Publication in Antiviral Research: Combining of Remdesivir, Molnupiravir and Ribavirin inhibits coronavirus replication

Further to CARE’S SARS-CoV-2 research, CARE partner KU Leuven has explored the inhibitory effect of a combination of 3 broad-spectrum antiviral nucleosides on the replication of coronaviruses as demonstrated through experiments with infected primary human airway epithelial cell (HAEC) cultures and subsequently through testing in SARS-CoV-2 infected hamsters in a prophylactic setup. This work indicates that co-administration of approved drugs for the treatment of coronavirus and other virus infections should be further explored.

To learn more, click here: The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model – ScienceDirect

The COVID-19 QTL-GWAS colocalization explorer application – allowing easy query of summary statistics and visualization of the genomic context of significant COVID-19 gene links

With the aim of understanding host response and prioritising treatment targets, the CARE partner AbbVie sought to identify human genes influencing genetically driven disease risk and severity, and to identify additional high-order phenotypes impacted by pleiotropic COVID-19-associated genomic loci.

The AbbVie team, led by Meri Oliva, published a paper aiming to identify genes and molecular phenotypes related to COVID-19 risk and severity. As part of the work, a web application has been made available at https://covidgenes.shinyapps.io/shiny/. The application allows us to easily query summary statistics and visualize the genomic context of all significant COVID-19 gene links, stratified relevant molecular categories, and aids the identification of COVID-19 causal genes.

Meri Oliva describes the aim of the work: “While many efforts are being devoted to the characterization of the genetic architecture of COVID-19 effects on the human host, its underlying molecular basis has not been exhaustively explored across multiple molecular layers. To understand host response and to prioritize treatment targets, we sought to identify human genes influencing genetically-driven disease risk and severity. To this end, we performed ancestry-aware, trans-layer, multi-omic analyses by integrating recent (April 8, 2022) COVID-19 Host Genetics Initiative GWAS data from six ancestry endpoints – African, Amerindian, South Asian, East Asian, European and meta-ancestry – with functional maps and QTL catalogues.

We explored 91 GWAS hits (P<5e-7), 28% of which were identified in a single ancestry. We analyzed a comprehensive set of >300 cis QTL maps from ~100 biotype sources for colocalization, including disease-relevant biotypes and contexts; blood of COVID-19 patients, large airway epithelium, and lung cell contexts. Across all GWASs, QTL maps and molecular phenotypes, we identified thousands of colocalizations (PP4>0.75) involving >100 genes.

This provided repository hosts a R shiny app to interact with the results of the colocalization analysis.”

To learn more, click here: Integration of GWAS and multi-omic QTLs identifies uncharacterized COVID-19 gene-biotype and phenotype associations | medRxiv

Dr. Daniel Hurdiss, Assistant Professor, Utrecht University

Cryogenic electron microscopy (cryo-EM) capability has been made available to CARE through the expertise of CARE partner, Utrecht University (UU). Virologists from UU’s Biomolecular Health Sciences department use cryo-EM as one of their core techniques to study viruses, under the leadership of Dr. Daniel Hurdiss who has used this method to study virus structures over the last 10 years.

Cryo-EM, or cryogenic electron microscopy, is a powerful imaging technique used to study the structures of biological molecules at high resolution. It involves flash-freezing samples in vitreous ice to preserve their natural state, followed by imaging with an electron microscope, and subsequent computational analysis to determine a three-dimensional structure.

Why Cryo-EM is so important for CARE’s research

The importance of cryo-EM for coronavirus pandemic preparedness and responsiveness cannot be overstated. The first human coronavirus spike structure was obtained using this technique in 2016, and this served as a roadmap for the development of pre-fusion stabilized vaccines. Similarly, this technique was essential to visualize other coronavirus proteins for the first time, such as the RNA-dependent RNA polymerase (RdRp) complex and membrane protein. Within CARE, all these proteins, and more, have been investigated as targets for pharmacological intervention. As such, cryo-EM plays an essential role in understanding the molecular mechanism of candidate antiviral molecules, as well as facilitating their optimization.

