Delivery of spike-RBD by bacterial type three secretion system for SARS-CoV-2 vaccine development.

COVID-19 pandemic continues to spread throughout the world with an urgent demand for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a bacterial vector COVID-19 vaccine (aPA-RBD) that carries the gene for the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Live-attenuated strains of Pseudomonas aeruginosa (aPA) were constructed which express the recombinant RBD and effectively deliver RBD protein into various antigen presenting cells through bacterial type 3 secretion system (T3SS) in vitro. In mice, two-dose of intranasal aPA-RBD vaccinations elicited the development of RBD-specific serum IgG and IgM. Importantly, the sera from the immunized mice were able to neutralize host cell infections by SARS-CoV-2 pseudovirus as well as the authentic virus variants potently. T-cell responses of immunized mice were assessed by enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays. aPA-RBD vaccinations can elicit RBD-specific CD4+and CD8+T cell responses. T3SS-based RBD intracellular delivery heightens the efficiency of antigen presentation and enables the aPA-RBD vaccine to elicit CD8+T cell response. Thus, aPA vector has the potential as an inexpensive, readily manufactured, and respiratory tract vaccination route vaccine platform for other pathogens.

Synthesis of maleimide-braced peptide macrocycles and their potential anti-SARS-CoV-2 mechanisms.

Macrocycles often exhibit good biological properties and potential druggability, which lead to versatile applications in the pharmaceutical industry. Herein, we report a highly efficient and practical methodology for the functionalization and macrocyclization of Trp and Trp-containing peptides via Pd(II)-catalyzed C-H alkenylation at the Trp C4 position. This method provides direct access to C4 maleimide-decorated Trp-containing peptidomimetics and maleimide-braced 17- to 30-membered peptide macrocycles. In particular, these unique macrocycles revealed low micro- to sub-micromolar EC50 values with promising anti-SARS-CoV-2 activities. Further explorations with computational methodologies and experimental validations indicated that these macrocycles exert antiviral effects through binding with the N protein of SARS-CoV-2.

Exploring and Learning the Universe of Protein Allostery Using Artificial Intelligence Augmented Biophysical and Computational Approaches.

Allosteric mechanisms are commonly employed regulatory tools used by proteins to orchestrate complex biochemical processes and control communications in cells. The quantitative understanding and characterization of allosteric molecular events are among major challenges in modern biology and require integration of innovative computational experimental approaches to obtain atomistic-level knowledge of the allosteric states, interactions, and dynamic conformational landscapes. The growing body of computational and experimental studies empowered by emerging artificial intelligence (AI) technologies has opened up new paradigms for exploring and learning the universe of protein allostery from first principles. In this review we analyze recent developments in high-throughput deep mutational scanning of allosteric protein functions; applications and latest adaptations of Alpha-fold structural prediction methods for studies of protein dynamics and allostery; new frontiers in integrating machine learning and enhanced sampling techniques for characterization of allostery; and recent advances in structural biology approaches for studies of allosteric systems. We also highlight recent computational and experimental studies of the SARS-CoV-2 spike (S) proteins revealing an important and often hidden role of allosteric regulation driving functional conformational changes, binding interactions with the host receptor, and mutational escape mechanisms of S proteins which are critical for viral infection. We conclude with a summary and outlook of future directions suggesting that AI-augmented biophysical and computer simulation approaches are beginning to transform studies of protein allostery toward systematic characterization of allosteric landscapes, hidden allosteric states, and mechanisms which may bring about a new revolution in molecular biology and drug discovery.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Pseudomonas aeruginosa modulates alginate biosynthesis and type VI secretion system in two critically ill COVID-19 patients.

BACKGROUND: COVID-19 pneumonia has caused huge impact on the health of infected patients and associated with high morbidity and mortality. Shift in the lung microbial ecology upon such viral infection often worsens the disease and increases host susceptibility to superinfections. Bacterial superinfection contributes to the aggravation of COVID-19 and poses a great challenge to clinical treatments. An in-depth investigation on superinfecting bacteria in COVID-19 patients might facilitate understanding of lung microenvironment post virus infections and superinfection mechanism. RESULTS: We analyzed the adaptation of two pairs of P. aeruginosa strains with the same MLST type isolated from two critical COVID-19 patients by combining sequencing analysis and phenotypic assays. Both P. aeruginosa strains were found to turn on alginate biosynthesis and attenuate type VI secretion system (T6SS) during short-term colonization in the COVID-19 patients, which results in excessive biofilm formation and virulence reduction-two distinct markers for chronic infections. The macrophage cytotoxicity test and intracellular reactive oxygen species measurement confirmed that the adapted P. aeruginosa strains reduced their virulence towards host cells and are better to escape from host immune clearance than their ancestors. CONCLUSION: Our study suggests that SARS-CoV-2 infection can create a lung environment that allow rapid adaptive evolution of bacterial pathogens with genetic traits suitable for chronic infections.

