Our project was aimed at the development of novel CB1 cannabinoid receptor antagonists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. In this study, we designed, synthesized, and established the CB1 affinity for the 1,5-diaryl-1,2,3- triazole esters, a series of 4,5-diaryl-1-substituted-1,2,3-triazole analogues and a series of 4,5- diaryl-2-substituted-1,2,3-triazoles. Our research group has been interested in the synthesis of amphibian alkaloids due to their interesting biological activities. We have recently developed a general synthetic strategy which can rapidly prepare a few amphibian alkaloids simply from the abundant natural product (-)- cocaine This strategy was first successfully applied to the synthesis of (-)-monomorine. More recently, this strategy has also been utilized in the syntheses of both of the enantiomers of cispyrrolidine 225H and (+)-gephyrotoxin 287C.

<p> This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide.</p><p> Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in </p><p>Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT₁-receptor had affinity for both receptor subtypes, while the compounds designed for the AT₂-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a <i>γ</i>-turn like conformation around Tyr-4 while interacting with the </p><p>Ang II receptor. Based on this hypothesis, three different <i>γ</i>-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing <i>γ</i>-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a <i>γ</i>-turn like conformation when activating the AT₁ receptor. The other Ang II analogues, incorporating bicyclic and aromatic <i>γ</i>-turn mimetics, did not display any binding to the AT₁ receptor.</p>

This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT1-receptor had affinity for both receptor subtypes, while the compounds designed for the AT2-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a γ-turn like conformation around Tyr-4 while interacting with the Ang II receptor. Based on this hypothesis, three different γ-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing γ-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a γ-turn like conformation when activating the AT1 receptor. The other Ang II analogues, incorporating bicyclic and aromatic γ-turn mimetics, did not display any binding to the AT1 receptor.

<p>Gene expression is regulated in response to metabolic necessities and environmental changes throughout the life of a cell.</p><p>A major part of this regulation is governed at the level of transcription, deciding whether messengers to specific genes are produced or not.</p><p>This decision is triggered by the action of transcription factors, proteins which interact with specific sites on DNA and thus influence the rate of transcription of proximal genes.</p><p>Mapping the organisation of these transcription factor binding sites sheds light on potential causal relations between genes and is the key to establishing networks of genetic interactions, which determine how the cell adapts to external changes.</p><p>In this work I review briefly the basics of genetics and summarise popular approaches to describe transcription factor binding sites, from the most straight forward to finally discuss a biophysically motivated representation based on the estimation of free energies of molecular interactions.</p><p>Two articles on transcription factors are contained in this thesis, one published (Aurell, Fouquier d'Hérouël, Malmnäs and Vergassola, 2007) and one submitted (Fouquier d'Hérouël, 2008).</p><p>Both rely strongly on the representation of binding sites by matrices accounting for the affinity of the proteins to specific nucleotides at the different positions of the binding sites.</p><p>The importance of non-specific binding of transcription factors to DNA is briefly addressed in the text and extensively discussed in the first appended article:</p><p>In a study on the affinity of yeast transcription factors for their binding sites, we conclude that measured in vivo protein concentrations are marginally sufficient to guarantee the occupation of functional sites, as opposed to unspecific emplacements on the genomic sequence.</p><p>A common task being the inference of binding site motifs, the most common statistical method is reviewed in detail, upon which I constructed an alternative biophysically motivated approach, exemplified in the second appended article.</p>

Influenza virus A and B are common pathogens that cause respiratory disease in humans. Recently, a highly virulent H5N1 subtype avian influenza virus caused disease outbreaks in poultry around the world. Drug resistant type A viruses rapidly emerged, and the recent H5N1 viruses were reported to be resistant to all current antiviral drugs. There is an urgent need for the development of new antiviral drugs target against both influenza A and B viruses. This dissertation describes work to identify small molecule inhibitors of influenza protein NS1 by a high throughput fluorescence polarization assay. The N-terminal GST fusion of NS1A (residue 1-215) and NS1B (residue 1-145) were chosen to be the NS1A and NS1B targets respectively for HT screening. In developing the assay, the concentrations of fluorophore and protein, and chemical additives were optimized. A total of 17,969 single chemicals from four compound libraries were screened using the optimized assay. Six true hits with dose-response activity were identified. Four of them show an IC₅₀ less than 1 [micromolar]. In addition, one compound, EGCG, has proven to reduce influenza virus replication in a cell based assay, presumably by interacting with the RNA binding domain of NS1. High throughput, computer based, virtual screenings were also performed using four docking programs. In terms of enrichment rate, ICM was the best program for virtual screening inhibitors against NS1-RBD. The compound ZINC0096886 was identified as an inhibitor showing an IC₅₀ around 19 [micromolars] against NS1A, and 13.8 [micromolars] against NS1B. In addition, the crystallographic structures of the NS1A effector domain (wild type, W187A, and W187Y mutants) of influenza A/Udorn/72 virus are presented. A hypothetical model of the intact NS1 dimer is also presented. Unlike the wild type dimer, the W187Y mutant behaved as a monomer in solution, but still was able to binding its target protein, CPSF30, with wild type binding affinity. This mutant may be a better target for the development of new antiviral drugs, as the CPSF30 binding pocket is more accessible to potential inhibitors. The structural information of those proteins would be very helpful for virtual screening and rational lead optimization. / text

