Bacterial Neuraminidase Research Articles

Bacterial neuraminidase inhibitory chalcones from flowers of Coreopsis lanceolata, their kinetic characterization and antibiofilm effect

BackgroundBacteria within biofilms are thousand times more resistant to antibiotics. Neuraminidase is a crucial enzyme for bacterial adhesion and biofilm formation, it hydrolyzes glycosidic residue of glycoproteins, glycolipids, and oligosaccharides. Coreopsis lanceolata L. flowers may have a significant potential of bacterial neuraminidase (BNA) inhibition because of high natural abundance of chalcones. PurposeThe investigation of bacterial biofilm inhibitors has emerged as a novel therapeutic strategy against antibiotic resistance. Therefore, individual chalcones were isolated from C. lanceolata and their capacity to inhibit BNA and formation of Escherichia coli biofilm were evaluated. MethodsDifferent chromatographic techniques were used to isolate the compounds (1-12). Enzyme inhibition and detailed kinetic behavior of compounds was determined by estimation of kinetic parameters (Michaelis-Menten constants (Km), maximum velocity (Vmax), dissociation constant for binding with the free enzyme (KI) and enzyme-substate complex (KIS)). Binding affinities (KSV) and binding modes of inhibitors were elucidated by fluorescence quenching and molecular docking, respectively. The natural abundance of chalcones was established through UPLC-Q-TOF/MS. The most potent inhibitor (1) was tested for its ability to inhibit the formation of E. coli biofilm, which was examined by crystal violet assay, scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). ResultsA series of eight chalcones (1-8) and four chalcone glucosides (9-12), inhibited BNA in a dose-dependent manner with IC50 of 8.3 ∼ 77.0 µM. The most potent chalcones were butein (1, IC50 = 8.3 µM) and its glucoside 9 (IC50 = 13.8 µM). The aglycones (1-8) showed non-competitive inhibition, while chalcone glucosides (9-12) displayed a mixed type I (KI < KIS). Inhibitory behaviors were doubly confirmed by KSV and matched with tendency of IC50. The functional group responsible for BNA inhibition were disclosed as 4ʹ-hydroxyl group on B-ring by structure activity relationship (SAR) and molecular docking experiments. Butein (1) suppressed E. coli biofilm formation by > 50 % at 100 µM according to crystal violet assay, which was confirmed by SEM and CLSM imaging. ConclusionThe results showed that chalcones (1-8) and chalcone glucosides (9-12), metabolites isolated from the flowers of C. lanceolata, had BNA inhibitory and antibiofilm formation effect on E. coli.

Surface-Controlled Sialoside-Based Biosensing of Viral and Bacterial Neuraminidases.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein-electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

High-resolution neuraminidase inhibition profiling of Arnebia euchroma (Royle) I.M. Johnst. based on HR-MS and target isolation: An example study of anti-infectious constituents in traditional Chinese medicine

Ethnopharmacological relevanceTraditional Chinese Medicines (TCMs) are an important source to discover new anti-infectious drugs. Neuraminidases (NAs) not only play a key role on human health, but also are promising targets for anti-infectious drugs. Arnebia euchroma which is a widely used traditional Chinese medicine with the effect of cooling blood and detoxifying showed potential inhibitory activities on both bacterial NA and virus NA, suggesting that the material basis of A. euchroma deserves in-depth study. Aim of the studyTo investigate the anti-infectious constituents of A. euchroma based on NA inhibition. Material and methodsA HPLC-DAD system incorporated an auto-sampler was used for micro-fractionation. A nanoliter liquid handler and a high sensitive multimode plate reader system were used for high throughput NA inhibition screening. Thus a high-resolution NA inhibition profiling platform was constructed. The structures of potential active components in A. euchroma obtained by the high-resolution bioassay profiling were identified by DAD and MS in parallel. Then, a target and rapid isolation of NAIs from A. euchroma was achieved under the guidance of the spectrum-effect relationship obtained above. Finally, the isolated compounds were elucidated by extensive spectroscopic methods and their bioactivities were validated by in vitro assay and molecular docking. Results16 potential active ingredients in A. euchroma were isolated and identified, including a new mero-monoterpenoid. The in vitro bioassay results revealed that 12 out of the 16 isolated compounds showed potent inhibitory activities on bacterial NA (IC50s = 1–6 μM) and five of them exhibited potent anti-microbial activities on methicillin-resistant Staphylococccus aureus (MRSA) with MICs in the range of 0.5–4 μg/mL. Furthermore, some isolated compounds showed equal or even better inhibitory activities on oseltamivir resistant viral NA than oseltamivir sensitive NA. The mechanism study in silicon revealed that these natural compounds possessed absolutely different binding modes on the bacterial and viral NAs. ConclusionsOur study gave a clear spectrum-effect relationship of A. euchroma, providing a scientific evidence for future study of the multi-components synergistic effect of TCMs.

