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Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

ABSTRACT: The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic acid, and are specifically bound to the ADP-ribose binding site of nsP3MD. Among the fragments, 2-oxo-5,6-benzopyrimidine-4-carboxylic acid showed anti-CHIKV activity with an IC50 of 23 ?M. Our fragment-based drug discovery approach provides valuable information to further develop a specific and potent nsP3 inhibitor of CHIKV viral replication based on the 2-pyrimidone-4-carboxylic acid scaffold. In silico studies suggest this pyrimidone scaffold could also bind to the macrodomains of other alphaviruses and coronaviruses and thus, have potential pan-antiviral activity.

SUBMITTER: Zhang S

PROVIDER: S-EPMC7822648 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

Zhang Sixue S Garzan Atefeh A Haese Nicole N Bostwick Robert R Martinez-Gzegozewska Yohanka Y Rasmussen Lynn L Streblow Daniel N DN Haise Mark T MT Pathak Ashish K AK Augelli-Szafran Corinne E CE Wu Mousheng M

PloS one 20210122 1

The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic aci ...[more]

PMID: 33482665

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Project description:Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.

Project description:UnlabelledAll coronaviruses encode a macrodomain containing ADP-ribose-1"-phosphatase (ADRP) activity within the N terminus of nonstructural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reductions in lethality, weight loss, viral titers, proinflammatory cytokine and chemokine expression, and immune cell infiltration in the brain compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 postinfection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus as all N1347A virus-infected mice survived the infection. However, the adaptive immune system was required for protection since N1347A virus was able to cause lethal encephalitis in RAG1(-/-) (recombination activation gene 1 knockout) mice although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis.ImportanceCoronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1"-phosphatase activity; however, its function during infection remains unclear as does the reason that coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies and may give further insight into the role of macrodomain proteins in the host cell since these proteins are found in all living organisms.

Project description:BACKGROUND:Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear. METHODOLOGY / PRINCIPAL FINDINGS:Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti. CONCLUSIONS / SIGNIFICANCE:The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies.

Dataset Information

Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

Publications

Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

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