Structure-affinity relationship
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In plants, oxysterols brassinosteroids (BR) and ecdysteroids (ES) can be found. BR act as plant hormones with a positive effect on growth, stress tolerance or senescence. Their mechanism of action is based on signal transduction via a membrane receptor cascade, with or without assistance of oxysterol-binding proteins. The role of ES as insect hormones in plants is unknown. ES are likely to show an antifeedant effect on herbivorous pests, but other as yet unknown roles in plants are not excluded. A range of affinity carriers with immobilized synthetic analogues of plant BS and ES were prepared. Using these carriers, some proteins with affinity to the oxysterols were separated from cytosol extracts of plants. The proteins were assessed by electrophoresis and identified by sequencing. One of them is an osmotin-like protein precursor known as a pathogenesis- related protein. Another protein, RuBisCO, is known as the key enzyme in the Calvine cycle of the dark phase of photosynthesis. The interactions of these proteins with the oxysterols remain unclear.
Acetylcholinesterase (AChE) reactivators (oximes) are generally used as antidotes in case of nerve agent poisoning. Because of their affinity to AChE, they may also act as weak inhibitors of AChE. Their inhibition potency against AChE was determined by an in vitro method based on the interaction between AChE and oxime reactivator in the concentration range 10-1 to 10 -8 M. We used eel AChE for these assays. We found that AChE inhibition strongly depends on the oxime structure. The aim of the present study is to describe the structure-activity relationship (SAR) between oxime structure and inhibition of AChE. AChE reactivators tested include both monoquaternary and bisquaternary structures with the oxime group in different positions on the pyridine ring and with changes in the connecting linker in the case of the bisquaternary compounds. We found AChE inhibition to be highest in bisquaternary oximes that have a longer linker length and have the oxime group in the ortho position. Increased AChE inhibition in monoquaternary oximes was highest when the meta position was occupied by the oxime nucleophile. In addition, different substituents in the connecting chain (in case of bisquaternary oximes) modulated their inhibition potency.
Blood flukes of the genus Schistosoma cause the disease schistosomiasis that infects over 200 million people worldwide. Treatment relies on just one drug, and new therapies are needed should drug resistance emerge. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease that digests host blood proteins as source of nutrients. It is under evaluation as a therapeutic target. Enzymatic activity of the SmCB1 zymogen is prevented by the pro-peptide that sterically blocks the active site until activation of the zymogen to the mature enzyme. We investigated the structure-inhibition relationships of how the SmCB1 pro-peptide interacts with the enzyme core using a SmCB1 zymogen model and pro-peptide-derived synthetic fragments. Two regions were identified within the pro-peptide that govern its inhibitory interaction with the enzyme core: an "active site region" and a unique "heparin-binding region" that requires heparin. The latter region is apparently only found in the pro-peptides of cathepsins B associated with the gut of trematode parasites. Finally, using the active site region as a template and a docking model of SmCB1, we designed a series of inhibitors mimicking the pro-peptide structure, the best of which yielded low micromolar inhibition constants. Overall, we identify a novel glycosaminoglycan-mediated mechanism of inhibition by the pro-peptide that potentially regulates zymogen activation and describe a promising design strategy to develop antischistosomal drugs.
- MeSH
- chromatografie afinitní MeSH
- heparin farmakologie MeSH
- inhibitory proteinkinas chemická syntéza chemie farmakologie MeSH
- katalytická doména účinky léků MeSH
- kathepsin B antagonisté a inhibitory metabolismus MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- peptidové mapování MeSH
- peptidy chemie farmakologie MeSH
- prasata MeSH
- racionální návrh léčiv MeSH
- Schistosoma mansoni enzymologie MeSH
- sekundární struktura proteinů MeSH
- střevní sliznice chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Misfolding of the neuronal protein α-synuclein into amyloid fibrils is a pathological hallmark of Parkinson's disease, a neurodegenerative disorder that has no cure. Inhibition of the fibril growth is considered a promising therapeutic approach. However, the majority of the existing inhibitors are either unspecific or work at high micromolar concentrations. Earlier, we created a protein-based inhibitor of α-synuclein fibril growth that consists of an α-synuclein moiety and a bulky group. It specifically binds to α-synuclein fibril ends and blocks them by creating steric hindrance to subsequent monomer binding. RESULTS: In this work, we prepared a series of inhibitors with modified α-synuclein moieties and bulky groups of different structure, size, and position. We studied the structure-activity relationship of these inhibitors and optimized them by improving affinity to the fibril end and blocking efficiency. The inhibitors were tested in a Thioflavin T-based kinetic assay, and their affinity to the fibril ends was measured by fluorescence anisotropy. We showed that decrease in electrostatic repulsion between inhibitor and fibril end improved the inhibitor efficiency. Inhibitors with rigid β-sheet-rich bulky groups bind to fibril ends stronger than monomeric α-synuclein and therefore have a high inhibition efficiency, showing a linear correlation between Kd and IC50. SIGNIFICANCE: We determined which properties of inhibitor molecules are the most important for good performance and found that the inhibitor affinity to the fibril end is a key feature that determines its inhibition efficiency. Applying this knowledge, we improved existing inhibitors and reached IC50 value of 300 nM.
