Browsing by Subject "protein-protein interaction"
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(2019)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.
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(2021)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.
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