Investigation of bacterial neuraminidase inhibition of xanthones bearing geranyl and prenyl groups from Cratoxylum cochinchinense.

Introduction: The root of Cratoxylum cochinchinense has been widely used as Chinese folk medicine to cure fevers, burns, and abdominal complications because it contains various bioactive metabolites such as xanthones, triterpenes, and flavonoids. In this study, we estimated bacterial neuraminidase inhibition with a series of xanthones from C. cochinchinense. BNA has connected to various biological functions such as pathogenic bacteria infection inflammatory process after infection and biofilm formation. Methods: The identification of xanthones (1-6) bearing geranyl and prenyl groups was established by spectroscopic data using UV, IR, NMR, and HREIMS. BNA inhibitory modes of isolated xanthones were investigated by Double-reciprocal plots. Moreover, the competitive inhibitor was evaluated the additional kinetic modes determined by kinetic parameters (k 3, k 4, and K i app). The molecular docking (MD) and molecular dynamics simulations (MDS) studies also provided the critical information regarding the role of the geranyl and prenyl groups against BNA inhibition. Results: A series of xanthones (1-6) appended prenyl and geranyl groups on the A-ring were isolated, and compounds 1-3 were shown to be new xanthones. The analogues within this series were highly inhibited with excellent affinity against bacterial neuraminidase (BNA). A subtle change in the prenyl or geranyl motif affected the inhibitory potency and behavior significantly. For example, the inhibitory potency and binding affinity resulting from the geranyl group on C4: xanthone 1 (IC50 = 0.38μM, KA = 2.4434 × 105 L·mol-1) were 100-fold different from those of xanthone 3 (IC50 = 35.8μM, KA = 0.0002 × 105 L·mol-1). The most potent compound 1 was identified as a competitive inhibitor which interacted with BNA under reversible slow-binding inhibition: K i app = 0.1440μM, k 3 = 0.1410 μM-1s-1, and k 4 = 0.0203 min-1. The inhibitory potencies (IC50) were doubly confirmed by the binding affinities (KA). Discussion: This study suggests the potential of xanthones derived from C. cochinchinense as promising candidates for developing novel BNA inhibitors. Further research and exploration of these xanthones may contribute to the development of effective treatments for bacterial infections and inflammatory processes associated with BNA activity.

Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase.

This study aimed to isolate bacterial neuraminidase (BNA) inhibitory O-methylated quercetin derivatives from the aerial parts of S. pubescens. All the isolated compounds were identified as O-methylated quercetin (1-4), which were exhibited to be noncompetitive inhibitors against BNA, with IC50 ranging from 14.0 to 84.1 μM. The responsible compounds (1-4) showed a significant correlation between BNA inhibitory effects and the number of O-methyl groups on quercetin; mono (1, IC50 = 14.0 μM) > di (2 and 3, IC50 = 24.3 and 25.8 μM) > tri (4, IC50 = 84.1 μM). In addition, the binding affinities between BNA and inhibitors (1-4) were also examined by fluorescence quenching effect with the related constants (KSV, KA, and n). The most active inhibitor 1 possessed a KSV with 0.0252 × 105 L mol-1. Furthermore, the relative distribution of BNA inhibitory O-methylated quercetins (1-4) in S. pubescens extract was evaluated using LC-Q-TOF/MS analysis.

