Browsing by study line "Pharmaceutical chemistry"

Prolyl oligopeptidase (PREP) is endopeptidase which cleaves short proline containing peptides. Abnormalities in brain PREP activity has been connected to neurodegenerative diseases. Recently it has been detected that besides its proteolytic activity PREP interacts directly with other proteins which might contribute to generation of neurodegenerative diseases. Further it has been discovered that certain small molecular PREP inhibitors are able to modify these protein-protein interactions (PPIs) and thus have a potential to alleviate the progression of neurodegenerative diseases. This has led to the development of novel second generation PREP ligands which lack the strong inhibitory activity but are potent compounds on modifying the PPIs. Thiazole structure containing PREP modulators has provided most promising class of compounds. It has been detected that these compounds mediate their effects via novel binding site on the enzyme and these effects are not connected to the inhibition of the enzymatic activity. The synthesis of these thiazole containing PREP modulators has proven to be demanding since it have involved a usage of laborious synthesis route and provided low yields. The aim of this research was to examine the synthesis of 2-(2-benzimidazol-1-yl)ethyl)- 4-methyl thiazole containing PREP modulators via previously reported synthesis route. Another aim was to design and develop a synthesis route for 2-(2-(benzimidazol-1- yl)ethyl)-5-bromo-4-methylthiazole, a molecule which serves as valuable intermediate for the lead optimization and generation of second-generation PREP modulators. A synthetic route for 2-(2-(benzimidazol-1-yl)ethyl)-5-bromo-4-methylthiazole was successfully developed. Despite that the total yield of the route remained low. When searching the reasons for the low obtained yield the chemistry behind a thiazole creating cycloaddition reaction and an aromatic halogenation was examined. This led to the discovery of a rare cationic compound which was found to be synthesized from previously undescribed starting materials.

Orexin receptors have gained more attention due to increased knowledge of their physiological significance. The successful development of orexin receptor antagonists treating insomnia has enlarged the scope of research leading to an interest in developing small molecule agonists that have drug-like properties and induce activation in the orexinergic system. Transforming this idea into reality has remained an unaccomplished challenge. In this work, molecular mechanism of activation in orexin receptor type 2 is studied by conducting molecular dynamics simulations after capturing coordinates of active and inactive state crystal structures. The crystal structure coordinates define the starting pose for the protein and the ligand in the simulations. Preliminary steps prior to simulation include building the surrounding system and model building in which homology modeling is employed to reconstruct missing loops. A 100-nanosecond simulation is executed for both models. Active and inactive state models display stable binding event characteristics when examining the coordinate changes of every atom during the simulation. No major conformational changes are observed. The most congruent feature relative to the literature is the difference in the interactive role of residue Q3x32 between the simulations. The agonist forms two consistent hydrogen bonds with the upward-shifted Q3x32 while an inactive downward-facing version is observed in the antagonist model. Some residues present unexpected features omitting comparative functions of literature, which along with general inconsistency of protein-ligand contacts undermine the reliability of models heavily. The simulations conducted need to be extended to produce a hypothesis for the activation mechanism because of the defects around simulation protocols and the low quantity of simulation runs.

