Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory metabolismus MeSH
- Candida parapsilosis enzymologie MeSH
- fungální proteiny antagonisté a inhibitory metabolismus MeSH
- inhibitory proteas chemická syntéza chemie farmakologie MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- peptidomimetika chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
β-secretase (BACE1) has been regarded as a prime target for the development of amyloid beta (Aβ) lowering drugs in the therapy of Alzheimer ́s disease (AD). Although the enzyme was discovered in 1991 and helped to formulate the Aβ hypothesis as one of the very important features of AD etiopathogenesis, progress in AD treatment utilizing BACE1 inhibitors has remained limited. Moreover, in the last years, major pharmaceutical companies have discontinued clinical trials of five BACE1 inhibitors that had been strongly perceived as prospective. In our review, the Aβ hypothesis, the enzyme, its functions, and selected substrates are described. BACE1 inhibitors are classified into four generations. Those that underwent clinical trials displayed adverse effects, including weight loss, skin rashes, worsening of neuropsychiatric symptoms, etc. Some inhibitors could not establish a statistically significant risk-benefit ratio, or even scored worse than placebo. We still believe that drugs targeting BACE1 may still hide some potential, but a different approach to BACE1 inhibition or a shift of focus to modulation of its trafficking and/or post-translational modification should now be followed.
Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
- MeSH
- afinitní značky chemická syntéza chemie účinky záření MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- biotin chemie MeSH
- diazomethan analogy a deriváty chemická syntéza účinky záření MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory chemie MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory enzymů chemická syntéza chemie účinky záření MeSH
- konfokální mikroskopie metody MeSH
- kyseliny polymethakrylové chemie MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie MeSH
- nádorové buněčné linie MeSH
- proteomika metody MeSH
- serinové endopeptidasy chemie MeSH
- ultrafialové záření MeSH
- želatinasy antagonisté a inhibitory chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this work a novel capillary electrophoresis-mass spectrometry (CE-MS) based method was developed and validated for the assay of β-secretase (BACE1) activity as a potential target for Alzheimer's disease (AD) treatment. In contrast with the typically used Förster resonance energy transfer (FRET) assays, an unlabelled decapeptide derived from the amyloid precursor protein BACE1 site with the "Swedish mutation" was used as the substrate. The CE usage enabled the enzymatic reaction to be carried out in as small a volume as 100μL in 60min with sufficient yields of proteolytic product, which was subsequently separated in a bare fused silica capillary using 12.5% acetic acid as a background electrolyte and detected by MS. The limits of detection and quantitation were estimated using the signal to noise ratio to be 5nM (S/N=3) and 15nM (S/N=10), respectively, both being well below the working range for kinetic and inhibition studies. Its applicability for the kinetic study of BACE1 was demonstrated using optimized enzyme assay conditions and the estimated kinetic parameter values were confirmed by classic CE-UV analyses. The method was finally used for the main purpose for which it was developed - to screen BACE1 inhibitors as potential AD therapeutics. The resulting kinetic and inhibition parameters values were compared to those published in the literature, which were almost exclusively obtained by FRET based assays. These comparisons brought up several issues that are further discussed below and favour the application of an unlabelled substrate. The proposed CE-MS based method offers a high-throughput capability for new drug development.
- MeSH
- Alzheimerova nemoc farmakoterapie MeSH
- aspartátové endopeptidasy antagonisté a inhibitory MeSH
- elektroforéza kapilární metody MeSH
- HEK293 buňky MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory proteas analýza terapeutické užití MeSH
- kinetika MeSH
- lidé MeSH
- preklinické hodnocení léčiv metody MeSH
- sekretasy antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Beta-site APP cleaving enzyme1 (BACE1) catalyzes the rate determining step in the generation of Aβ peptide and is widely considered as a potential therapeutic drug target for Alzheimer's disease (AD). Active site of BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein with the help of flap. Currently, there are no marketed drugs available against BACE1 and existing inhibitors are mostly pseudopeptide or synthetic derivatives. There is a need to search for a potent inhibitor with natural scaffold interacting with flap and Asp dyad. This study screens the natural database InterBioScreen, followed by three-dimensional (3D) QSAR pharmacophore modeling, mapping, in silico ADME/T predictions to find the potential BACE1 inhibitors. Further, molecular dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bond throughout the simulation. This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrates have been earlier used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility, and easy permeability through the blood-brain barrier. Our finding will be helpful in identify the potential leads to design novel BACE1 inhibitors for AD therapy.
