Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni

Schistosomóza je závažné infekční onemocnění postihující více než 200 milionů lidí v oblasti tropů a subtropů. Původcem je parazit krevnička, který žije v cévách člověka a živiny získává degradací hemoglobinu z krve hostitele působením trávicích proteas. V současnosti je k dispozici pouze jediný lék proti schistosomóze a hrozba vzniku rezistence vyvolává potřebu vývoje nových terapeutik. Katepsin B1 (SmCB1) je klíčovou trávicí proteasou krevničky střevní (Schistosoma mansoni) a představuje cílovou molekulu pro terapeutický zásah. Tato práce podává komplexní charakterizaci SmCB1 zaměřenou na vztah struktury a aktivity a na inhibiční regulaci s využitím šesti vyřešených krystalografických struktur molekulárních forem a komplexů SmCB1. SmCB1 je syntetizován ve formě neaktivního zymogenu, ve kterém N-koncový propeptid působí jako přirozený intramolekulární inhibitor blokující aktivní místo. Detailní biochemickou a strukturní analýzou byl identifikován nový unikátní mechanismus procesu aktivace zymogenu, při kterém je propeptid odštěpen, a byla popsána regulace tohoto procesu pomocí glykosaminoglykanů. Studium proteolytické aktivity SmCB1 prokázalo, že se jedná o enzym působící jako endopeptidasa i exopeptidasa a představuje tak účinný nástroj pro trávení hemoglobinu hostitele. Významná část práce se...Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni

Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host..

Digestive cysteine proteases of the blood fluke parasite Schistosoma mansoni

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

The effect of private law re-codification on property and transfer taxation of immovables

The thesis engages in a influence of re-codification of the Civil Law on the property taxation and transfers of real estates. This thesis deals with legal tax changes which effect property transfers and passages of real estates. I have included to the new regulation Private Law in the new Civil Code which has come into effect from January 1 st, 2014. I pay attention especially to a concept and a definiton of real estates, an institute of property and other ways of use of real estates. In respect of the subject the text mentions the current system of taxes in the Czech Republic. The thesis focuses especially on the legal adjustment real estates tax that taxes the property to the real estates, value added tax, income tax and tax on real estate acquisition which tax transactions with real estates. I would mark as the main chapter the tax on real estate acquisition that has substituted the last property transfer tax. In the ending of the thesis there is introduced the comparison of taxes relevant to real estates in foreign legal adjustement. The thesis is treated upon the valid legal adjustment at the date of May 30, 2014

International adoption in the Czech Republic

Diplomová práce se zabývá problematikou mezinárodního osvojení a jeho aktuální právní úpravou v České republice. Za nejvýznamnější náhradní rodinnou péči je považováno osvojení, institut rodinného práva. Pokud se nepodaří pro dítě najít vhodnou rodinu ve státě původu, nastupuje mezinárodní osvojení. Zprostředkování osvojení do ciziny má v České republice na starost Úřad pro mezinárodněprávní ochranu dětí se sídlem v Brně.Katedra soukromého práva a civilního procesuObhájenoThis thesis deals with international adoption and current legislation in Czech Republic. An adoption, the institute of the family law is consideredthe most important alternative family care. If it is not find a suitable family for a child in the state of his origin, then it is enteringan international adoption. In the Czech Republic there is the Office for International Legal Protection of Children in Brno, that enters adoptions abroad

Digestive cysteine proteases of the blood fluke parasite Schistosoma mansoni

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

ProlyL oligopeptidase from the blood fluke schistosoma mansoni: from functional analysis to anti-schistosomal inhibitors

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs

Druggable hot spots in the schistosomiasis cathepsin B1 target identified by functional and binding mode analysis of potent vinyl sulfone inhibitors

© 2020 American Chemical Society. Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1′ and S2 subsites. The most potent inhibitor targets the S1′ subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis

Druggable Hot Spots in the Schistosomiasis Cathepsin B1 Target Identified by Functional and Binding Mode Analysis of Potent Vinyl Sulfone Inhibitors

© 2020 American Chemical Society. Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1′ and S2 subsites. The most potent inhibitor targets the S1′ subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis