BIT036- a likely candidate for curing COVID-19, page-922

Hi BMD, I tried to clean it up a BIT if that's OK



Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity

The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose trans membrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle,including virion assembly, budding, release, and pathogenesis.
The binding affinity of the amilorides to Eprotein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein.
The biological relevance of E protein comes from its involvement in key aspects of the virus lifecycle, including infection, replication, assembly, budding, and pathogenesis [12]. Furthermore, recombinant coronaviruses lacking E protein exhibit significantly reduced viral titers, crippled viral maturation, and yield propagation incompetent progeny [13–15]. SARS-CoV-2 E protein is a hydrophobic75-residue protein with an amino acid sequence nearly identical to that of SARS-CoV E protein (S1 Fig) [12]. Since E protein is a viral membrane-spanning miniprotein [16], a recurring question is whether it is aviroporin. Although ion-channel activity has been detected in a variety of preparations it lacks sequence homology with any of the well-established viroporins, and there is a notable absence of charged side chains on the interior of a pore formed by pentamers of the protein in membrane environments[9,10,17,18].
The importance of E protein for viral replication and maturation is well established, making it an attractive target for antiviral drugs. Drug design requires high-resolution structures of the protein receptor in its bound and free states. Small membrane proteins are notoriously difficult to crystallize in their native states in liquid crystalline membrane bilayers for X-ray crystallography and are too small for cryoEM to be effective. While generally suited for NMR spectroscopy, careful consideration of the membrane-like environment of the samples and the NMR experimental methods are essential [19]. Even the earliest NMR studies of membrane proteins showed that caution is called for when using micelle environments [20], because of the potential for aggregation and structural distortions [21,22]. Nonetheless, careful optimization of sample conditions has enabled solution NMR to provide valid structural information about membrane proteins that could be obtained in no other way. Moreover, we have found it essential to prepare samples of membrane proteins in micelles that yield high-resolution solution NMR spectra in order to verify that they integrate into an amphipathic membrane-like environment, are chemically pure,not mis-folded, and not aggregated before initiating significantly more demanding solid-state NMR studies of phospholipid bilayer samples. In order to ensure that solid-state NMR experiments are performed under near-native conditions, both the protein and the bilayers must be fully characterized to ensure that the protein is in its biologically active conformation and stably embedded in liquid crystalline, fully hydrated phospholipid bilayers at high lipid to protein ratios.
To date, no structural data have been presented for any full-length coronavirus E protein …. sequences from the SARS-CoV Eprotein…..are highly relevant to studies of the SARS-CoV-2 E protein because the amino acid sequences of these two proteins are identical between residues 1and 68.
These studies provide strong biological and mechanistic justification for considering coronavirus E protein as a potential drug target.
The channel activity of E protein has been suggested to play a role in viral replication [29]. A well-characterized channel blocker,hexamethylene amiloride (HMA), inhibits ion channel conductance of E proteins from HCoV-229E and MHV as well as virus replication in cultured cells [30]. HMA also inhibits the channel conductance of transmembrane-containing synthetic and expressed polypeptides from the SARS-CoVE protein [17,18]. Although interactions of HMA with E protein of SARS-CoV have been detected in prior studies, the residues in the HMA binding site identified by NMR chemical shift perturbations varied quite a bit depending upon the specific E protein constructs and experimental conditions [9,10,17,18].
We also map out the complete binding sites of amiloride and three amiloride derivatives (dimethyl amiloride (DMA), ethylisopropyl amiloride (EIPA), and HMA) and compare their binding properties.Importantly, the antiviral potency of the amiloride derivatives against SARS-CoV-2 infection of Vero E6 cells correlates well with their strength of binding as observed in the NMR experiments.
We were unable to overcome the difficulties inherent in dealing with hydrophobic membrane proteins in the case of E protein using approaches that we had previously applied successfully to viral,bacterial, and human membrane proteins with between one and seven trans membranehelices [31–36].
Qualitatively, the data in Fig 5 confirm that HMA (amiloride) interacts with the N-terminal domain of E protein.
The amiloride derivatives were tested for their ability to inhibit replication of SARS-CoV-2in Vero E6 cells. The similarity of the trends for inhibition of replication and of binding to E protein suggests that this protein may very well be a target for the antiviral activity of amiloride compounds. Of note, the most active compound examined here, HMA, shows considerable cytotoxicity (therapeutic index = 21.23), therefore, EIPA may be a better choice for potential therapeutic use (therapeutic index = 84.83) or as a starting point for further drug development.
These data suggest that the amiloride compounds act late in the viral replication cycle and affect the spread of virus from cell-to-cell, although they do not exclude the possibility of a modest effect on the establishment of infection by cell-free virus.