Adhesion to host cell membrane microdomains in cornea as an antimicrobial target to prevent corneal ulceration

This award, made jointly to The University of Sheffield, UK and LV Prasad Eye Institute, India, isaimed at the development of inhibitors of microbial adhesion to host cells based on the sequence ofa human protein, tetraspanin CD9. A key discovery that we have made during the tenure of thisaward, is that the mechanism of action of the peptides is highly likely to be the dispersal of heparansulphate-bearing proteins at the host cell surface. These proteins (e.g. syndecan-1) are usuallylocated in tetraspanin-enriched microdomains (TEM). CD9 is found at high levels on most epithelialsurfaces and is a critical component of TEM in these cells. We have shown that many types ofpathogenic bacteria, and some fungi, use heparan sulphates as an adhesion platform, allowing theirattachment to host cells to enable later events, such as internalisation and transcytosis acrossepithelial layers. CD9 peptides and heparin, a soluble analogue of heparan sulphate, give identicalinhibition of bacterial attachment.We note that some viruses, e.g. herpes simplex, also use heparan sulphates during cell attachment.Coronaviruses such as human NL63, OC43 and SARS-CoV, have also been reported to adhere toheparan sulphates. The furin cleavage motif (RRXRR) found in several human coronaviruses stronglyresembles a heparan sulphate binding motif (XBBXBX or XBBBXXBX). In SARS-CoV-2, the putativefurin site is PRRARSV and is thought to be an important adaption that allowed the transfer ofvirulence between animals and humans.In preliminary work, we have shown that SARS-CoV-2 spike protein (containing S1 and S2 domains)binds strongly to the human RT4 epithelial cell line that expresses low levels of receptor ACE2 andproteases TMPRSS2 and ADAM17. In contrast, recombinant S1 domain, a smaller fragment of S1containing only the receptor-binding domain and a mutant form of S1S2 spike protein that lacks thefurin cleavage site, bind at very low levels. Unfractionated heparin, at 0.1U/ml, can inhibit wild typeS1S2 binding, reducing it to the very low levels detected for the other spike proteins. Surfen, aheparan sulphate binding aminoquinoline, can also inhibit S1S2 binding.We interpret these data as an indication that SARS-CoV-2 has a high affinity for heparan sulphates,and that this interaction with the host cell surface can occur in cell lines with only minimal ACE2expression. Heparan sulphates may therefore be important in the retention of virus at an epithelialsurface prior to internalisation and infection via ACE2, a protein paradoxically found only at lowlevels in airway epithelia. Heparin may inhibit this interaction, and so lower the rate of infection. Wenote that low molecular weight heparin (LMWH) is already used therapeutically with some successin late stage Covid-19 patients to treat hypercoagulation.We would therefore like to repurpose this award to allow us to make a fuller investigation of SARSCoV-2 use of heparan sulphates before alerting clinicians to the possibility that heparin or LMWHtreatment earlier in Covid-19, perhaps even before blood clotting disorders develop, would bebeneficial. There are also heparin analogues and heparan sulphate antagonists that have been usedclinically for other indications (e.g. Fondaparinux, Ciraparantag) that we would like to test forinhibition of S1S2 binding. Finally, we would like to be able to test (by collaboration) the effects ofheparin and analogues in a live virus infection assay. We envisage that this work will take 4-6 weeks,enabling the full resources of the MRC award to be used in Sheffield: at least part-time effort byPDRA, technician and the associated PhD student, funding for drug synthesis and the purchase ofrecombinant viral proteins. During this period, we would apply to the UKRI Rapid Response Call forfurther funding to help transfer this research into clinical practice.This work would make minimal impact on the original aims of the award, as we are currently unableto make progress in the UK or India. It will also feedback into the original award because we wouldalso be able to test Surfen, Fondaparinux and Ciraparantag, etc as inhibitors of bacterial infection.