Structural biology of flavivirus NS1 protein and its antibody complexes.

The genus of flavivirus includes many mosquito-borne human pathogens, such as Zika (ZIKV) and the four serotypes of dengue (DENV1-4) viruses, that affect billions of people as evidenced by epidemics and endemicity in many countries and regions in the world. Among the 10 viral proteins encoded by the viral genome, the nonstructural protein 1 (NS1) is the only secreted protein and has been used as a diagnostic biomarker. NS1 has also been an attractive target for its biotherapeutic potential as a vaccine antigen. This review focuses on the recent advances in the structural landscape of the secreted NS1 (sNS1) and its complex with monoclonal antibodies (mAbs). NS1 forms an obligatory dimer, and upon secretion, it has been reported to be hexametric (trimeric dimers) that could dissociate and bind to the epithelial cell membrane. However, high-resolution structural information has been missing about the high-order oligomeric states of sNS1. Several cryoEM studies have since shown that DENV and ZIKV recombinant sNS1 (rsNS1) are in dynamic equilibrium of dimer-tetramer-hexamer states, with tetramer being the predominant form. It was recently revealed that infection-derived sNS1 (isNS1) forms a complex of the NS1 dimer partially embedded in a High-Density Lipoprotein (HDL) particle. Structures of NS1 in complexes with mAbs have also been reported which shed light on their protective roles during infection. The biological significance of the diversity of NS1 oligomeric states remains to be further studied, to inform future research on flaviviral pathogenesis and the development of therapeutics and vaccines. Given the polymorphism of flavivirus NS1 across sample types with variations in antigenicity, we propose a nomenclature to accurately define NS1 based on the localization and origin.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein.

Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we obtained infection-derived sNS1 (isNS1) from dengue virus (DENV)-infected Vero cells through immunoaffinity purification instead of recombinant sNS1 (rsNS1) overexpressed in insect or mammalian cell lines. We found that isNS1 appeared as an approximately 250 kDa complex of NS1 and ApoA1 and further determined the cryoEM structures of isNS1 and its complex with a monoclonal antibody/Fab. Indeed, we found that the major species of isNS1 is a complex of the NS1 dimer partially embedded in a high-density lipoprotein (HDL) particle. Crosslinking mass spectrometry studies confirmed that the isNS1 interacts with the major HDL component ApoA1 through interactions that map to the NS1 wing and hydrophobic domains. Furthermore, our studies demonstrated that the sNS1 in sera from DENV-infected mice and a human patient form a similar complex as isNS1. Our results report the molecular architecture of a biological form of sNS1, which may have implications for the molecular pathogenesis of dengue.

SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.