NECESSITY-New chemical entities modulating SARSCoV2 activity

Enzymes play a pivotal role in the propagation of viruses, their inhibition is a hallmark in established therapies e.g. against HIV-Infection. Among thousands of enzyme binding molecules, only a limited number is suitable as a drug. Under this international proposal three research sites teamed up to take the challenge of finding inhibitors based on the exactly known 3d structure of a SARS-CoV-2 enzyme. Vladimir Cryštof and his team (Olmütz/CZ) synthesize about 9,000 compounds which are tested by Christian Filer at the Helmholtz Center (Berlin/D) via differential X-ray diffraction in a high throughput mode for binding to a SARS-CoV-2 enzyme. In the FWF-funded project part the co-authors and research partners Theresia Dunzendorfer- Matt (Innsbruck Medical University) and Eduard Stefan (University of Innsbruck) will analyze preselected molecules in a reiterative process for interaction and inhibition in test tubes and in cultivated cells. Substances are filtered according to efficacy in vitro, specificity in a biosensor system, and development of resistance and finally screened by the virologist Christian Drosten (Charité Berlin/D). Aim of this project is the allocation of selected molecules as starting material for therapeutic compounds. The designed partially automated pipeline can in principle also be adapted to the development of other drugs. 2022-05-11created by TDMpage 1 1

As part of this project, we have taken an important step toward developing new drugs against coronaviruses so that we are prepared to treat severe cases of infection with established therapies as the number of resistant viruses increases. We have discovered a new drug candidate that specifically binds to a key enzyme of SARS-CoV-2, validated this finding with X-ray structural data, and significantly improved the binding by making targeted modifications to the candidate. Specifically, we screened an in-house, diverse collection of 336 small molecules using X-ray crystallography to identify compounds that bind to the SARS-CoV-2 main protease (the virus's vital "scissors enzyme"). In this way, we identified a previously undescribed molecule that binds highly specifically to the active site of the enzyme and functionally blocks this protein, which is vital for the virus. Based on this structure, we designed and synthesized a small series of targeted variants to strengthen the binding and better understand which regions of the enzyme pocket are particularly important for specific binding. Through this structure-guided optimization, we were able to significantly increase the binding affinity-even to the point of developing variants that are effective at very low concentrations or dosages, an important prerequisite for the development of therapies with few side effects. Our measurements and computer simulations show that for this molecular blockade, it is not only "static" contact points that matter, but above all how stably and coordinately a molecule binds to multiple sites on the enzyme center over time. In particular, the interaction between three adjacent sub regions of the active site is crucial for a sustained binding. This insight helps in designing future drugs specifically to exploit these contact sites simultaneously and stably. Although our optimized drug candidate binds relatively well, it has so far only shown weak inhibition of the enzyme and, for the time being, has had little effect in cells. This is common in early development phases, but it makes it clear that further improvements are needed-for example, even greater utilization of central contact zones or extension into adjacent regions of the enzyme center. Our structural data provides a solid starting point for this. Chemically novel, well-understood binding sites on viral enzymes are valuable-both for refining existing therapies and for preparing for future coronavirus threats. In practical terms, this could lead to more effective, well tolerated, and resilient drugs in the medium term; from a societal perspective, it strengthens pandemic preparedness; and from an economic standpoint, it can accelerate the development of targeted therapies.