Genetics is among the most rewarding fields of biology for the theoretically inclined, offering both room and need for modeling approaches in the light of an abundance of experimental data of different kinds. Many aspects of the field are today understood in terms of physical and chemical models, joined by information theoretical descriptions. This thesis discusses different mechanisms and phenomena related to genetics, employing tools from statistical physics along with experimental biomolecular methods. Five articles support this work. Two articles deal with interactions between proteins and DNA. The first one reports on the properties of non-specific binding of transcription factors proteins in the yeast Saccharomyces cerevisiae, due to an effective background free energy which describes the affinity of a single protein for random locations on DNA. We argue that a background pool of non-specific binding sites is filled up before specific binding sites can be occupied with high probability, thus presenting a natural filter for genetic responses to spurious transcription factor productions. The second article describes an algorithm for the inference of transcription factor binding sites for proteins using a realistic physical model. The functionality of the method is verified on a set of known binding sequences for Escherichia coli transcription factors. The third article describes a possible genetic feedback mechanism between human cells and the ubiquitous Epstein-Barr virus (EBV). 40 binding regions for the major EBV transcription factor EBNA1 are identified in human DNA. Several of these are located nearby genes of particular relevance in the context of EBV infection and the most interesting ones are discussed. The fourth article describes results obtained from a positional autocorrelation analysis of the human genome, a simple technique to visualize and classify sequence repeats, constituting large parts of eukaryotic genomes. Applying this analysis to genome sequences in which previously known repeats have been removed gives rise to signals corroborating the existence of yet unclassified repeats of surprisingly long periods. The fifth article combines computational predictions with a novel molecular biological method based on the rapid amplification of cDNA ends (RACE), coined 5’tagRACE. The first search for non-coding RNAs encoded in the genome of the opportunistic bacterium Enterococcus faecalis is performed here. Applying 5’tagRACE allows us to discover and map 29 novel ncRNAs, 10 putative novelm RNAs and 16 antisense transcriptional organizations. Further studies, which are not included as articles, on the monitoring of secondary structure formation of nucleic acids during thermal renaturation and the inference of genetic couplings of various kinds from massive gene expression data and computational predictions, are outlined in the central chapters. / QC 20110316

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA. Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases. The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

The rapidly increasing number of existing drugs with genomic, biomedical, and pharmacological data make computational analyses possible, which reduces the search space for drugs and facilitates drug repositioning (DR). Thus, artificial intelligence, machine learning, and data mining have been used to identify biological interactions such as drug-target interactions (DTI), drug-disease associations, and drug-response. The prediction of these biological interactions is seen as a critical phase needed to make drug development more sustainable. Furthermore, late-stage drug development failures are usually a consequence of ineffective targets. Thus, proper target identification is needed. In this dissertation, we tried to address three crucial problems associated with the DR pipeline and presents several novel computational methods developed for DR. First, we developed three network-based DTI prediction methods using machine learning, graph embedding, and graph mining. These methods significantly improved prediction performance, and the best-performing method reduces the error rate by more than 33% across all datasets compared to the best state-of-the-art method. Second, because it is more insightful to predict continuous values that indicate how tightly the drug binds to a specific target, we conducted a comparison study of current regression-based methods that predict drug-target binding affinities (DTBA). We discussed how to develop more robust DTBA methods and subsequently developed Affinity2Vec, the first regression-based method that formulates the entire task as a graph-based method and combines several computational techniques from feature representation learning, graph mining, and machine learning with no 3D structural data of proteins. Affinity2Vec outperforms the state-of-the-art methods. Finally, since drug development failure is associated with sub-optimal target identification, we developed the first DL-based computational method (OncoRTT) to identify cancer-specific therapeutic targets for the ten most common cancers worldwide. Implementing our approach required creating a suitable dataset that could be used by the computational method to identify oncology-related DTIs. Thus, we created the OncologyTT datasets to build and evaluate our OncoRTT method. Our methods demonstrated their efficiency by achieving high prediction performance and identifying therapeutic targets for several cancer types. Overall, in this dissertation, we developed several computational methods to solve biomedical domain problems, specifically drug repositioning, and demonstrated their efficiencies and capabilities.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.