Cryo-EM and small molecule development in CARE

In Work Package 2 (Target-based drug discovery and design), cryo-EM was used to study how a macrocyclic peptide inhibitor, developed by UU, binds to the SARS-CoV-2 spike protein. This three-dimensional information is now being used to guide the rational improvement of this molecule.

Cryo-EM was also essential for determining the mechanism of action of a first-in-class SARS-CoV-2 membrane protein-targeting compound, developed by CARE partner KU Leuven. This data can now be used to further improve the potency and breath of these molecules.

In the same vein, Cryo-EM was key in determining the mechanism of action of Bemnofosbuvir, a nucleotide analogue prodrug initially developed against the Hepatitis C virus and repositioned in 2020 against SARS-CoV-2. The drug binds to two independent sites in nsp12, namely the RNA dependent RNA polymerase and the NiRAN domain, opening avenues to further improve the design of novel inhibitors.

Cryo-EM and antibody development in CARE

Cryo-EM structural analysis is a powerful tool for binding epitope mapping of antibodies targeting the viral spike protein and inhibiting SARS-CoV-2 cell entry. In Work Package 4 (Antibody development), many cryo-EM structures of spike-antibody complexes were determined by UU and CHUV. Defining the different epitopes of neutralizing antibodies allows us to understand their mechanism of action and to increase our knowledge of how antibodies can confer protective immunity against SARS-CoV-2 infection. Furthermore, precise knowledge of an antibody’s epitope allows a scientist to select antibody combinations that effectively target the viral spike protein without the antibodies interfering with each other, and to select antibodies that bind to more conserved regions of the spike that are less susceptible to mutations that could confer viral resistance to the neutralizing antibodies.  Computational antibody design is another emerging field that requires structural data to generate therapeutic antibodies with improved potency and neutralizing breadth against the emerging variants of concern.

How might it be used in the future?

Given how important the knowledge derived from structural biology has been prior to, and during, the COVID-19 pandemic, it is safe to assume that this method will continue to be used to provide fundamental insights into the structure and function of viral proteins and facilitate the development of vaccines and antiviral molecules. Conceivably, with the ongoing improvements in AI for small molecule and antibody design, these methods can be used synergistically to design and validate future antiviral therapies. Thinking even further ahead, developments in cryo-EM which allow viral proteins to be visualised inside the infected cell may reveal new druggable targets that can be exploited in the future.

What added value has Cryo-EM brought to CARE?

Beyond the valuable biological insights cryo-EM provides for drug discovery efforts within CARE, these data also provide a wonderful instrument for communicating scientific results and concepts to a wider audience. After all, a picture paints a thousand words.

Published in PLoS Pathogens: picornaviruses modulate nucleotide metabolism

The CARE partner Utrecht University (UU) assessed the modulation of host metabolism by two picornaviruses using steady state as well as 13C-glucose tracing metabolomics. The family Picornaviridae, a large family of small, non-enveloped viruses with a single stranded positive sense RNA genome, includes many well-known human and animal pathogens. Upon infection of their host, these viruses modulate several cellular processes for efficient replication and spreading, such as host cell gene expression, intracellular protein and membrane transport, and cell death pathways. However, little is known about the effects of picornaviruses on cellular metabolism, an important aspect in virus replication. Indeed, viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks to be able to replicate and spread.

UU showed that both coxsackievirus B3 (CVB3), an enterovirus, and encephalomyocarditis, a cardiovirus, increase the levels of pyrimidine and purine metabolites. This increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis.

Moreover, by integrating the metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, UU identified alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of this during infection.

Insight into picornaviral modulation of cellular metabolism is important to increase our understanding of picornavirus-host interactions and may uncover novel therapeutic strategies. This work was important to establish assays and methodology to evaluate the impact of coronaviruses on host cells in CARE.

To learn more, click here: Modulation of nucleotide metabolism by picornaviruses

CARE – Infographic – Work Package 6 – From lead to pre-clinical candidate and proof-of-concept in small-animal and non-human primate models

CARE has 8 Work Packages but do you know what each one does? Here, you can learn about the Work Package 6 team, their objectives, their partners, their breakthrough moments and more.

The infographic is also available here