Periodic Reporting for period 1 - EV-2C (Structural and functional studies of enterovirus 2C proteins: promising targets for antiviral therapy.)
Periodo di rendicontazione: 2019-04-01 al 2021-03-31
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.
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.
During this project, an engineering strategy to produce soluble, hexameric 2C proteins was developed. This represents a significant breakthrough in 2C research because a hexameric 2C structure has been pursued for decades but remained elusive due to the unfavourable biochemical properties of the protein. Following an extensive period of optimization, the limitations of the full-length 2C protein were overcome by replacing the insoluble N-terminal region of the protein with a heterologous, soluble hexameric coiled-coil. Compared to monomeric constructs used previously for X-ray crystallography, the engineered 2C hexamer has robust enzymatic activity. Using this engineered 2C protein, researchers were able to show that several compounds inhibit 2C ATPase activity in a dose-dependent manner. Moreover, incubation of the engineered 2C hexamer with fluoxetine or dibucaine demonstrated that these drugs stabilize the oligomeric complex. This allowed researchers to capture the first three-dimensional structure of the 2C hexamer by cryo-EM and provide insights into the inhibitory mechanism for these molecules. Furthermore, researchers were able to obtain a high-resolution crystal structure of the monomeric 2C fragment in complex with fluoxetine, representing the first 2C-inhibtor complex to be reported. The structure reveals a highly conserved hydrophobic pocket, distal to the ATP binding site, into which the fluoxetine trifluoro-phenoxy moiety inserts. Taken together, these data provide new mechanistic insights into the mode-of-action of 2C targeting compounds and offer unique tools for the design and validation of 2C inhibitors. Indeed, the 2C engineering strategy developed in this project is currently being developed further and will jumpstart numerous structural and functional studies of enterovirus 2C proteins. These results have been collated into a scientific manuscript and posted on the bioRxiv preprint server, so that academic and industry researchers can access and utilize these structures and protocols without delay. In addition to the above-mentioned results, researchers also performed structure-activity relationship studies of two previously identified 2C targeting compounds. The resulting compounds were tested for their ability to bind 2C in vitro and their antiviral efficacy was tested against several pathogenic enteroviruses. These novel compounds were shown to have increased antiviral potency and/or increased broad-spectrum activity. The results of these studies have been published as peer-reviewed articles and will promote further investigation into 2C inhibitors.
This work describes the first 3D reconstruction of a hexameric 2C complex and represents a significant step towards understanding this enigmatic protein. Going forward, this engineering strategy should facilitate further structural and functional studies of oligomeric 2C proteins, and their interaction with inhibitors, viral RNA, and other viral/host factors. Conceivably, the engineering strategy developed in this project could be applied to the 2C protein of any current, or emerging pandemic, enterovirus and be used to develop high-throughout, in vitro drug screening assays. Furthermore, the high-resolution crystal structure of the monomeric 2C fragment in complex with fluoxetine is the first 2C-inhibitor complex to be determined. As such, it is a valuable resource for serve in silico screening for novel 2C inhibitors. Ultimately, this basic research described in this project can ultimately lead to development of antiviral medications to treat enterovirus-associated diseases, and thus be of great benefit to society.