Hit-to-lead optimisation of fragment hits targeting SARS-CoV-2 non structural protein 10 using structure-based drug design

Coronaviruses have probably haunted humans for several centuries. They are responsible for approximately 25% of all cases of the common cold. In the last 20 years there have been various coronavirus outbreaks representing serious threats such as the SARS outbreak in China with 8000 infections and a mortality rate of ca. 10%. This was followed by another outbreak occurring in Saudi Arabia in 2012 and South Korea in 2015 causing ca. 2500 infections so far with a mortality rate of 34%. This novel coronavirus was named MERS, as it originated from the Middle East. The most recent novel coronavirus, named SARS-CoV-2 that is causing the current Covid-19 pandemic has caused more than 550 million infections and more than 6.2 million deaths worldwide. However, unofficial estimates report much higher infection and mortality rates. It is difficult to predict whether we will face further coronavirus peak this winter and beyond, and how much the virus will mutate and escape the protection afforded by vaccines. The risk is also high that additional coronavirus outbreaks will occur in the future, through transmission of related viruses from animals to humans. It is therefore important to study coronaviruses to understand how they infect humans and how these infections can be treated or prevented, either by the development of vaccines or the development of medications such as small molecules that bind to and inhibit coronavirus proteins, and prevent them from multiplying in the human body.

SARS-CoV-2 has more than 25 distinct proteins, which can be divided into three distinct classes. One class of proteins are called the non-structural proteins, and these are involved in creating many new copies of the virus and infection of other humans. We therefore decided to work on one of the non-structural proteins, named non-structural protein 10 (nsp10). Although a relatively small protein it activates at least two other non-structural viral proteins (nsp14 and nsp16), without which the virus cannot multiply. By blocking nsp10, we can equally block these two other viral proteins, and the virus ceases to be 'viable'.

In this project we propose to build upon our previous work to develop inhibitors that bind to and block the action of nsp10. In the future, these inhibitors could be combined with other drugs that bind to and block other proteins present in SARS-CoV-2, leading to even more potent drug combinations. With this approach, we hope to not only combat SARS-CoV-2 but also closely related novel coronaviruses that may cause new outbreaks in the foreseeable future.

SARS-CoV-2, a new coronavirus that causes Covid-19, is responsible for the recent pandemic that has had widespread public health, societal and economic impacts across the globe. Vaccines were developed rapidly and have a strong protective effect in the first few months after administration. However, a significant proportion of the human population is not yet vaccinated and some clinically vulnerable patients are ineligible for vaccination. Treatment development through drug repurposing has had mixed outcomes; dexamethasone has been shown to reduce mortality in patients with severe Covid-19, but large trials with remdesivir showed little benefit. Recent clinical studies with molnupiravir and nirmatrelvir/ritonavir, appear to be effective when administered early after symptom onset. There is clearly an urgent need to develop new antiviral drugs by targeting specific SARS-CoV-2 proteins, to address the current pandemic and prepare for future coronavirus outbreaks.

In this proposal we target non-structural protein 10 (nsp10), which is part of the Replication-Transcription Complex and stimulates the activities of two other essential viral proteins, nsp14 and nsp16. Consequently, targeting nsp10 has been shown to stall replication leading to non "viable" virus. We identified a series of fragment hits using state of the art fragment-based screening via x-ray crystallography and verified nsp10 binding using orthogonal biophysical techniques. An initial SAR-by-catalogue study coupled with structure-based drug design is currently in progress, using fragment growing, linking and merging techniques. We aim to generate potent fragment analogues with balanced drug-like properties and efficacy in cellular SARS-CoV-2 models through a program of chemical synthesis and biological evaluation. By including MERS and SARS-CoV-1 coronavirus nsp10s in our analysis, our hits may also be optimised against other medically relevant coronaviruses and therefore be used in future outbreaks.