the viral constructs to drive the transcription of beneficial transgenes, are able to function. Currently it is unknown whether E3, the native promoter of adenovirus 5 of the E3 region, is capable of functioning in murine cell lines. In this thesis we study whether human cytomegalovirus promoter (CMV) and E3 differ in their efficacy to drive the transcription of the mOX40L and mCD40L transgenes. In the experimental part of this thesis, we compared the efficacies of two viral promoters, AdE3 and AdCVM, in transcribing mOX40Land mCD40L in vitro. Efficacy of transcription was assessed through immunofluorescence and flow cytometry in human and murine cell lines. Furthermore, the effects of promoters on viral infection, killing and replication were evaluated in burst assay and the colorimetric MTS proliferation assay. MTS and burst assay were conducted to confirm if viral infection, killing and replication occurs in human and murine cell lines. Both AdE3 and AdCMV were able to infect and kill human cell lines and cell viability decreased in correlation to the number of viral particles used. In murine cell lines, no decrease in cell viability was detected in the 4T1 cell line. In burst assay, viral replication was observed for both AdE3 and AdCMV in the human MDA-MB-436 cell line. In murine CT26 cell line, no replication was observed for AdE3 or AdCMV constructs. Immunofluorescence assay was performed to visualize transgene expression and localization. Results indicated that mOX40L was localized on cell surface while mCD40L was detected both outside and inside of the cytosolic compartment. Flow cytometry results revealed that both AdE3 and AdCMV constructs are capable of efficiently transcribing mOX40L in human cell lines. In the flow cytometry results for AdE3, two large cell populations with different fluorescence intensities were detected. AdCMV lacked this feature which is postulated to be due to higher lytic activity of the viral construct. In murine cell lines, HCMV could produce mOX40L, but production in murine cell lines was severely attenuated compared to human cell lines. mOX40L produced by the AdE3 construct did not differ from the baseline and was deemed incapable of producing mOX40L in murine cell lines. For the purpose of studying novel virotherapeutics the results of this thesis would indicate that human CMV can be used to drive expression of transgenes in murine cell lines. Despite this, it is preferable to use host specific viruses and promoter sequences for a better translation between mice and humans. Viruksen promoottorit ovat keskeisessä osassa toimintakykyisessä viruksessa. Virus promoottorin päätarkoituksena on geenien transkriptio, mitkä vastaavat solun keskeisten toimintojen kaappaamisesta ja virus partikkeleiden replikaatiosta. Näiden toimintojen lisäksi promoottoreita voidaan käyttää transgeenien transkriptiossa, mitä voidaan hyödyntää sairauksien hoidossa. Onkolyyttisissä terapioissa transgeenejä voidaan käyttää virittämään kehon immuunipuolustus taistelemaan kasvainkudosta vastaan. Ihmisen adenovirusta käytetään usein onkolyyttisten viroterapioiden alustana. Ihmisen adenovirus (Ad) replikoituu hyvin heikosti hiiren syöpäsoluissa, mutta osa adenovirukseen sisälletyistä eksogeenisistä promoottoreista, joita käytetään terapeuttisten transgeenien transkription ajamiseen, kykenee toimimaan ja tuottamaan haluttua proteiinia. Tällä hetkellä ei tiedetä, kykeneekö E3, joka on adenoviruksen E3 lokuksen promoottori, toimimaan hiiren solulinjoissa. Tässä tutkielmassa selvitämme ihmisen sytomegalovirus promoottorin (CMV) ja E3 eroa niiden tehossa ajaa mOX40L ja mCD40L transgeenien transkriptiota. Kokeellisessa osuudessa vertailimme kahden virus promoottorin, E3 ja CMV, eroa niiden tehossa ajaa mCD40L ja mOX40L transkriptiota in vitro. Transkription tehoa tutkittiin immunofluoresenssin ja virtaussytometrian avulla ihmisen ja hiiren syöpäsolulinjoissa. Tämän lisäksi promoottorien vaikutusta virus infektioon, replikaatioon ja kykyyn tappaa soluja arvioitiin burst kokeella ja kolorimetrisellä MTS menetelmällä. MTS ja burst kokeiden avulla varmistettiin AdE3 ja AdCMV virusten kyky infektoida, tappaa ja replikoitua ihmisen ja hiiren syöpäsolulinjoissa. Sekä Ad3 ja AdCMV todettiin kykenevän infektoimaan ja tappamaan ihmissyöpäsoluja ja solujen viabiliteetin lasku korreloi virus partikkeleiden määrän kanssa. Hiiren 4T1 syöpäsoluissa ei todettu solujen viabiliteetin laskevan. Burst kokeessa havaitsimme sekä AdE3 että AdCMV kykenevän replikoitumaan ihmisen MDA-MB-436 solulinjassa. Hiiren CT26 solulinjassa kummankaan viruksen ei havaittu kykenevän replikoitumaan. Immunofluoresenssi kokeessa visualisoimme transgeenien ilmentymisen ja paikantumisen. Tulokset osoittivat, että mOX40L paikantui solun pinnalle. mCD40L havaittiin puolestaan sekä solun ulkopuolella että sytosolissa. Virtaussytometria kokeen tulokset osoittivat, että sekä AdE3 ja AdCMV pystyivät tehokkaasti ilmentämään mOX40L ihmisen solulinjoissa. AdE3 virtausytometria tuloksissa löydettiin kaksi solupopulaatiota, joilla oli toisistaan poikkeavat fluoresenssi intensiteetit. Tätä ilmiötä ei havaittu AdCMV:lla infektoiduilla soluilla, mikä saattoi johtua korkeammasta lyyttisestä aktiivisuudesta. Hiirisolulinjoissa CMV kykeni ilmentämään mOX40L, mutta transkription teho oli selvästi alhaisempi verrattuna ihmissolulinjoihin. E3 promoottorin ilmentämä mOX40L ei eronnut kontrollista ja sen todettiin olevan kykenemätön tuottamaan mOX40L hiirisolulinjoissa. Tuloksemme osoittavat, että ihmisen CMV promoottori kykenee ilmentämään transgeenejä hiiren 4T1 ja CT26 solulinjoissa. On kuitenkin huomattava, että isäntälajille natiivien virusten ja promoottorien käyttö olisi tarkoituksenmukaisempaa tulosten käännettävyyden kannalta hiiristä ihmisiin.

