The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.
- MeSH
- antivirové látky * farmakologie chemie MeSH
- fluorometrie metody MeSH
- lidé MeSH
- piperidiny farmakologie MeSH
- pyridiny MeSH
- pyrimidiny MeSH
- pyrroly MeSH
- RNA čepičky metabolismus MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory metabolismus MeSH
- rychlé screeningové testy * metody MeSH
- virové proteiny * antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Precancerous lesions of human cervix uteri have a tendency for regression or progression. In cervical intraepithelial neoplasia grade 2 (CINII) case there is an uncertainty if a lesion will progress or regress. The carbonic anhydrase IX (CAIX) enzyme is overexpressed in cervical cancer which is more sensitive to radiotherapy. CAIX is associated with poor prognosis in solid hypoxic tumors. The aim of this study was to determine factors related to elevated soluble CAIX (s-CAIX) in high-grade intraepithelial lesion (HSIL) cases.METHODS: Patients diagnosed with HSIL (N = 77) were included into the research group whereas without HSIL (N = 72)-the control group. Concentration of the soluble CAIX (s-CAIX) in plasma was determined by the DIANA ligand-antibody-based method. C. trachomatis was detected from cervical samples by PCR. Primary outcomes were risk factors elevating s-CAIX level in HSIL group. Non-parametric statistical analysis methods were used to calculate correlations. RESULTS: The s-CAIX level in patients with HSIL was elevated among older participants (rs = 0.27, p = 0.04) and with C. trachomatis infection (p = 0.028). Among heavy smokers with HSIL, the concentration of s-CAIX was higher in older women (rs = 0.52, p = 0.005), but was not related to the age of heavy smokers' controls (τ = 0.18 p = 0.40). CONCLUSION: The concentration of s-CAIX was higher among older, heavy smoking and diagnosed with C. trachomatis patients. All these factors increased the risk for HSIL progression.
The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.
- MeSH
- antigeny nádorové genetika MeSH
- biotest * MeSH
- DNA účinky léků genetika MeSH
- inhibitory karboanhydras izolace a purifikace terapeutické užití MeSH
- karboanhydrasa IX antagonisté a inhibitory genetika MeSH
- katalytická doména účinky léků MeSH
- léčivé přípravky MeSH
- lidé MeSH
- rychlé screeningové testy * MeSH
- simulace molekulového dockingu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Posuzujeme-li rozvoj jakéhokoliv oboru v historických souvislostech, nelze oddělit Českou a Slovenskou republiku. Společných 75 let (kromě období 1939–1945) charakterizuje úzká spolupráce českých a slovenských univerzit a vědeckých ústavů, a radiobiologie nepředstavovala výjimku – proto hovoříme o československé radiobiologii. Ta ve své historii prošla třemi významnými etapami, ovlivněnými i světovou politicko-vojenskou situací, a je nutné zdůraznit, že její výsledky byly a jsou srovnatelné se zahraničními pracovišti. První etapa mezi roky 1895–1939 přinesla poznání základních mechanismů a důsledků působení ionizujícího záření na organismus. Druhá etapa v letech 1939–1990 představovala období rozvoje mírového využití atomové energie, ale i jejího zneužití pro vojenské účely. Podrobně se studovala nemoc z ozáření, ochrana před radiačním poškozením a možnosti radioprotekce. Převážná většina výsledků zůstávala utajena a nebylo možné je publikovat. Třetí etapa po roce 1990 posunuje radiobiologii k podrobnému studiu poradiačních změn na intracelulární úrovni s cílem zefektivnění radioterapie a ochrany před zářením, a to rovněž v kontextu nebezpečí zneužití ionizujícího záření teroristy. Zvýšená pozornost se věnuje rovněž vlivu ionizujícího záření na rostliny a možnému následnému přínosu.
