Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro.

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

Targeting RNA G-Quadruplex in SARS-CoV-2: A Promising Therapeutic Target for COVID-19?

The COVID-19 pandemic caused by SARS-CoV-2 has become a global threat. Understanding the underlying mechanisms and developing innovative treatments are extremely urgent. G-quadruplexes (G4s) are important noncanonical nucleic acid structures with distinct biofunctions. Four putative G4-forming sequences (PQSs) in the SARS-CoV-2 genome were studied. One of them (RG-1), which locates in the coding sequence region of SARS-CoV-2 nucleocapsid phosphoprotein (N), has been verified to form a stable RNA G4 structure in live cells. G4-specific compounds, such as PDP (pyridostatin derivative), can stabilize RG-1 G4 and significantly reduce the protein levels of SARS-CoV-2 N by inhibiting its translation both in vitro and in vivo. This result is the first evidence that PQSs in SARS-CoV-2 can form G4 structures in live cells, and that their biofunctions can be regulated by a G4-specific stabilizer. This finding will provide new insights into developing novel antiviral drugs against COVID-19.

Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design.

COVID-19, caused by SARS-CoV-2, has resulted in severe and unprecedented economic and social disruptions in the world. Nucleocapsid (N) protein, which is the major structural component of the virion and is involved in viral replication, assembly and immune regulation, plays key roles in the viral life cycle. Here, we solved the crystal structures of the N- and C-terminal domains (N-NTD and N-CTD) of SARS-CoV-2 N protein, at 1.8 and 1.5 Å resolution, respectively. Both structures show conserved features from other CoV N proteins. The binding sites targeted by small molecules against HCoV-OC43 and MERS-CoV, which inhibit viral infection by blocking the RNA-binding activity or normal oligomerization of N protein, are relatively conserved in our structure, indicating N protein is a promising drug target. In addition, certain areas of N-NTD and N-CTD display distinct charge distribution patterns in SARS-CoV-2, which may alter the RNA-binding modes. The specific antigenic characteristics are critical for developing specific immune-based rapid diagnostic tests. Our structural information can aid in the discovery and development of antiviral inhibitors against SARS-CoV-2 in the future.

Autophagy involved in antiviral activity of sodium tanshinone IIA sulfonate against porcine reproductive and respiratory syndrome virus infection in vitro.

BACKGROUND: In previous research, we have demonstrated that sodium tanshinone IIA sulfonate (STS) has anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity, but whether autophagy is involved in this process is still unknown. In this study, the autophagy effect of STS against PRRSV infection was investigated in vitro. METHODS: Quantitative real-time PCR (qRT-PCR) and western blot was used to evaluate the inhibition ability of STS on the mRNA expression levels on cell autophagy genes, that is Beclin1, ATG5 and ATG7. Simultaneously, the effect of STS on N protein/gene expression was assessed by indirect immuno-fluorescence assay (IFA), qRT-PCR and western blot. RESULTS: The results indicated that STS inhibits autophagy induced by PRRSV. In addition, STS effectively suppresses PRRSV's N protein replication and N gene expression in Marc-145 cells infected with PRRSV in a time-dependent manner. CONCLUSIONS: Our results suggest that STS exhibits anti-PRRSV activity in vitro by suppressing autophagy-related genes, which may provide a theoretical basis for further pharmacological agent development regarding PRRSV infection.

Structural basis for the identification of the N-terminal domain of coronavirus nucleocapsid protein as an antiviral target.

Coronaviruses (CoVs) cause numerous diseases, including Middle East respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. CoVs encode the nucleocapsid (N) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral RNA through the N protein's N-terminal domain (N-NTD). Using human CoV-OC43 (HCoV-OC43) as a model for CoV, we present the 3D structure of HCoV-OC43 N-NTD complexed with ribonucleoside 5'-monophosphates to identify a distinct ribonucleotide-binding pocket. By targeting this pocket, we identified and developed a new coronavirus N protein inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)(N,N-dimethylamino)acetamide hydrochloride (PJ34), using virtual screening; this inhibitor reduced the N protein's RNA-binding affinity and hindered viral replication. We also determined the crystal structure of the N-NTD-PJ34 complex. On the basis of these findings, we propose guidelines for developing new N protein-based antiviral agents that target CoVs.