Heart failure is a disease of major social and economic impact. The disease is most commonly onset by extensive cardiomyocyte death following a myocardial infarction. Five-year mortality of heart failure is higher than some cancers. Loss of cardiac muscle tissue leads to pathological thickening and fibrosis of the left ventricular wall, which eventually further diminish cardiac function. Cardiomyocytes hardly proliferate, which also exacerbates the problem. Several cell signalling pathways are indicated in pathological reprogramming of the heart and the exact significance of these pathways remains to be demonstrated. Treatment strategies based on renewing cardiac muscle, such as direct injection of stem cells into the myocardium, have failed to reach clinically significant effects on heart failure patients. Direct inhibition of pathological cardiac reprogramming by using small molecule modulators remains as an auspicious strategy to treat heart failure. GATA4, or GATA binding protein 4, is a transcription factor expressed mainly in heart, lung, intestine, gonad and liver tissues, which regulates tissue renewal and cell proliferation by controlling protein transcription. GATA4 binds to GATA sequences in DNA with two zinc finger moieties and enables transcription of target genes. Interactions of GATA4 and several other transcription factors are in central role of guiding heart development, hypertrophy and fibrosis. One of these transcription factors is NKX2-5, which synergistically interacts with GATA4. Inhibition of this interaction in rat myocardial infarction model has been shown to protect against development of heart failure. A screening campaign against the transcriptional synergy of GATA4 and NKX2-5 found potent small molecule inhibitors of this interaction, but these compounds are characterised with stem cell toxicity. The aim of the study was to design and synthesise novel derivatives of GATA4-NKX2-5 synergy inhibitor hit molecule with reduced stem cell toxicity. Modifications on the phenyl ring of the hit molecule were designed, which either increase electron density of the ring or possibly alter the torsional angle between the phenyl and isoxazole ring moieties. Activity of the compounds was studied on a luciferase reporter gene system in COS-1 cells and toxicity was analysed on IMR90 human induced pluripotent stem cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide and lactate dehydrogenase (LDH) assays were selected to measure toxicity on stem cells. Stem cell toxicity of several previously synthesised compounds was assayed in parallel with the novel derivatives. Ten novel derivatives were designed, synthesised and assayed. Four of the new compounds, a mono-ortho-methyl, a di-ortho-methyl, a di-meta-methoxy and cyclohexyl derivatives were found to be equipotent inhibitors of reporter gene activity compared to the hit compound. Additionally, the mono-ortho-methyl, di-ortho-methyl and di-meta-methoxy derivatives were less toxic to stem cells than the hit molecule in the MTT assay. Several other derivatives were found to be less toxic, but also non-active in the luciferase assay. None of the studied compounds exhibited notable necrotic toxicity on stem cells, as examined by the LDH assay. According to this study it may be concluded that substituents of the hit molecule phenyl ring may be altered to decrease stem cell toxicity of the compound. Some of the alterations, most notably substituents in the para-position of the phenyl ring and substitution of the phenyl ring with smaller saturated hydrocarbon rings, diminish the activity of the hit compound. Remarkable toleration of ortho-substitution reinforces the hypothesis of phenyl-isoxazole torsional angle significance for toxicity. On the other hand, addition of two methoxy groups to both meta positions most likely lacks any substantial effect on the torsional angle, which implies another mechanism of toxicity avoidance. Activity and improved safety of the novel inhibitors should be confirmed in animal models before any decisive conclusions on the effects of structural modifications on the hit molecule can be made. In addition, other mechanisms of toxicity should be studied with relevant cell-based assays.

Prolyl oligopeptidase (PREP) is a serine protease that is widely found throughout the human body and especially in the brain. The primary function of PREP is thought to be the hydrolysis of the carboxyl side bond of proline residues in oligopeptides. PREP is also shown to increase the dimerization and aggregation of α-synuclein and downregulate the protein phosphatase 2A mediated autophagy in the cell via direct protein-protein interactions (PPI). The accumulation of α-synuclein aggregates in cell is known to cause α-synucleinopathies such as Parkinson’s disease. This makes the PPIs of PREP an attractive target for drug research. The mechanisms of the PPIs of PREP are still poorly understood. Recent studies have shown that these PPIs can be modulated with ligands lacking high inhibitory activity for the proteolytic activity. These studies show that the IC50-value of the ligand does not correlate with ligands ability to affect the PPIs of PREP. Ligands that could selectively modulate the PPIs of PREP without inhibiting the proteolytic activity of PREP could give valuable information on the mechanisms of the PPIs and on how to modulate them. It is hypothesized that the ligands could bind to PREP at a site that does not interfere with its proteolytic activity, and ligand binding is assumed to restrict the dynamic structure of PREP and thereby also modulating the PPIs of PREP. The aim of this study was to synthetize novel peptidic PREP ligands and study their effects on the proteolytic activity of PREP and the PPIs of PREP. The aim was to find and identify ligands and structures that would modulate the PPIs of PREP and observe how the IC50-values of the ligands would correlate. L-Alanyl-pyrrolidine was selected as the scaffold for the compound series and the five-membered heteroaromatics, imidazole, triazole and tetrazole, were added to the 2-position of the pyrrolidine ring. In this position there is an electrophilic group in many PREP inhibitors, although these heteroaromatics are not electrophiles. The scaffold was also expanded by adding phenyalkyl groups with different linker lengths were added to the N-terminal side of the alanine. The ligands were synthesized using four synthesis routes which were based on synthesis methods found in literature. The IC50-values and the effects on α-synuclein dimerization and autophagic flux were determined for five synthetized compounds. The tested compounds were all weak PREP inhibitors and showed no strong activity in the α-synuclein dimerization and autophagy assays. Despite the weak activities in the assays, the importance of the linker length in the phenyalkyl group was shown. Changing the linker by one methylene group had noticeable effect on the overall activity. The results also demonstrate a lack of correlation between the IC50-values and the effects on α-synuclein dimerization and autophagic flux, which further confirms the lack of correlation between the proteolytic function and the PPIs of PREP which was observed also in previous studies.

