Below is a link to a recent paper, and I have copied directly some extracts further below.

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

Typically, it is quite technical, but those who have been reading this type of stuff since their investment here - if in the case of a Covid cure - will be familiar with some of the concepts. Also, it is noteworthy that the researchers have utilized amilorides, of which BIT225 is a deriviative. (no need for peeps to state that BIT225 is not in the SARS2 soup). Any relevance of amiloride to SARS2 is still important for Biotron.

I've included paragraph four, a layout of the techniques they have used, for the simple reason to show how intricate working with E is, which should give some reason to appreciate why results from Biotron are taking considerable time to produce.

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

The envelope (E) protein, one of fourstructural proteins encoded in the viral genome, is a 75-residue integralmembrane protein whose transmembrane domain exhibits ion channel activity andwhose cytoplasmic domain participates in protein-protein interactions. Theseactivities 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 isindeed the likely target of their antiviral activity. We found that residueasparagine15 plays an important role in maintaining the conformation of theamiloride binding site, providing molecular guidance for the design ofinhibitors targeting E protein.

The biological relevance of E protein comesfrom its involvement in key aspects of the virus lifecycle, includinginfection, replication, assembly, budding, and pathogenesis [12]. Furthermore, recombinant coronaviruseslacking E protein exhibit significantly reduced viral titers, crippled viralmaturation, 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 ofSARS-CoV E protein (S1 Fig) [12]. Since E protein is a viral membrane-spanningminiprotein [16], a recurring question is whether it is aviroporin. Although ion-channel activity has been detected in a variety ofpreparations it lacks sequence homology with any of the well-establishedviroporins, and there is a notable absence of charged side chains on theinterior of a pore formed by pentamers of the protein in membrane environments[9,10,17,18].

The importance of E protein for viralreplication and maturation is well established, making it an attractive targetfor antiviral drugs. Drug design requires high-resolution structures of theprotein receptor in its bound and free states. Small membrane proteins arenotoriously difficult to crystallize in their native states in liquidcrystalline membrane bilayers for X-ray crystallography and are too small forcryoEM to be effective. While generally suited for NMR spectroscopy, carefulconsideration of the membrane-like environment of the samples and the NMRexperimental methods are essential [19]. Even the earliest NMR studies of membraneproteins showed that caution is called for when using micelle environments [20], because of the potential for aggregation andstructural distortions [21,22]. Nonetheless, careful optimization of sampleconditions has enabled solution NMR to provide valid structural informationabout membrane proteins that could be obtained in no other way. Moreover, wehave found it essential to prepare samples of membrane proteins in micellesthat yield high-resolution solution NMR spectra in order to verify that theyintegrate into an amphipathic membrane-like environment, are chemically pure,not mis-folded, and not aggregated before initiating significantly moredemanding solid-state NMR studies of phospholipid bilayer samples. In order toensure that solid-state NMR experiments are performed under near-nativeconditions, both the protein and the bilayers must be fully characterized toensure that the protein is in its biologically active conformation and stablyembedded in liquid crystalline, fully hydrated phospholipid bilayers at highlipid to protein ratios.

To date, no structural data have been presentedfor any full-length coronavirus E protein …. sequences from the SARS-CoV Eprotein…..are highly relevant to studies of the SARS-CoV-2 E protein becausethe amino acid sequences of these two proteins are identical between residues 1and 68.

These studies provide strong biological andmechanistic justification for considering coronavirus E protein as a potentialdrug target.

The channel activity of E protein has been suggestedto play a role in viral replication [29]. A well-characterized channel blocker,hexamethylene amiloride (HMA), inhibits ion channel conductance of E proteinsfrom HCoV-229E and MHV as well as virus replication in cultured cells [30]. HMA also inhibits the channel conductance oftransmembrane-containing synthetic and expressed polypeptides from the SARS-CoVE protein [17,18]. Although interactions of HMA with E proteinof SARS-CoV have been detected in prior studies, the residues in the HMAbinding site identified by NMR chemical shift perturbations varied quite a bitdepending upon the specific E protein constructs and experimental conditions [9,10,17,18].

We also map out the complete binding sites ofamiloride 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 againstSARS-CoV-2 infection of Vero E6 cells correlates well with their strength ofbinding as observed in the NMR experiments.

We were unable to overcome the difficultiesinherent in dealing with hydrophobic membrane proteins in the case of E proteinusing approaches that we had previously applied successfully to viral,bacterial, and human membrane proteins with between one and seven transmembranehelices [31–36].

Qualitatively, the data in Fig 5 confirm that HMA (amiloride)interacts with the N-terminal domain of E protein.

The amiloridederivatives 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 compoundsact late in the viral replication cycle and affect the spread of virus fromcell-to-cell, although they do not exclude the possibility of a modest effecton the establishment of infection by cell-free virus.

Not long now.