- MeSH
- algoritmy MeSH
- aspartátové endopeptidasy antagonisté a inhibitory metabolismus MeSH
- biologické přípravky chemie farmakologie MeSH
- enoxaparin farmakologie MeSH
- heparitinsulfát farmakologie MeSH
- inhibiční koncentrace 50 MeSH
- inhibitory enzymů chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- kvantitativní vztahy mezi strukturou a aktivitou * MeSH
- lidé MeSH
- ligandy MeSH
- oligosacharidy chemie MeSH
- preklinické hodnocení léčiv * MeSH
- sekretasy antagonisté a inhibitory metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In our ongoing study focused on Corydalis cava (Fumariaceae), used in folk medicine in the treatment of memory dysfunctions, we have investigated fifteen previously isolated alkaloids for their potential multifunctional activity on Alzheimer's disease (AD) targets. Determination of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition was carried out using a BACE1-Immobilized Enzyme Reactor (IMER) by validating the assay with a multi-well plate format Fluorescence Resonance Energy Transfer (FRET) assay. Seven alkaloids out of fifteen were found to be active, with (-)-corycavamine (3) and (+)-corynoline (5) demonstrating the highest BACE1 inhibition activity, in the micromolar range, in a concentration dependent manner. BACE1-IMER was found to be a valid device for the fast screening of inhibitors and the determination of their potency. In a permeation assay (PAMPA) for the prediction of blood-brain barrier (BBB) penetration, the most active compounds, (-)-corycavamine (3) and (+)-corynoline (5), were found to be able to cross the BBB. Not all compounds showed activity against glycogen synthase kinase-3β (GSK-3β) and casein kinase-1δ (CK-1δ). On the basis of the reported results, we found that some C. cava alkaloids have multifunctional activity against AD targets (prolyl oligopeptidase, cholinesterases and BACE1). Moreover, we tried to elucidate the treatment effectivity (rational use) of its extract in memory dysfunction in folk medicine.
- MeSH
- alkaloidy chemie izolace a purifikace MeSH
- Alzheimerova nemoc MeSH
- aspartátové endopeptidasy antagonisté a inhibitory MeSH
- berberinové alkaloidy chemie izolace a purifikace MeSH
- Corydalis chemie MeSH
- enzymy imobilizované antagonisté a inhibitory MeSH
- hematoencefalická bariéra MeSH
- lidé MeSH
- rekombinantní proteiny MeSH
- sekretasy antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer disease (AD) is the most common cause of progressive dementia in the elderly population, with prevalence of 5% after 65 year of age and is increasing to about 30% in people over 85 year. AD is a neurodegenerative and incurable disease. Currently, three inhibitors of acetylcholinesterase (AChE), galantamine, donepezil and rivastigmine, and one inhibitor of N-methyl-d-aspartate (NMDA) receptor are available as drugs for amelioration of the disease. Demand to prepare drugs for the therapy providing at least relieve of symptoms remains. In this experiment, the ability of standards (donepezil, galantamine, huperzine A, tacrine and 7-methoxytacrine) and precursors used for synthesis of new AD drugs (l-tryptophan, pyridoxine B6, tryptamine, acridine, chinoline, isochinoline, indole, pyridine and piperidine) to inhibit AChE, BChE and BACE or to have the antioxidant properties were determined. The results were compared using statistical expression of the relationship between the performed tests. In this experiment, IC50 for every one method and every compound were found. Beside this, prediction of free energy in a link to ln(IC50) was assessed using in silico tests. This article focuses on possibility to find the most suitable chemical precursors to be used in the next development of drugs for AD
- MeSH
- akridiny farmakologie MeSH
- Alzheimerova nemoc * farmakoterapie MeSH
- antioxidancia farmakologie MeSH
- aspartátové endopeptidasy antagonisté a inhibitory MeSH
- chinoliny farmakologie MeSH
- cholinesterasové inhibitory farmakologie MeSH
- farmaceutická chemie * metody statistika a číselné údaje MeSH
- indoly farmakologie MeSH
- isochinoliny farmakologie MeSH
- neurodegenerativní nemoci farmakoterapie MeSH
- piperidiny farmakologie MeSH
- počítačová simulace MeSH
- pyridiny farmakologie MeSH
- pyridoxin farmakologie MeSH
- sekretasy antagonisté a inhibitory MeSH
- techniky in vitro statistika a číselné údaje MeSH
- tryptaminy farmakologie MeSH
- tryptofan farmakologie MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K(i) of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- Candida enzymologie MeSH
- fungální proteiny antagonisté a inhibitory chemie MeSH
- inhibitory HIV-proteasy chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- ritonavir chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We previously found that endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).
- MeSH
- antagonisté endotelinového receptoru A MeSH
- aspartátové endopeptidasy antagonisté a inhibitory metabolismus MeSH
- časové faktory MeSH
- cyklické peptidy farmakologie MeSH
- endotelin-1 analogy a deriváty farmakologie MeSH
- financování organizované MeSH
- glykopeptidy farmakologie MeSH
- inhibitory proteas farmakologie MeSH
- kardiomyocyty enzymologie metabolismus účinky léků MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- malondialdehyd metabolismus MeSH
- metaloendopeptidasy antagonisté a inhibitory metabolismus MeSH
- novorozená zvířata MeSH
- peptidové fragmenty farmakologie MeSH
- potkani Sprague-Dawley MeSH
- receptor endotelinu A metabolismus MeSH
- superoxiddismutasa metabolismus MeSH
- vápníková signalizace účinky léků MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