If we consider the development of any specialization in our region in an historical context, we cannot separate the Czech and Slovak Republics. The 75 years together (with the exception of 1939-1945) were a period of narrow cooperation between Czech and Slovak Universities and Scientific Institutes, and Radiobiology is no exception, which is why the discussion falls to Czechoslovak Radiobiology, as opposed to Czech Radiobiology alone. Czechoslovak Radiobiology has gone through three important stages in its history, influenced also by international political-military situations. Here it is necessary to emphasize that the results obtained by Czechoslovak Radiobiology were and are comparable with foreign institutions. The first period, which dates back to 1895-1939, represents the initial discovery period of the mechanism and consequences of ionizing radiation on the body. The second period in 1939-1990 is considered to be the phase of peaceful uses of nuclear energy, their misuse for military purposes, along with detailed studies of radiation sickness and radiation protection, the majority of the results which were kept secret and could not be published at the time. The third period after 1990 is dedicated to the detailed study of post-radiation changes at the intracellular level with the aim to be used especially in radiotherapy and radiation protection, as well as the dangers of misuse of ionizing radiation by terrorists. Increased attention is also devoted to the effect of ionizing radiation on plants and their possible subsequent use.
- MeSH
- dějiny 19. století MeSH
- dějiny 20. století MeSH
- ionizující záření MeSH
- jaderná energie MeSH
- lidé MeSH
- radiační poranění MeSH
- radiobiologie * dějiny MeSH
- radioterapie dějiny MeSH
- Check Tag
- dějiny 19. století MeSH
- dějiny 20. století MeSH
- lidé MeSH
- Publikační typ
- historické články MeSH
- Geografické názvy
- Československo MeSH
This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite ("150-cavity") adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported Ki values determined at non-physiological pH should be carefully scrutinized.
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
- MeSH
- antivirové látky chemie farmakologie MeSH
- chřipka lidská farmakoterapie enzymologie virologie MeSH
- DNA chemie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- neuraminidasa antagonisté a inhibitory metabolismus MeSH
- oseltamivir analogy a deriváty chemie MeSH
- preklinické hodnocení léčiv metody MeSH
- reprodukovatelnost výsledků MeSH
- virové proteiny antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků enzymologie fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telerehabilitace využívá informační a komunikační technologie (IKT) k poskytnutí rehabilitace lidem, kteří jsou ve svých domovech, na služebních cestách nebo z jiných důvodů vzdáleni péči odborníků ve zdravotnickém zařízení. Telerehabilitace může znamenat impulz ve výrazném zlepšení klientova přístupu k péči. IKT úspěšně nahrazují fyzický kontakt lékaře s pacientem mimo stěny zdravotnického zařízení. Masově se tak rozšiřuje kontinuita péče pro osoby s hendikepem pohyblivosti. Koncept telepéče spolu s telerehabilitací umožňuje a dovoluje jedinci získat kontrolu nad řízením svých lékařských potřeb. Komunikace s terapeutem umožňuje přizpůsobenou péči, možnost volby a osobní kontrolu. Léčebné programy mají různé možnosti. Využívají dálkové monitorovací systémy, robotické a virtuální technologie. Je zde krátce prezentována historie telerehabilitace a telepéče a nejčastější technologie používané pro dálkové rehabilitační služby. K této problematice jsou prezentovány studie zahraniční i domácí.
Telerehabilitation uses information and communication technology (ICT) to provide rehabilitation to people who are away from healthcare professionals in their homes, on business trips or for other reasons. Telerehabilitation can lead to a significant improvement in client care access. ICT successfully replaces the physician´s physical contact with the patient outside the walls of a healthcare facility. The continuity of care for patients with disabilities is increasing. The telepathic concept along with telerehabilitation allows individuals to gain control over the management of their own medical needs. Communicating with a therapist allows personalized care, and personal control through choice. Therapeutic programs have different options. They use remote monitoring systems, robotic and virtual technologies. This work briefly presents the history of telerehabilitation and telephetics, and the most common technology used for remote rehabilitation services. Foreign and domestic studies are also presented on this issue.
Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.
- MeSH
- biotest * MeSH
- DNA * MeSH
- enzymy metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- objevování léků * MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.
- MeSH
- antigeny povrchové chemie metabolismus MeSH
- glutamátkarboxypeptidasa II chemie metabolismus MeSH
- glutamáty chemie metabolismus MeSH
- karboxypeptidasy chemie metabolismus MeSH
- katalytická doména MeSH
- lidé MeSH
- molekulární struktura MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
