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Nové inhibitory HIV proteasy: návrh, synthesa a testování aktivity
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
More than 20 years after its discovery HIV protease still remains one of the primary targets in HIV treatment. Currently there are 9 approved protease inhibitors on the market. However, due to immense replication rate and the high error prone nature of reverse transcriptase, resistance to each of them has already been described. Therefore, the search for new protease inhibitors with different binding mode is still active. A novel type of protease inhibitors (1, 4-benzodiazepine analogs) was recently discovered in our laboratory. Even though this new class of inhibitors is highly potent (Ki' in range of 10-9 ), it also has several undesirable qualities, such as low solubility and a high number of stereogenic centers. Primary objective of this study was to try to prepare more soluble compounds with lower number of possible stereoisomers, enzymologically characterize its binding to the wild-type and mutated HIV protease and to determine its structure in the complex with the enzyme. A small library of 1, 4-benzodiazepine inhibitors of HIV protease was synthesized and fully characterized using NMR spectroscopy and mass spectroscopy. The number of stereogenic centers was successfully reduced from 4 to 2 without loosing activity of the inhibitor. The improvement in solubility was always associated with a...
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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...
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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...
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Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Ruml, Tomáš (referee)
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...
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Mechanism of action of non-peptide inhibitors of HIV protease
Began, Jakub ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...
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Nové inhibitory HIV proteasy: návrh, synthesa a testování aktivity
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
More than 20 years after its discovery HIV protease still remains one of the primary targets in HIV treatment. Currently there are 9 approved protease inhibitors on the market. However, due to immense replication rate and the high error prone nature of reverse transcriptase, resistance to each of them has already been described. Therefore, the search for new protease inhibitors with different binding mode is still active. A novel type of protease inhibitors (1, 4-benzodiazepine analogs) was recently discovered in our laboratory. Even though this new class of inhibitors is highly potent (Ki' in range of 10-9 ), it also has several undesirable qualities, such as low solubility and a high number of stereogenic centers. Primary objective of this study was to try to prepare more soluble compounds with lower number of possible stereoisomers, enzymologically characterize its binding to the wild-type and mutated HIV protease and to determine its structure in the complex with the enzyme. A small library of 1, 4-benzodiazepine inhibitors of HIV protease was synthesized and fully characterized using NMR spectroscopy and mass spectroscopy. The number of stereogenic centers was successfully reduced from 4 to 2 without loosing activity of the inhibitor. The improvement in solubility was always associated with a...
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A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, M. ; Hradílek, M. ; Lepšík, M. ; Konvalinka, J.
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance developoment. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permits to assess the donor/acceptor realtions of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired ihnibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
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Vazba fenylnorstatinového inhibitoru na proteázu HIV-1: geometrie, protonace a interakce kapes podřadných míst analyzované při atomovém rozlišení
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, Milan ; Hradilek, Martin ; Lepšík, Martin ; Konvalinka, Jan
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permints to assess the donor/acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses
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