Theoretical framework: The consolidated pharmaceutical market is becoming increasingly global and the same international pharmaceutical companies operate around the world in different countries, responsible for drug development and production. The high costs of developing novel medicines and the motive for higher profits has led to elevating price level of pharmaceuticals and health care services. Finland and the U.S. offer two extremes at the pharmaceutical market. The pharmaceutical market field in Finland is very structural and rigid, and medicine prices are regulated by law. In the U.S. the prices are based on the laws of supply and demand and the prices differ by different states, retailers and insurance policies. A small-scale longitudal price comparison is also reviewed to showcase the effect of continuously rising medicine prices. Study objective: The idea of this study is to describe and compare pricing mechanisms of pharmaceuticals and price differences between two very different market structures and review how these might affect the cost-effectiveness of national health care spending. These divergences are also mirrored to survey recent global pharmaceutical market problems such as drug shortages, possibly due to less appealing markets of higher price regulation policies. Materials and methods: Price data were collected from national, official, open-source databases. National health care expenditure and comparison to GDP was collected from publications by the OECD. All monetary values have been presented in both currencies (EUR and USD) to present more comparable values. Results: When compared to other OECD-countries the U.S. spent distinctly the largest amount of funds on health care per capita. Finland’s national health care costs were thousand times minor in total spending and less than a half per capita when compared to those of the U.S. With lower expenditure Finland manages to offer access to public, government-funded health insurance program. Meanwhile the prices of prescription medicines in Finland have decreased significantly, the prices for have continuously elevated in the U.S. Conclusions: The outcome of this study is that free markets and a complex supply chain, compared to more regulated markets with more transparency, have higher overall price level in pharmaceuticals and health care services. Free markets and sufficient intellectual property rights are more enticing to pharmaceutical companies. They promote new innovations and developing of much-needed novel therapies to modern health problems, such as AIDS and the global threat of worsening situation of antibiotic resistance. More regulated markets may create problems such as drug shortages and are often considered complex and less appealing market systems due to high level of administrative work but conserve the cost-effectiveness of the use of public funds.

The orexinergic system is a central regulator for sleep-wake rhythm and energy homeostasis. Dysfunction of the system is at least one of the reasons behind narcolepsy, in addition to which insomnia, obesity and certain cancers could be treated by targeting orexin receptors. The orexin system in human comprises two receptor subtypes, orexin receptor 1 and 2 (OX₁R and OX₂R respectively) as well as two cognate ligands, peptides orexin-A and -B. In this study the focus is on OX₁R and orexin-A. The aims of the study are (1) to propose a binding mode for orexin-A to OX₁R and (2) to understand the molecular interactions of OX₁R leading to receptor activation. I order to create 3D molecular models of OX₁R, a sequence alignment of the eight G proteincoupled receptors (GPCRs) that have been crystallized up to date was first generated by ClustalX and adjusted based on the superimposition by SYBYL-X. Structurally conserved regions were deduced from the alignment and used to add the orexin receptors. Five different models built with MODELLER were selected for their large binding cavity among a large pool of models. These models were constructed based on the chemokine receptor 4 (PDB Id:3ODU), as such and a modified version where TM3 was moved by 1 Å further from the center of the binding cavity, from the β₂-adrenoceptor (PDB Id: 2RH1) and from the adenosine receptor A2A (PDB Id: 2YD0), as such and with rotamer changes to few binding site residues. Orexin-A with straight conformation found by NMR (PDB Id:1WSO) was docked to these models using ZDOCK and RDOCK. In addition, an in-house docking protocol was implemented, but could not be validated. Docking poses were scored by purpose built knowledge based scoring function and clustered. High scoring clusters were then used to converge to three different binding modes. As a result, we suggest that the binding site of OX₁R consists of two hydrophobic walls, one from TM3 and TM5, the other from TM6 and TM7. Binding modes include a hydrogen bond network between the ligand and especially binding site residues Gln1263.32, Thr2235.46, Asn3186.55, Lys3216.58 and Tyr3117.43. Based on the binding modes, it is suggested that the OX₁R is activated by similar binding site contraction as β-adrenoceptors and adenosine A2A. The contraction in could result from the hydrogen bonds between ligand, Gln1263.32, Thr2235.46 and Asn3186.55. The hydrogen bonding of Thr2235.46 can also disrupt interactions between TM5 and TM3, an interaction which is identified as an important factor in keeping the receptor in the inactive state. The role of other ligand residues would be to direct ligand binding and keep the ligand in the helical conformation.