What is the problem/issue being addressed?
Enteroviruses are a large group of non-enveloped viruses which contains many important pathogens for humans. Enterovirus genomes encodes four structural proteins (VP1-4) and seven non-structural proteins (2A-C, 3A-D). The enterovirus 2C protein is a particularly attractive target for broad-spectrum antiviral development because it performs several essential roles in the virus lifecycle. However, a molecular-level understanding of these functions is lacking. In addition, several molecules have been suggested to target and inhibit 2C, but the mechanism-of-action of these compounds is not understood, hampering their further development into antiviral drugs. Efforts to understand the antiviral inhibition of 2C requires an in-depth knowledge of its structure and function. Unfortunately, the functional oligomeric form of 2C has been notoriously difficult to study due to its poor biochemical properties. In this project, researchers will develop an oligomeric and enzymatically active 2C protein and use this to study the structure, function, and inhibition of the hexameric complex.
Why is it important for society?
The genus Enterovirus comprises many clinically relevant human pathogens, such as poliovirus, coxsackievirus, rhinovirus and emerging viruses such as EV-A71 and EV-D68. Diseases associated with these pathogens range from mild illnesses to debilitating, and occasionally life-threatening, conditions such as meningitis, encephalitis, and acute flaccid paralysis. Young children are most at risk of developing severe illness. In addition, seemingly harmless enteroviruses can gain pathogenicity and spread rapidly in the human population. For example, EV-D68, discovered to be a respiratory pathogen in 1962, changed into an acute flaccid paralysis-associated virus causing world-wide outbreaks in 2014. Moreover, a variant of coxsackievirus A24 emerged as a pandemic pathogen and spread worldwide, causing millions of cases of viral conjunctivitis. Vaccination to the hundreds of enterovirus serotypes is not possible and there are currently no licensed antivirals to treat enterovirus-associated diseases. As painfully demonstrated by the COVID-19 pandemic, availability of potent, broad-spectrum antivirals is critically important before the emergence of novel viral pathogens. Therefore, the time to start development of anti-enteroviral therapeutics is now. The structural and functional analysis of the viral 2C protein, described in this project, will facilitate the development of antiviral drugs against enteroviruses.
What are the overall objectives?
This project aims to study the structure, function, and inhibition of the hexameric 2C protein and thus provide a structural roadmap for the development drugs to treat enteroviruses-associated diseases.
Conclusions of the action
In this project, researchers engineered a soluble, hexameric and ATPase competent 2C protein. They used this novel protein construct to show that the compounds fluoxetine, dibucaine, HBB and guanidine hydrochloride all inhibit 2C ATPase activity in a dose-dependent manner. Using cryo-electron microscopy analysis, it was shown that fluoxetine and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing the first three-dimensional reconstruction of the oligomeric complex to be captured. In addition, a high resolution crystal structure of the soluble, monomeric fragment of the 2C protein in complex with fluoxetine was obtained, which revealed a conserved, hydrophobic drug-binding pocket that is distal to the ATP binding site. Alongside this work, researcher also performed structure-activity relationship studies of previously identified 2C targeting compounds to increase their potency and broad-spectrum activity.