Target-Based Virtual Screening and LC/MS-Guided Isolation Procedure for Identifying Phloroglucinol-Terpenoid Inhibitors of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 5 million deaths worldwide to date. Due to the limited therapeutic options so far available, target-based virtual screening with LC/MS support was applied to identify the novel and high-content compounds 1-4 with inhibitory effects on SARS-CoV-2 in Vero E6 cells from the plant Dryopteris wallichiana. These compounds were also evaluated against SARS-CoV-2 in Calu-3 cells and showed unambiguous inhibitory activity. The inhibition assay of targets showed that compounds 3 and 4 mainly inhibited SARS-CoV-2 3CLpro, with effective Kd values. Through docking and molecular dynamics modeling, the binding site is described, providing a comprehensive understanding of 3CLpro and interactions for 3, including hydrogen bonds, hydrophobic bonds, and the spatial occupation of the B ring. Compounds 3 and 4 represent new, potential lead compounds for the development of anti-SARS-CoV-2 drugs. This study has led to the development of a target-based virtual screening method for exploring the potency of natural products and for identifying natural bioactive compounds for possible COVID-19 treatment.

Probing the Allosteric Inhibition Mechanism of a Spike Protein Using Molecular Dynamics Simulations and Active Compound Identifications.

The receptor recognition of the novel coronavirus SARS-CoV-2 relies on the "down-to-up" conformational change in the receptor-binding domain (RBD) of the spike (S) protein. Therefore, understanding the process of this change at the molecular level facilitates the design of therapeutic agents. With the help of coarse-grained molecular dynamic simulations, we provide evidence showing that the conformational dynamics of the S protein are globally cooperative. Importantly, an allosteric path was discovered that correlates the motion of the RBD with the motion of the junction between the subdomain 1 (SD1) and the subdomain 2 (SD2) of the S protein. Building on this finding, we designed non-RBD binding modulators to inhibit SARS-CoV-2 by prohibiting the conformational change of the S protein. Their inhibition effect and function stages at inhibiting SARS-CoV-2 were evaluated experimentally. In summary, our studies establish a molecular basis for future therapeutic agent design through allosteric effects.

Identification of Potential Binding Sites of Sialic Acids on the RBD Domain of SARS-CoV-2 Spike Protein.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still an emergent pandemic for humans. The virus infection is achieved by penetrating its spike protein to host cells via binding with ACE2. Moreover, recent studies show that SARS-CoV-2 may have multiple receptors that need to be further revealed. SARS-CoV-2 shares similar sequences of the spike protein with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which can invade host cells by binding to either DPP4 or sialic acids. Sialic acids can be linked to the terminal of glycoproteins and gangliosides are used as one of the receptors of many types of viruses. Therefore, it is very interesting to determine whether sialic acid is a potential receptor of SARS-CoV-2. To address this question, we took N-Acetylneuraminic acid (Neu5Ac), a type of predominant sialic acid found in human cells, as the molecular probe to computationally search the surface of the spike protein to locate the potential binding sites of Neu5Ac. SPR analysis and mass spectrum analysis confirmed the interaction between Neu5Ac and spike protein. This study shows that sialic acids can moderately interact with the spike protein of SARS-CoV-2 by binding between the two RBDs of the spike protein, indicating it could be a potential secondary or auxiliary receptor of SARS-CoV-2.

Discovery of potential small molecular SARS-CoV-2 entry blockers targeting the spike protein.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

A multi-targeting drug design strategy for identifying potent anti-SARS-CoV-2 inhibitors.

The COVID-19, caused by SARS-CoV-2, is threatening public health, and there is no effective treatment. In this study, we have implemented a multi-targeted anti-viral drug design strategy to discover highly potent SARS-CoV-2 inhibitors, which simultaneously act on the host ribosome, viral RNA as well as RNA-dependent RNA polymerases, and nucleocapsid protein of the virus, to impair viral translation, frameshifting, replication, and assembly. Driven by this strategy, three alkaloids, including lycorine, emetine, and cephaeline, were discovered to inhibit SARS-CoV-2 with EC50 values of low nanomolar levels potently. The findings in this work demonstrate the feasibility of this multi-targeting drug design strategy and provide a rationale for designing more potent anti-virus drugs.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the etiological agent responsible for the global COVID-19 (coronavirus disease 2019) outbreak. The main protease of SARS-CoV-2, Mpro, is a key enzyme that plays a pivotal role in mediating viral replication and transcription. We designed and synthesized two lead compounds (11a and 11b) targeting Mpro Both exhibited excellent inhibitory activity and potent anti-SARS-CoV-2 infection activity. The x-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a or 11b, both determined at a resolution of 1.5 angstroms, showed that the aldehyde groups of 11a and 11b are covalently bound to cysteine 145 of Mpro Both compounds showed good pharmacokinetic properties in vivo, and 11a also exhibited low toxicity, which suggests that these compounds are promising drug candidates.