Inhibition of S-protein RBD and hACE2 Interaction for Control of SARSCoV- 2 Infection (COVID-19).

COVID-19 has become a pandemic with higher morbidity and mortality rates after its start from Wuhan city of China. The infection by RNA virus, also known as SARS-CoV-2 or 2019-nCoV, from the beta class of coronaviruses, has been found to be responsible for COVID-19. Structural analysis and evidences have been indicated that interaction between a segment of receptor binding domain (RBD) from S protein of the virus and human angiotensin-converting enzyme 2 (hACE2) is essential for cellular entry of the virus. The current review sheds light on structural aspects for the inhibition of RBD-hACE2 interaction mediated cellular entry of SARS-CoV-2. The present study provides a critical review of recently published information on RBDhACE2 interaction and its inhibitors to control SARS-CoV-2 infection. The review highlighted the structural aspects of the interaction between RBD-hACE2 and involved amino acid residues. Recently, several studies are being conducted for the inhibition of the SARS-CoV-2 attachment and entry to the human cellular system. One of the important targets for viral invasion is its binding with cell surface receptor, hACE2, through RBD on S-protein. Mimicking of three residues on ACE2 (Lys31, Glu35 and Lys353 on B chain) provided a hot target directed strategy for the inhibition of early attachment of the virus to the cell. Early screening of peptidic or non-peptidic molecules for the inhibition of RBD-hACE2 interaction has raised the hope for potential therapeutics against COVID-19. The higher affinity of molecules toward RBD than ACE2 is an important factor for selectivity and minimization of ACE2 related adverse events on the cardiovascular system, brain, kidney, and foetus development during pregnancy. Inhibition of RBD-hACE2 interaction by different molecular scaffolds can be used as a preferred strategy for control of SARS-CoV-2 infection. Recently, published reports pointed out Lys31, Glu35 and Lys353 on the B chain of ACE2 as crucial residues for mimicking and design of novel molecules as inhibitors SARS-CoV-2 attachment to human cells. Moreover, some recently identified RBD-hACE2 interaction inhibitors have also been described with their protein binding pattern and potencies (IC50 values), which will help for further improvement in the selectivity.

Nayak SK 《-》

Multidisciplinary Approaches Identify Compounds that Bind to Human ACE2 or SARS-CoV-2 Spike Protein as Candidates to Block SARS-CoV-2-ACE2 Receptor Interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 μM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.

Day CJ ，Bailly B ，Guillon P ，Dirr L ，Jen FE ，Spillings BL ，Mak J ，von Itzstein M ，Haselhorst T ，Jennings MP ... -  《mBio》

Tinocordiside from Tinospora cordifolia (Giloy) May Curb SARS-CoV-2 Contagion by Disrupting the Electrostatic Interactions between Host ACE2 and Viral S-Protein Receptor Binding Domain.

SARS-CoV-2 has been shown to bind the host cell ACE2 receptor through its spike protein receptor binding domain (RBD), required for its entry into the host cells. We have screened phytocompounds from a medicinal herb, Tinospora cordifolia for their capacities to interrupt the viral RBD and host ACE2 interactions. We employed molecular docking to screen phytocompounds in T. cordifolia against the ACE2-RBD complex, performed molecular dynamics (MD) simulation, and estimated the electrostatic component of binding free energy. 'Tinocordiside' docked very well at the center of the interface of ACE2-RBD complex, and was found to be well stabilized during MD simulation. Tinocordiside incorporation significantly decreased the electrostatic component of binding free energies of the ACE2-RBD complex (23.5 and 17.10 kcal/mol in the trajectories without or with the ligand, respectively). As the basal rate constant of protein association is in the order of 5 (105 to 106 M-1S-1), there might be no big conformational change or loop reorganization, but involves only local conformational change typically observed in the diffusion-controlled association. Taken together, the increase in global flexibility of the complex clearly indicates the start of unbinding process of the complex. It indicates that such an interruption of electrostatic interactions between the RBD and ACE2, and the increase in global flexibility of the complex would weaken or block SARSCoV- 2 entry and its subsequent infectivity. We postulate that natural phytochemicals like Tinocordiside could be viable options for controlling SARS-CoV-2 contagion and its entry into host cells.

Balkrishna A ，Pokhrel S ，Varshney A 《-》

Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model.

SARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals. This study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone. Through molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction. Withanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein. In conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.

Balkrishna A ，Pokhrel S ，Singh H ，Joshi M ，Mulay VP ，Haldar S ，Varshney A ... -  《Drug Design Development and Therapy》

De novo design, retrosynthetic analysis and combinatorial synthesis of a hybrid antiviral (VTAR-01) to inhibit the interaction of SARS-CoV2 spike glycoprotein with human angiotensin-converting enzyme 2.

SARS-like coronavirus (SARS-CoV2) has emerged as a global threat to humankind and is rapidly spreading. The infectivity, pathogenesis and infection of this virus are dependent on the interaction of SARS-CoV2 spike protein with human angiotensin converting enzyme 2 (hACE2). Spike protein contains a receptor-binding domain (RBD) that recognizes hACE-2. In the present study, we are reporting a de novo designed novel hybrid antiviral 'VTAR-01' molecule that binds at the interface of RBD-hACE2 interaction. A series of antiviral molecules were tested for binding at the interface of RBD-hACE2 interaction. In silico screening, molecular mechanics and molecular dynamics simulation (MDS) analysis suggest ribavirin, ascorbate, lopinavir and hydroxychloroquine have strong interaction at the RBD-hACE2 interface. These four molecules were used for de novo fragment-based antiviral design. De novo designing, docking and MDS analysis identified a 'VTAR' hybrid molecule that has better interaction with this interface than all of the antivirals used to design it. We have further used retrosynthetic analysis and combinatorial synthesis to design 100 variants of VTAR molecules. Retrosynthetic analysis and combinatorial synthesis, along with docking and MDS, identified that VTAR-01 interacts with the interface of the RBD-ACE2 complex. MDS analysis confirmed its interaction with the RBD-ACE2 interface by involving Glu35 and Lys353 of ACE2, as well as Gln493 and Ser494 of RBD. Interaction of spike protein with ACE2 is essential for pathogenesis and infection of this virus; hence, this in silico designed hybrid antiviral molecule (VTAR-01) that binds at the interface of RBD-hACE2 may be further developed to control the infection of SARS-CoV2.

Tiwari V 《Biology Open》