Licochalcone A plays dual antiviral roles by inhibiting RSV and protecting against host damage.

Respiratory syncytial virus (RSV) causes lower respiratory tract diseases and bronchiolitis in children and elderly individuals. There are no effective drugs currently available to treat RSV infection. In this study, we report that Licochalcone A (LCA) can inhibit RSV replication and mitigate RSV-induced cell damage in vitro, and that LCA exerts a protective effect by reducing the viral titer and inflammation in the lungs of infected mice in vivo. We suggest that the mechanism of action occurs through pathways of antioxidant stress and inflammation. Further mechanistic results demonstrate that LCA can induce nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus, activate heme oxygenase 1 (HO-1), and inhibit reactive oxygen species-induced oxidative stress. LCA also works to reverse the decrease in I-kappa-B-alpha (IкBα) levels caused by RSV, which in turn inhibits inflammation through the associated nuclear factor kappa B and tumor necrosis factor-α signaling pathways. The combined action of the two cross-talking pathways protects hosts from RSV-induced damage. To conclude, our study is the first of its kind to establish evidence of LCA as a viable treatment for RSV infection.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

Diversity in Proprotein Convertase Reactivity among Human Papillomavirus Types.

The cleavage of viral surface proteins by furin is associated with some viruses' high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

3,4-Seco-Isopimarane Diterpenes from the Twigs and Leaves of Isodon Flavidus.

Three isopimarane diterpenes [fladins B (1), C (2), and D (3)] were isolated from the twigs and leaves of Chinese folk medicine, Isodon flavidus. The chemical structures were determined by the analysis of the comprehensive spectroscopic data, and the absolute configuration was confirmed by X-ray crystallographic analysis. The structures of 1-3 were formed from isopimaranes through the rearrangement of ring A by the bond break at C-3 and C-4 to form a new δ-lactone ring system between C-3 and C-9. This structure type represents the first discovery of a natural isopimarane diterpene with an unusual lactone moiety at C-9 and C-10. In the crystal of 1, molecules are linked to each other by intermolecular O-H···O bonds, forming chains along the b axis. Compounds 1-3 were evaluated for their bioactivities against different diseases. None of these compounds displayed cytotoxic activities against HCT116 and A549 cancer cell lines, antifungal activities against Trichophyton rubrum and T. mentagrophytes, or antiviral activities against HIV entry at 20 µg/mL (62.9-66.7) µM. Compounds 1 and 3 did not show antiviral activities against Ebola entry at 20 µg/mL either; only 2 was found to show an 81% inhibitory effect against Ebola entry activity at 20 µg/mL (66.7 µM). The bioactivity evidence suggested that this type of compound could be a valuable antiviral lead for further structure modification to improve the antiviral potential.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Current therapy and development of therapeutic agents for lung cancer.

In the past decades, great progress has been made for the prevention and treatment of lung cancer. Yet, lung cancer remains as the leading cause of cancer death worldwide. In this manuscript, we describe the current genetic and molecular characterization of lung cancer subtypes, review up-to-date treatment options for lung cancer patients, summarize the antibodies and small molecule drugs under clinical development, and elaborate on the expression and characteristics of important RTK primary targets and representative preclinical agents which may provide new opportunities for lung cancer treatment. Since gefitinib was first introduced to non-small-cell lung carcinoma (NSCLC) patients in 2002, remarkable progress has been made in targeted therapy for NSCLC patients with the development of multiple generations of small molecule inhibitors targeting relevant driver mutations. However, very little achievement has been made in the development of targeted drugs for small-cell lung carcinoma (SCLC). The successful harness of immune checkpoint inhibitors against PD-1/PD-L1 has marked a major advancement in recent lung cancer treatment. Looking forward, therapeutic strategies that tackle brain metastasis are highly desirable, the combination of molecular testing and strategies tailored to tackle tumor heterogeneity and resistance mechanisms is the key direction for future development.

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors.

Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to inform infectivity, causing misdiagnoses and spread of the viruses. Here, we report a method for direct detection and differentiation of infectious from noninfectious human adenovirus and SARS-CoV-2, as well as from other virus types, without any sample pretreatment. DNA aptamers are selected from a DNA library to bind intact infectious, but not noninfectious, virus and then incorporated into a solid-state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1 pfu/ml for human adenovirus and 1 × 104 copies/ml for SARS-CoV-2. Applications of the aptamer-nanopore sensors in different types of water samples, saliva, and serum are demonstrated for both enveloped and nonenveloped viruses, making the sensor generally applicable for detecting these and other emerging viruses of environmental and public health concern.

Identification of the SARS-CoV-2 Entry Receptor ACE2 as a Direct Target for Transcriptional Repression by Miz1.

Multiple lines of evidence have demonstrated that cigarette smoke or Chronic Obstructive Pulmonary Disease upregulates angiotensin-converting enzyme 2, the cellular receptor for the entry of the severe acute respiratory syndrome coronavirus 2, which predisposes individuals to develop severe Coronavirus disease 2019. The reason for this observation is unknown. We recently reported that the loss of function of Miz1 in the lung epithelium in mice leads to a spontaneous COPD-like phenotype, associated with upregulation of angiotensin-converting enzyme 2. We also reported that cigarette smoke exposure downregulates Miz1 in lung epithelial cells and in mice, and Miz1 is also downregulated in the lungs of COPD patients. Here, we provide further evidence that Miz1 directly binds to and represses the promoter of angiotensin-converting enzyme 2 in mouse and human lung epithelial cells. Our data provide a potential molecular mechanism for the upregulation of angiotensin-converting enzyme 2 observed in smokers and COPD patients, with implication in severe Coronavirus disease 2019.

Current Development of Glioblastoma Therapeutic Agents.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Over the past several decades, despite improvements in neurosurgical techniques, development of powerful chemotherapeutic agents, advances in radiotherapy, and comprehensive genomic profiling and molecular characterization, treatment of GBM has achieved very limited success in increasing overall survival. Thus, identifying and understanding the key molecules and barriers responsible for the malignant phenotypes and treatment resistance of GBM will yield new potential therapeutic targets. We review the most recent development of receptor tyrosine kinase targeted therapy for GBM and discuss the current status of several novel strategies with the emphasis on blood-brain barrier penetration as a major obstacle for small-molecule drugs to achieve their therapeutic goals. Likewise, a major opportunity for the treatment of GBM lies in the use of biomarkers for the discovery and development of new receptor tyrosine kinase targeted therapy.

A recombinant Cedar virus based high-throughput screening assay for henipavirus antiviral discovery.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic, bat-borne paramyxoviruses in the genus Henipavirus that cause severe and often fatal acute respiratory and/or neurologic diseases in humans and livestock. There are currently no approved antiviral therapeutics or vaccines for use in humans to treat or prevent NiV or HeV infection. To facilitate development of henipavirus antivirals, a high-throughput screening (HTS) platform was developed based on a well-characterized recombinant version of the nonpathogenic Henipavirus, Cedar virus (rCedV). Using reverse genetics, a rCedV encoding firefly luciferase (rCedV-Luc) was rescued and its utility evaluated for high-throughput antiviral compound screening. The luciferase reporter gene signal kinetics of rCedV-Luc in different human cell lines was characterized and validated as an authentic real-time measure of viral growth. The rCedV-Luc platform was optimized as an HTS assay that demonstrated high sensitivity with robust Z' scores, excellent signal-to-background ratios and coefficients of variation. Eight candidate compounds that inhibited rCedV replication were identified for additional validation and demonstrated that 4 compounds inhibited authentic NiV-Bangladesh replication. Further evaluation of 2 of the 4 validated compounds in a 9-point dose response titration demonstrated potent antiviral activity against NiV-Bangladesh and HeV, with minimal cytotoxicity. This rCedV reporter can serve as a surrogate yet authentic BSL-2 henipavirus platform that will dramatically accelerate drug candidate identification in the development of anti-henipavirus therapies.

