Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

General practitioners’ views on (long-term) prescription and use of problematic and potentially inappropriate medication for oldest-old patients—A qualitative interview study with GPs (CIM-TRIAD study)

Background: Potentially inappropriate medication (PIM) is defined as medication with uncertain therapeutic effects and/ or potential adverse drug reactions outweighing the clinical benefits. The prescription rate of PIM for oldest- old patients is high despite the existence of lists of PIM (e. g. the PRISCUS list) and efforts to raise awareness. This study aims at identifying general practitioners' views on PIM and aspects affecting the (long- term) use of PIM. Methods: As part of the CIM- TRIAD study, we conducted semi- structured, qualitative interviews with 47 general practitioners, discussing 25 patients with and 22 without PIM (according to the PRISCUS list). The interview guideline included generic and patient- specific questions. Interviews were digitally recorded and transcribed verbatim. We content analyzed the interviews using deductive and inductive category development. Results: The majority of the general practitioners were not aware of the PRISCUS list. Agents deemed potentially inappropriate from the general practitioners' point of view and the PRISCUS list are not completely superimposable. General practitioners named their criteria to identify appropriate medication for elderly patients (e. g. renal function, cognitive state) and emphasized the importance of monitoring. We identified prescription- (e. g. benzodiazepines on alternative private prescription), medication- (e. g. subjective perception that PIM has no alternative), general practitioner- (e. g. general practitioner relies on specialists), patient( e. g. demanding high- user, positive subjective benefit- risk- ratio) and system- related aspects (e. g. specialists lacking holistic view, interface problems) related to the (long term) use of PIM. Conclusions: While the PRISCUS list does not seem to play a decisive role in general practice, general practitioners are well aware of risks associated with PIM. Our study identifies some starting points for a safer handling of PIM, e. g. stronger dissemination of the PRISCUS list, better compensation of medication reviews, positive lists, adequate patient information, multifaceted interventions and improved communication between general practitioners and specialists