Implementation of a crystallographic fragment screening pipeline to advance preclinical drug discovery efforts toward the development of COVID-19 antivirals

Despite the ever-present possibility of a global viral pandemic, the world was caught off guard by the sudden appearance and rapid spread of SARS-CoV-2. Given the virulence and infectivity of the virus, an immediate global response is required to meet the current and any future coronavirus pandemics. While classical vaccinations are perhaps the ideal form of intervention, they are not always sufficient to deliver long term protection. Small molecular drugs targeting multiple non-structural proteins (NSPs), however, have already been proven to be efficient treatments in the case of RNA viruses such as HCV, but there are no effective treatments for coronaviruses currently available. Therefore, development of multi-target small molecule drugs is needed to combat current, recurring and future CoVs. Although the development cycle for small molecule drug discovery is relatively long (5-15 years), recent advances in high-throughput crystallography could expedite the structure-guided drive to lead compounds and high-affinity drug-like inhibitors. The power of HT crystallography has been convincedly demonstrated by the very recent international efforts in structure determinations of NSPs of SARS-CoV-2. X-ray structures of nine of 15 NSPs have been solved and ~100 fragment-bound main proteinase structures have determined in just a few months. However, the structure of the RNA-helicase (NSP13) - a central component of the viral replication organelle that is essential for RNA synthesis remains elusive, probably because of its multi-domain architecture. The main applicants have been working for years in the structural and functional study of helicases. We determined the very first CoV RNA helicase structure - MERS-CoV NSP13 - in 2017, and three more RNA helicase structures from other RNA viruses in the following years. Since the outbreak of Covid-19, we have naturally geared our research effort towards SARS-CoV-2 RNA helicase, aiming to make a unique contribution to the international efforts in developing multi-target drugs against SARS-CoV-2.This proposal aims to reveal novel binding sites in multiple enzymatic states of NSP13 by a multi-channel approach that is designed to utilize macromolecular crystallography methods both at cryogenic and room-temperatures. The first of these channels is to determine high-resolution X-ray structures of NSP13 in various enzymatic states. The second channel is to assess, through biophysics and X-ray crystallography, if any currently available inhibitors also inhibit SARS-CoV-2. The third channel is to attempt to discover novel molecular entities with anti-viral properties. Fragment-screening, both under cryo (100 K) and at more physiological temperatures (293 K), has the potential to explore comprehensively the molecular space for both direct and allosteric inhibition of NSP13 in different conformational states. The information gained from these three channels will feed into one another, for example an understanding of inhibitor binding can aid the stabilization of other enzymatic states, or fragment binding data can inform the improvement of existing inhibitors.To tackle this ambitious project, we have assembled a unique team with broad expertise from structural virology to advanced macromolecular crystallography. The key to the success of this proposal is fast and regular access to large scale X-ray crystallographic infrastructure, and the Paul Scherrer Institut is ideally placed to carry out the central aspects of the proposed research. Given the similarity of coronaviruses, the technologies and methods developed in this proposal will facilitate and expedite a response to any future pandemic.