Inhibition of the cytochrome P450 enzymes is one of the most significant factors causing drug-drug-interactions, and thus one of the most important objects of study at preclinical drug development. CYP-inhibition can be either reversible or irreversible. Although different inhibition mechanisms are well known, their evaluation in vitro is still challenging. Thus, the development of more accurate and efficient in vitro methods is important and as a continuous target of interest. Immobilized enzyme microreactors (IMER) have presumably several advantages over traditional in vitro methods and have been presented as a promising tool for drug metabolism studies in vitro. The purpose of this work was to evaluate the suitability of a novel flow-through based immobilized enzyme microreactor in determining the CYP enzyme kinetic parameters. The developed immobilization protocol is based on attaching biotinylated human liver microsomes to a thiolene-based microreactor coated with Streptavidin. To validate the developed method, the activity of the CYP2C9 enzyme was assessed using the recommended model reaction by authorities, that is 4-hydroxylation of diclofenac. The enzyme kinetic parameters i.e., enzyme affinity (Km) and activity (Vmax), determined with the developed IMER were comparable to the values previously published in the literature and determined in static in vitro conditions. In addition, the inhibition of CYP2C9 enzyme by four model inhibitors (fluconazole, nicardipine, sulfaphenazole and miconazole), was examined by determining the IC50 (half-maximal inhibitory constant) values for each compound and by monitoring the reversibility of the CYP2C9 enzyme for 90 minutes after the inhibitor was removed from the feed solution. The IC50 values determined with the developed method for all inhibitors were well in line with previous publications, showing fluconazole (IC50 22 µM) to be the weakest inhibitor of CYP2C9 enzyme and the other examined inhibitors caused more potent inhibition (IC50 for sulfaphenazole 1.3 µM; IC50 for miconazole 1.3 µM; IC50 for nicardipine 0.67-1.1 µM). The reversibility of the CYP2C9 enzyme was examined by removing the inhibitor from the feed solution and monitoring the recovery of the enzyme activity via diclofenac 4-hydroxylation. Based on the results obtained with developed IMER, the inhibition of fluconazole and sulfaphenazole was reversible and thus well in line with previous studies. In contrast, on account of data obtained with IMER, inhibition by miconazole and nicardipine was not reversible, although these compounds have previously been reported to be reversible CYP2C9 inhibitors in vitro, which may be due to the strong aggregation tendency of these compounds. The study shows that the developed flow-through based IMER is well suited for studying inhibition of CYP enzymes However, to utilize the developed technology in CYP enzyme inhibition research, it’s applicability in determining enzyme inhibition should still be evaluated with more comprehensively with several CYP isoenzymes.

