|
|
Analysis of the mechanism of action of metallacarborane inhibitors of HIV PR
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...
|
|
|
Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases
Hympánová, Michaela ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...
|
|
|
Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
|
|
|
Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří
This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...
|
|
|
Effect of cytochrome b5 on enzyme kinetics of Sudan I hydroxylation catalyzed by human cytochrome P450 1A1
Netolický, Jakub ; Martínek, Václav (advisor) ; Černá, Věra (referee)
Cytochromes P450 are the major xenobiotics converting enzymes. They are classified as mixed function monooxygenases (MFO). Isoform 1A1 is a extrahepatic form found mainly in the lung and other tissues. It is strongly induced by polycyclic aromatic hydrocarbons and their derivatives via the Ah receptor. As a marker reaction for this enzyme can be used hydroxylation of Sudan I, which has previously been widely used as a azo dye in industry, but since 1980s it is banned for coloring food and cosmetics for its negative influence on the organism. NADPH:cytochrome P450 reductase is the major electron donor for cytochrome P450 catalyzed monooxygenation reactions. Another electron carrier for cytochrome P450 catalyzed reactions is cytochrome b5. It was shown that cytochrome b5 can stimulate, inhibit or have no effect on P450 catalyzed reactions. This thesis aims to evaluate the influence of the ration between NADPH:cytochrome P450 reductase and cytochrome b5 on cytochrome P450 1A1 catalyzed Sudan I hydroxylation. The main goal is to characterize the influence of electron donor and electron transfer ratios on hydroxylation of Sudan I, and to determine the kinetic parameters KM and VMAX for selected protein ratios. Partial aims of the thesis were to characterize the recombinant proteins used in this study...
|
|
|
Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.
|
|
|
Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
|
|
|
Significance of the S1 subsite of rhomboid intramembrane proteases for catalysis and inhibitor design
Kučerová, Jolana ; Stříšovský, Kvido (advisor) ; Hodek, Petr (referee)
This thesis focuses on the development of specific inhibitors of rhomboid intramembrane proteases. These inhibitors are needed for the cell-biological investigation of rhomboid proteases and their potential pharmacological targeting, as rhomboid proteases have been associated with various diseases, such as malaria, Parkinson's disease, cancer or toxoplasmosis. The thesis advisor's laboratory has recently discovered the first such group of compounds, the peptidyl ketoamides. To exploit them fully, it is necessary to examine their properties and the possibilities of their modifications. In this work, synthetic fluorogenic substrates and enzyme kinetics were used to examine the possibilities of exploiting the S1 subsite in the rhomboid active site for rhomboid inhibitor design. Furthermore, using variants of these substrates modified by unnatural amino acids in the P1 position, the mechanism of water transfer to the rhomboid active site was investigated. Comparison of cleavage rates of ten fluorogenic substrates modified in the P1 position revealed that the E. coli rhomboid protease GlpG strongly prefers side chains -CH2-CH3 and -CH3 in the P1 position (i.e. binding into the S1 subsite). This trend was apparent also with peptidyl ketoamide inhibitors. The present substrate and inhibitor study suggests...
|
|
|
Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases
Hympánová, Michaela ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...
|
|
|
Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří
This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...
|
guest ::