Co-infection of the respiratory epithelium, scene of complex functional interactions between viral, bacterial, and human neuraminidases.

The activity of sialic acids, known to play critical roles in biology and many pathological processes, is finely regulated by a class of enzymes called sialidases, also known as neuraminidases. These are present in mammals and many other biological systems, such as viruses and bacteria. This review focuses on the very particular situation of co-infections of the respiratory epithelium, the scene of complex functional interactions between viral, bacterial, and human neuraminidases. This intrinsically multidisciplinary topic combining structural biology, biochemistry, physiology, and the study of host-pathogen interactions, opens up exciting research perspectives that could lead to a better understanding of the mechanisms underlying virus-bacteria co-infections and their contribution to the aggravation of respiratory pathology, notably in the context of pre-existing pathological contexts. Strategies that mimic or inhibit the activity of the neuraminidases could constitute interesting treatment options for viral and bacterial infections.

X-linked C1GALT1C1 mutation causes atypical hemolytic uremic syndrome.

Hemolytic-uremic syndrome (HUS), mostly secondary to infectious diseases, is a common cause of acute kidney injury in children. It is characterized by progressive acute kidney failure due to severe thrombotic microangiopathy, associated with nonimmune, Coombs-negative hemolytic anemia and thrombocytopenia. HUS is caused mostly by Shiga toxin-producing E. Coli, and to a lesser extent by Streptococcus pneumonia. In Streptococcus pneumonia HUS (pHUS), bacterial neuraminidase A exposes masked O-glycan sugar residues on erythrocytes, known as the T antigen, triggering a complement cascade causing thrombotic microangiopathy. Atypical HUS (aHUS) is a life-threatening genetic form of the disease, whose molecular mechanism is only partly understood. Through genetic studies, we demonstrate a novel X-linked form of aHUS that is caused by a de-novo missense mutation in C1GALT1C1:c.266 C > T,p.(T89I), encoding a T-synthase chaperone essential for the proper formation and incorporation of the T antigen on erythrocytes. We demonstrate the presence of exposed T antigen on the surface of mutant erythrocytes, causing aHUS in a mechanism similar to that suggested in pHUS. Our findings suggest that both aHUS caused by mutated C1GALT1C1 and pHUS are mediated by the lectin-complement-pathway, not comprehensively studied in aHUS. We thus delineate a shared molecular basis of aHUS and pHUS, highlighting possible therapeutic opportunities.

Pyrrolo[2,3-e]indazole as a novel chemotype for both influenza A virus and pneumococcal neuraminidase inhibitors.

Influenza infections are often exacerbated by secondary bacterial infections, primarily caused by Streptococcus pneumoniae. Both respiratory pathogens have neuraminidases that support infection. Therefore, we hypothesized that dual inhibitors of viral and bacterial neuraminidases might be an advantageous strategy for treating seasonal and pandemic influenza pneumonia complicated by bacterial infections. By screening our in-house chemical library, we discovered a new chemotype that may be of interest for a further campaign to find small molecules against influenza. Our exploration of the pyrrolo[2,3-e]indazole space led to the identification of two hit compounds, 6h and 12. These molecules were well-tolerated by MDCK cells and inhibited the replication of H3N2 and H1N1 influenza A virus strains. Moreover, both compounds suppress viral and pneumococcal neuraminidases indicating their dual activity. Given its antiviral activity, pyrrolo[2,3-e]indazole has been identified as a promising scaffold for the development of novel neuraminidase inhibitors that are active against influenza A virus and S. pneumoniae.

Traditional Use, Phytochemical Profiles and Pharmacological Properties of Artemisia Genus from Central Asia.