Prolyloligopeptidase (PREP) and alphasynuclein are linked to various neurological and psychiatric conditions of which the most relevant considering this study is parkinsonism. PREP cleaves small peptides after a proline residue. It has also protein-protein ineractions with alphatubulin, GAP-43 and alphasynuclein. PREP inhibitors have been shown to have an effect to elimination of alphasyuclein via autophagy. Thiazole is a heteroaromatic compound with two heteroatoms (sulphur and nitrogen). Thiazole can be found as a structural component among various active pharmaceutical ingredients with wide array of indications. Synthetic route for thiazole was published in 1887 and a considerable amount of literature regarding the use of thiazole in medicinal and synthetic chemistry has been published. The aim of the study was to extend the the scope of research done in the research group on small-molecular thiazole-based PREP inhibitors. The goal was to develop a synthetic route to access a series of molecules and gain information of the possible biological activities of the produced compounds by determining their IC50-values in vitro, effect on dimerization of alphasynuclein and removal of alphasynuclein via autophagy in a cell culture. Optimization of the synthetic route and search of alternative reactions were among the aims to some extent. During the course of study yields of some steps of the synthesis were improved and some new molecules had biological activity.

The aim of this master’s thesis was to investigate whether drug-induced inhibition of cytochrome P450 enzymes (CYP), especially time-dependent inhibition (TDI), could be the reason for bioaccumulation of the pharmaceuticals present in the aquatic environment in fish and whether the in vitro method could identify pharmaceuticals causing an environmental risk, which should primarily be investigated more closely. The half-maximal inhibitory concentrations (IC50) of seven antimicrobial drugs detected in the environment (erythromycin, clarithromycin, ketoconazole, clotrimazole, miconazole, ciprofloxacin, and sulfamethoxazole) and three known human time-dependent inhibitors (furafylline, diltiazem and verapamil) chosen for the validation of the method, were determined by EROD (7-ethoxy-resorufin-O-deethylase) and BFCOD (7-benzyloxy-4-trifluoromethyl-coumarin-O-debenzyloxylase) activities. The IC50 shift method and commercially available rainbow trout (Oncorhynchus mykiss) liver microsomes were used in determinations. The known human time-dependent inhibitors chosen for the validation of the method, furafylline (EROD) and diltiazem (BFCOD) proved to be possible time-dependent inhibitors also in rainbow trout in vitro, but this was not observed for verapamil (BFCOD). All antimicrobial drugs, except ciprofloxacin, inhibited more selectively BFCOD-reaction, as in human. In the case of sulfamethoxazole, inhibition was not observed at the concentrations used (0–500 µmol/L). Both enzyme activities (EROD and BFCOD) were inhibited in rainbow trout by ketoconazole, clotrimazole and miconazole. Among antimicrobial drugs acting as time-dependent inhibitors in human, erythromycin inhibited BFCOD activity in a time-dependent manner also in rainbow trout, but this was not observed for clarithromycin. Strongest inhibitors for CYP enzymes of rainbow trout in vitro were ketoconazole (EROD, IC50=4,19 µM and BFCOD, IC50=2,31 µM) and clotrimazole (EROD, IC50=33,78 µM and BFCOD, IC50=1,55 µM). The IC50 values of diltiazem, erythromycin, clarithromycin, ciprofloxacin, and verapamil were of the same order of magnitude as in human. The IC50 values of furafylline, ketoconazole, clotrimazole and miconazole were several times higher in rainbow trout than in human. Based on the results of this study, the IC50-shift method is also valid for fish, but there are differences in the inhibition potencies between human and fish, and the inhibition potency of human CYP enzymes cannot therefore directly predict enzyme inhibition of fish or the mechanism of inhibition. The In vitro measured IC50 values of rainbow trout were several orders of magnitude higher than the average concentrations of the pharmaceutical residues measured in the environment. Exposure to pharmaceutical mixtures is long-term, so interactions and bioaccumulation may still be possible due to inhibition of CYP enzymes. Developing a valid in vitro method for environmental risk assessment would be important, as animal experiments are ethically challenging.