[Effect of the antiviral drug Ingaviruin on intracellular transformations and import into the nucleus of influenza A virus nucleocapsid protein].

The paper presents the results of studying the effect of the antiviral drug Ingavirin on different stages of intracellular transformations of influenza A virus nucleocapsid protein (NP). Ingavirin 400-1000 microg/ml has been found to impair the biogenesis of influenza virus NP, to lower the efficiency of formation of conformationally mature compact NP oligomers, and to retard the migration of newly-synthesized NP from the cytoplasm to the nucleus. It is shown that there is an association of tritium-labeled Ingavirin with the nuclear membranes of MDCK cells. The investigations of the mechanisms of antiviral activity of Ingavirin are not only important for the characterization of this drug, but also promote the detection of potential targets to design novel antiviral agents.

Targeting the viral nucleocapsid protein in anti-HIV-1 therapy.

The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.

RSV604, a novel inhibitor of respiratory syncytial virus replication.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein.

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.

The HIV-1 nucleocapsid zinc finger protein as a target of antiretroviral therapy.

Despite advances made in its therapeutic management, human immunodeficiency virus (HIV) infection has remained an intractable problem, and complete eradication of the virus an unrealized goal. Experience in the clinical application of combination therapy using a variety of reverse transcriptase and protease inhibitors have revealed a number of challenges, in spite of the observed albeit temporary success in reduction of patient viral loads. Problems with current protocols include poor patient compliance, and the presence of latent reservoirs of virus that ultimately result in the appearance of phenotypic resistance. These considerations necessitate continued research and development into alternative strategies to circumvent the aforementioned problems. One approach to minimizing and/or eliminating the appearance of escape mutants and latent viral reservoirs is the targeting of essential and mutationally intolerant enzymes such as the nucleocapsid protein, which contains two highly conserved zinc knuckles. Concerns have been raised regarding the targeting of this protein, since the ubiquitous occurrence of important mammalian zinc finger proteins implies that drug specificity towards the nucleocapsid protein may be difficult to attain. In this review, strong evidence supporting the hypothesis that this protein can be targeted to the exclusion of other cellular zinc finger proteins is presented. The effects of small molecule induced abrogation of nucleocapsid protein mediated activities, as well as efforts to develop nucleocapsid protein inhibitors as antiretrovirals are also discussed.

2,2'-Dithiobisbenzamides and 2-benzisothiazolones, two new classes of antiretroviral agents: SAR and mechanistic considerations.

Substituted 2,2'-dithiobisbenzamides and 2-benzisothiazolones were prepared and shown to possess low microM activity with high therapeutic indices against HIV-1, HIV-2 and SIV in cell culture. The mechanism of antiviral action was determined to be directed toward the nucleocapsid protein (NCp7), which contains two zinc fingers and plays vital roles in the viral life cycle. The "active sulfides" of this study cause the extrusion of zinc from these zinc fingers. Structure-activity relationships of the 2,2'-dithiobisbenzamides reveal that the disulfide bond and the ortho benzamide functional groups are essential for activity, with the best compounds having a carboxylic acid, carboxamide, or sulfonamide substituent. The 2-benzisothiazolones are formed from the disulfides both chemically and in vivo and their SAR mimics that of the 2,2'-dithiobisbenzamides. The antiviral activity of the disulfides may require cyclization to the isothiazolones. Two agents, PD 159206 and PD 161374, which showed good antiviral activity, physical properties, and excellent pharmacokinetics in mice, were selected for advanced studies.