The flora of Kazakhstan is characterized by its wide variety of different types of medicinal plants, many of which can be used on an industrial scale. The Traditional Kazakh Medicine (TKM) was developed during centuries based on the six elements of ancient Kazakh theory, associating different fields such as pharmacology, anatomy, pathology, immunology and food nursing as well as disease prevention. The endemic Artemisia L. species are potential sources of unique and new natural products and new chemical structures, displaying diverse bioactivities and leading to the development of safe and effective phytomedicines against prevailing diseases in Kazakhstan and the Central Asia region. This review provides an overview of Artemisia species from Central Asia, particularly traditional uses in folk medicine and the recent numerous phytochemical and pharmacological studies. The review is done by the methods of literature searches in well-known scientific websites (Scifinder and Pubmed) and data collection in university libraries. Furthermore, our aim is to search for promising and potentially active Artemisia species candidates, encouraging us to analyze Protein Tyrosine Phosphatase 1B (PTP1B), α-glucosidase and bacterial neuraminidase (BNA) inhibition as well as the antioxidant potentials of Artemisia plant extracts, in which endemic species have not been explored for their secondary metabolites and biological activities so far. The main result of the study was that, for the first time, the species Artemisia scopiformis Ledeb. Artemisia albicerata Krasch., Artemisia transiliensis Poljakov, Artemisia schrenkiana Ledeb., Artemisia nitrosa Weber and Artemisia albida Willd. ex Ledeb. due to their special metabolites, showed a high potential for α-glucosidase, PTP1B and BNA inhibition, which is associated with diabetes, obesity and bacterial infections. In addition, we revealed that the methanol extracts of Artemisia were a potent source of polyphenolic compounds. The total polyphenolic contents of Artemisia extracts were correlated with antioxidant potential and varied according to plant origin, the solvent of extraction and the analytical method used. Consequently, oxidative stress caused by reactive oxygen species (ROS) may be managed by the dietary intake of current Artemisia species. The antioxidant potentials of the species A. schrenkiana, A. scopaeformis, A. transiliensis and Artemisia scoparia Waldst. & Kitam. were also promising. In conclusion, the examination of details between different Artemisia species in our research has shown that plant materials are good as an antioxidant and eznyme inhibitory functional natural source.

Discovery and Characterization of Chemical Compounds That Inhibit the Function of Bacterial Neuraminidase from Codonopsis ussuriensis

Codonopsis ussuriensis (Rupr. and Maxim.) Hemsl is a medicinal herb commonly found in Korea, Japan, and the Russian Far East. However, its chemical composition and bioactivity have not been extensively studied. Thus, in this study, we aimed to examine and characterize the major components of the plant’s roots. Liquid chromatography with high-resolution mass spectrometry (LC-HRMS) was used to identify the components of the crude extracts. The compounds that were identified were named ussurienoside I (1) and tangshenoside I (2). The identified compounds were tested in vitro for inhibitory action against bacterial neuraminidase, and the mechanisms of inhibition were revealed. Compound 1 significantly inhibited bacterial neuraminidase activity in a dose-dependent manner (IC50 = 56.0 μM). The neuraminidase inhibitor (compound 1) exhibited mixed type-I kinetic characteristics. The LC-HRMS data showed that the root extracts contained eight compounds. The findings of this study may aid in the development of bacterial neuraminidase inhibitors with medicinal potential.

New dihydrobenzoxanthone derivatives with bacterial neuraminidase inhibitory activity isolated from Artocarpus elasticus

Artocarpus elasticus is a popular fruit tree in the tropical regions. Primary screenings of methanol extracts of the root bark confirmed its potent inhibition of bacterial neuraminidase (BNA), which plays an essential role in the pathogenesis of many microbial diseases. Assessments of the responsible phytochemicals were conducted by isolating eight compounds (1–8) and two of them (6 and 8) were identified as new compounds. Among the isolates, the dihydrobenzoxanthones attained the highest BNA inhibition with IC50 values of 0.5 ∼ 3.9 µM. Further investigation of the inhibitory mechanism by Lineweaver-Burk plots revealed the phytochemicals to function as reversible noncompetitive inhibitors. Fluorescence quenching showed their binding affinities were highly correlated with their inhibitory potential dose-dependently. Molecular docking experiments suggested the dihydrobenzoxanthones (4 and 6) as noncompetitive inhibitors of BNA with unique interaction with Tyr435 of BNA in comparison with the mother flavonoid (7).

