Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- methyltransferasy chemie MeSH
- pandemie MeSH
- RNA MeSH
- SARS-CoV-2 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.
Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action.
- MeSH
- buněčné linie MeSH
- chuť * MeSH
- chuťová percepce * MeSH
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- myši inbrední C57BL fyziologie MeSH
- myši MeSH
- oxid deuteria analýza MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- simulace molekulární dynamiky MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Surfactant protein B (SP-B) is essential in transferring surface-active phospholipids from membrane-based surfactant complexes into the alveolar air-liquid interface. This allows maintaining the mechanical stability of the surfactant film under high pressure at the end of expiration; therefore, SP-B is crucial in lung function. Despite its necessity, the structure and the mechanism of lipid transfer by SP-B have remained poorly characterized. Earlier, we proposed higher-order oligomerization of SP-B into ring-like supramolecular assemblies. In the present work, we used coarse-grained molecular dynamics simulations to elucidate how the ring-like oligomeric structure of SP-B determines its membrane binding and lipid transfer. In particular, we explored how SP-B interacts with specific surfactant lipids, and how consequently SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. Based on these studies, there are specific lipid-protein interactions leading to perturbation and reorganization of pulmonary surfactant layers. Especially, we found compelling evidence that anionic phospholipids and cholesterol are needed or even crucial in the membrane binding and lipid transfer function of SP-B. Also, on the basis of the simulations, larger oligomers of SP-B catalyze lipid transfer between adjacent surfactant layers. Better understanding of the molecular mechanism of SP-B will help in the design of therapeutic SP-B-based preparations and novel treatments for fatal respiratory complications, such as the acute respiratory distress syndrome.
- MeSH
- fosfolipidy chemie MeSH
- konformace proteinů MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- plicní surfaktanty chemie MeSH
- protein B asociovaný s plicním surfaktantem chemie metabolismus MeSH
- simulace molekulární dynamiky MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