Discovery of chebulagic acid and punicalagin as novel allosteric inhibitors of SARS-CoV-2 3CLpro.

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures.

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Ginkgolic acid and anacardic acid are specific covalent inhibitors of SARS-CoV-2 cysteine proteases.

BACKGROUND: In the urgent campaign to develop therapeutics against SARS-CoV-2, natural products have been an important source of new lead compounds. RESULTS: We herein identified two natural products, ginkgolic acid and anacardic acid, as inhibitors using a high-throughput screen targeting the SARS-CoV-2 papain-like protease (PLpro). Moreover, our study demonstrated that the two hit compounds are dual inhibitors targeting the SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) in addition to PLpro. A mechanism of action study using enzyme kinetics further characterized the two compounds as irreversible inhibitors against both 3CLpro and PLpro. Significantly, both identified compounds inhibit SARS-CoV-2 replication in vitro at nontoxic concentrations. CONCLUSIONS: Our finding provides two novel natural products as promising SARS-CoV-2 antivirals.

Targeting SARS-CoV-2 viral proteases as a therapeutic strategy to treat COVID-19.

The 21st century has witnessed three outbreaks of coronavirus (CoVs) infections caused by severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, spreads rapidly and since the discovery of the first COVID-19 infection in December 2019, has caused 1.2 million deaths worldwide and 226,777 deaths in the United States alone. The high amino acid similarity between SARS-CoV and SARS-CoV-2 viral proteins supports testing therapeutic molecules that were designed to treat SARS infections during the 2003 epidemic. In this review, we provide information on possible COVID-19 treatment strategies that act via inhibition of the two essential proteins of the virus, 3C-like protease (3CLpro ) or papain-like protease (PLpro ).

Screening and Reverse-Engineering of Estrogen Receptor Ligands as Potent Pan-Filovirus Inhibitors.

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.

Development of coumarine derivatives as potent anti-filovirus entry inhibitors targeting viral glycoprotein.

Filoviruses, including Ebolavirus (EBOV), Marburgvirus (MARV) and Cuevavirus, cause hemorrhagic fevers in humans with up to 90% mortality rates. In the 2014-2016 West Africa Ebola epidemic, there are 15,261 laboratory confirmed cases and 11,325 total deaths. The lack of effective vaccines and medicines for the prevention and treatment of filovirus infection in humans stresses the urgency to develop antiviral therapeutics against filovirus-associated diseases. Our previous study identified a histamine receptor antagonist compound CP19 as an entry inhibitor against both EBOV and MARV. The preliminary structure-activity relationship (SAR) studies of CP19 showed that its piperidine, coumarin and linker were related with its antiviral activities. In this study, we performed detailed SAR studies on these groups with synthesized CP19 derivatives. We discovered that 1) the piperidine group could be optimized with heterocycles, 2) the substitution groups of C3 and C4 of coumarin should be relatively large hydrophobic groups and 3) the linker part should be least substituted. Based on the SAR analysis, we synthesized compound 32 as a potent entry inhibitor of EBOV and MARV (IC50 = 0.5 µM for EBOV and 1.5 µM for MARV). The mutation studies of Ebola glycoprotein and molecular docking studies showed that the coumarin and its substituted groups of compound 32 bind to the pocket of Ebola glycoprotein in a similar way to the published entry inhibitor compound 118a. However, the carboxamide group of compound 32 does not have strong interaction with N61 as compound 118a does. The coumarin skeleton structure and the binding model of compound 32 elucidated by this study could be utilized to guide further design and optimization of entry inhibitors targeting the filovirus glycoproteins.

Discovery and Structural Optimization of 4-(Aminomethyl)benzamides as Potent Entry Inhibitors of Ebola and Marburg Virus Infections.

The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33, and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses. Representative compounds (20, 32, and 35) have shown good metabolic stability in plasma and liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.