Inhibition of Bacterial Neuraminidase and Biofilm Formation by Ugonins Isolated From Helminthostachys Zeylanica (L.) Hook

Bacterial neuraminidase (BNA) plays a pivotal role in the pathogenesis of several microbial diseases including biofilm formation. The aim of this study is to reveal the neuraminidase inhibitory potential of metabolites from Helminthostachys zeylanica (L.) Hook. which have diverse biological activities including PTP1B and α-glucosidase. The six ugonins (1–6) from the target plant showed significant neuraminidase inhibition. The inhibitory potencies were observed at a nanomolar level of 35–50 nM, which means they are 100 times more active than their corresponding mother compounds (eriodyctiol and luteolin). A detailed kinetic study revealed that all ugonins were reversible noncompetitive inhibitors. An in-depth investigation of the most potent compound 1 showed its time-dependent inhibition with the isomerization model having k 5 = 0.0103 min−1, k 6 = 0.0486 min−1, and K i app = 0.062 μM. The binding affinities (K sv) were agreed closely with our prediction based on the inhibitory potencies. Particularly, ugonin J (1) blocked the biofilm formation of E. coli dose-dependently up to 150 µM without the inhibition of bacteria. The major compounds (1–6) in the extract were characterized by UPLC-ESI-Q-TOF/MS.

Anomeric 1,2,3-triazole-linked sialic acid derivatives show selective inhibition towards a bacterial neuraminidase over a trypanosome trans-sialidase.

Sialic acid is the natural substrate for sialidases and its chemical modification has been a useful approach to generate potent and selective inhibitors. Aiming at advancing the discovery of selective Trypanosoma cruzi trans-sialidase (TcTS) inhibitors, we have synthesised a small series of anomeric 1,2,3-triazole-linked sialic acid derivatives in good yields and high purity via copper-catalysed azide–alkyne cycloaddition (CuAAC, click chemistry) and evaluated their activity towards TcTS and neuraminidase. Surprisingly, the compounds showed practically no TcTS inhibition, whereas ca. 70% inhibition was observed for neuraminidase in relation to the analogues bearing hydrophobic substituents and ca. 5% for more polar substituents. These results suggest that polarity changes are less tolerated by neuraminidase due to the big difference in impact of hydrophobicity upon inhibition, thus indicating a simple approach to differentiate both enzymes. Moreover, such selectivity might be reasoned based on a possible steric hindrance caused by a bulky hydrophobic loop that sits over the TcTS active site and may prevent the hydrophobic inhibitors from binding. The present study is a step forward in exploiting subtle structural differences in sialidases that need to be addressed in order to achieve selective inhibition.

Soybean phytochemicals responsible for bacterial neuraminidase inhibition and their characterization by UPLC-ESI-TOF/MS.

Ethanol extract of soybean (Glycine max (L.) Merr.) showed good inhibitory activity against bacterial neuraminidase (BNA), which plays a pivotal role in the pathogenesis of a number of microbial diseases. The saponin portion fractionated through preparative HPLC (IC50 = 2.25 μg mL-1) was found to be responsible for the observed BNA inhibition. Estimation of the inhibitory effects by individual compounds showed that the soyasaponins of group B (Ba, Bb, Bb', Bc, and Bd) exhibited extremely high inhibitions (IC50 = 0.25-0.48 μM), whereas group A (Aa, Ab, and Ac) was almost inactive. Kinetic studies determined that group B soyasaponins were noncompetitive inhibitors. Furthermore, molecular docking experiments confirmed that soyasaponin Ba (group B) could undergo binding interactions with various residues in the binding pocket. In contrast, soyasaponin Aa (group A) failed to enter the binding pocket due to its extra scaffold structure of oligosaccharides bonded to the 22-hydroxyl position. The metabolites in the soybean extract were fully characterized using UPLC-ESI-TOF/MS.

