RESUMO
The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5'-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5'-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5'-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5'-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.
RESUMO
Early vascular thrombotic complications in renal transplantation frequently result in loss of the allograft with an increased long-term incidence of renal dysfunction from venous and arterial thrombosis. Hypercoagulable states are recognized risk factors of allograft thrombosis but are seldom discussed in the literature. This is the first report of preemptive heparin anticoagulation of two hypercoagulable patients undergoing renal transplantation, resulting in long term allograft success with warfarin anticoagulation. We recommend that any hemodialysis patient who has had recurrent dialysis graft thrombosis with a few months' time be evaluated for possible hypercoagulable state. Our experience suggests that appropriate management of such patients includes warfarin therapy. Renal transplantation can be successful in such patients provided heparin is given before, during, and after surgery followed by reinstitution of warfarin prior to discharge from the hospital.