- MeSH
- alfa-synuklein chemie metabolismus MeSH
- amyloid chemie metabolismus MeSH
- benzothiazoly chemie metabolismus MeSH
- fluorescenční polarizace MeSH
- kinetika MeSH
- lidé MeSH
- proteinové agregáty MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protilátky obsahující pouze dva těžké řetězce (hcAbs – “heavy chain antibodies”) byly objeveny u lam v r. 1993 a později u jednohrbých i dvouhrbých velbloudů. Článek informuje čtenáře o jejich vzniku a výskytu během evoluce, jejich přednostech a aplikačních možnostech v terapii, imunodiagnostice a výzkumu.
Antibodies composed of two heavy chains only, named “heavy chain antibodies” (hcAbs), were discovered at first in llamas in 1993 and found later on in camels and dromedaries. The short review paper informs the readers about their origin and occurrence during evolution, their advantages and application options in therapy, immunodiagnostics and research.
- Klíčová slova
- Camalidae,
- MeSH
- afinita protilátek imunologie MeSH
- biologická evoluce MeSH
- nanočástice diagnostické užití terapeutické užití MeSH
- protilátky bispecifické diagnostické užití terapeutické užití MeSH
- rekombinantní proteiny biosyntéza terapeutické užití MeSH
- specificita protilátek imunologie MeSH
- těžké řetězce imunoglobulinů biosyntéza farmakologie izolace a purifikace MeSH
- vztahy mezi strukturou a aktivitou MeSH
Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.
- MeSH
- biodegradace * MeSH
- fylogeneze MeSH
- izotopové značení metody MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- metagenomika metody trendy MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
Inhibition of glutamate carboxypeptidase II (GCPII) is effective in preclinical models of neurological disorders associated with excessive activation of glutamatergic systems. Here we report synthesis, structural characterization, and biological activity of new hydroxamic acid-based inhibitors with nanomolar affinity for human GCPII. Crystal structures of GCPII/hydroxamate complexes revealed an unprecedented binding mode in which the putative P1' glutarate occupies the spacious entrance funnel rather than the conserved glutamate-binding S1' pocket. This unique binding mode provides a mechanistic explanation for the structure-activity relationship data, most notably the lack of enantiospecificity and the tolerance for bulky/hydrophobic functions as substituents of a canonical glutarate moiety. The in vivo pharmacokinetics profile of one of the inhibitors will be presented along with analgesic efficacy data from the rat chronic constrictive injury model of neuropathic pain.
- MeSH
- antigeny povrchové metabolismus MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory metabolismus MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- kyseliny hydroxamové chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A structure-activity relationship (SAR) study in terms of G-quadruplex binding ability and antiproliferative activity of six fluorescent perylenemonoimide (PMIs) derivatives is reported. A positive charge seems to be the key to target G4. This study also reveals the importance of the element substitution in the potential biological activity of PMIs, being the polyethylene glycol (PEG) chains in the peri position responsible for their antiproliferative activity. Among them, the cationic PMI6 with two PEG chains is the most promising compound since its fluorescence is enhanced in the presence of G-quadruplex structures. Moreover, PMI6 binds to the human telomeric G-quadruplex hTelo with high affinity and displays a high antiproliferative potential towards HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma) and A2780 (ovarian adenocarcinoma) cells. Its fate can be followed inside cells thanks to its fluorescent properties: the compound is found to accumulate in the mitochondria.
- MeSH
- G-kvadruplexy účinky léků MeSH
- imidy chemická syntéza chemie farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mitochondrie účinky léků MeSH
- molekulární struktura MeSH
- perylen analogy a deriváty chemická syntéza chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stable isotope probing (SIP) provides the opportunity to label decomposer microorganisms that build their biomass on a specific substrate. In combination with high-throughput sequencing, SIP allows for the identification of fungal community members involved in a particular decomposition process. Further information can be gained through gene-targeted metagenomics and metatranscriptomics, opening the possibility to describe the pool of genes catalyzing specific decomposition reactions in situ and to identify the diversity of genes that are expressed. When combined with gene descriptions of fungal isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual fungal taxa. Here we describe the use of these methods to explore the cellulolytic fungal community in forest litter and soil.