New Dihydrobenzoxanthone Derivatives with Bacterial Neuraminidase Inhibitory Activity Isolated from Artocarpus Elasticus

New Dihydrobenzoxanthone Derivatives with Bacterial Neuraminidase Inhibitory Activity Isolated from Artocarpus Elasticus

Surface Glycans Regulate Salmonella Infection-Dependent Directional Switch in Macrophage Galvanotaxis Independent of NanH.

Salmonella invades and disrupts gut epithelium integrity, creating an infection-generated electric field that can drive directional migration of macrophages, a process called galvanotaxis. Phagocytosis of bacteria reverses the direction of macrophage galvanotaxis, implicating a bioelectrical mechanism to initiate life-threatening disseminations. The force that drives direction reversal of macrophage galvanotaxis is not understood. One hypothesis is that Salmonella can alter the electrical properties of the macrophages by modifying host cell surface glycan composition, which is supported by the fact that cleavage of surface-exposed sialic acids with a bacterial neuraminidase severely impairs macrophage galvanotaxis, as well as phagocytosis. Here, we utilize N-glycan profiling by nanoLC-chip QTOF mass cytometry to characterize the bacterial neuraminidase-associated compositional shift of the macrophage glycocalyx, which revealed a decrease in sialylated and an increase in fucosylated and high mannose structures. The Salmonella nanH gene, encoding a putative neuraminidase, is required for invasion and internalization in a human colonic epithelial cell infection model. To determine whether NanH is required for the Salmonella infection-dependent direction reversal, we constructed and characterized a nanH deletion mutant and found that NanH is partially required for Salmonella infection in primary murine macrophages. However, compared to wild type Salmonella, infection with the nanH mutant only marginally reduced the cathode-oriented macrophage galvonotaxis, without canceling direction reversal. Together, these findings strongly suggest that while neuraminidase-mediated N-glycan modification impaired both macrophage phagocytosis and galvanotaxis, yet to be defined mechanisms other than NanH may play a more important role in bioelectrical control of macrophage trafficking, which potentially triggers dissemination.

Rapid identification of isoprenylated flavonoids constituents with inhibitory activity on bacterial neuraminidase from root barks of paper mulberry (Broussonetia papyrifera)

This study was to assess the possibility of using competitive and slow binding experiments with affinity-based ultrafiltration UPLC-QTof-MS analysis to identify potent bacterial neuraminidase (bNA) inhibitors from the Broussonetia papyrifera roots extract. To isolate unbound compounds from the enzyme-binding complex, the root bark extracts were either incubated in the absence of bNA, in the presence of bNA, or with the time-dependent bNA before the ultrafiltration was performed. Thirteen flavonoids were separated from the target extract, and their inhibitory activities were tested against bNA. The isolated flavonoids exhibited potent inhibition against NA (IC50 = 0.7–54.0 μM). Our kinetic analysis of representative active flavonoids (1, 2, and 6) showed slow and time-dependent reversible inhibition. Additionally, chalcones exhibited noncompetitive inhibition characteristics, whereas flavonols and flavans showed mixed-type behavior. The computational results supported the experimental behaviors of flavonoids 2, 6, 10, and 12, indicating that bounded to the active site, but flavonoids 6 and 10 binds near but not accurately at the active site. Although this is mixed-type inhibition, their binding can be considered competitive.

Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria\u2013host interactions

Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air–liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases

Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin – glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC50 values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC50 = 10.1 μM for V. cholerae, 13.4 μM for C. perfringens and 402.9 μM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC50 = 101.9 μM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

Syntheses and biological investigations of kirkamide and oseltamivir hybrid derivatives

Abstract The C7N-aminocyclitol kirkamide was recently isolated from the plant obligate symbiont Candidatus Burkholderia kirkii and was hypothesized to be beneficial to the plant host due to its cytotoxic activity against insects and arthropods. To study its mechanism of action and inspired by its structural similarity with N-acetylglucosamine (GlcNAc) and oseltamivir, kirkamide-oseltamivir hybrid derivatives were synthesized and investigated for their biological activity. Interestingly, kirkamide analogues were reasonably potent against a known bacterial neuraminidase.