Rational Design and In-Vivo Validation of Capsid Inhibitors for Enterovirus D68

The viral capsid has emerged as a validated target for Antiviral Drug Discovery (AViDD), due to the recent regulatory approval of the long-acting anti-HIV drug Lenacapavir (marketed asYeytuo & Sunlenca). This extraordinary medicine is provided to patients that don’t respond to standard anti-retroviral treatments as a log-acting injectable (LAI) formulation, which amounts to a single injection every 6 months.

Enterovirus-D68 (EV-D68) is an emerging non-polio enterovirus (NPEV) that, unlike HIV-1, is transmissible through respiratory droplets and contaminated surfaces. As well as being highly contagious, EV-D68 infections can result in polio-like paralysis symptoms that are seen primarily in very young/old/infirm patients. Despite the risk posed by pandemic EV-D68 or a novel mutant thereof, there are no approved drugs or vaccines capable of treating or preventing EV-D68 infections. Numerous compounds that display antiviral activity againstEV-D68 in-vitroexist in the AViDD literature, with various hypotheses as to their mechanismof action, these targets include: viral protease enzymes (2A-, 2C- and 3C-protease), the RNA-dependent RNA polymerase (RdRp) and the capsid Viral Protein 1 (VP1). The EV-D68 capsid is made up of 4 protein subunits: VP1, VP2, VP3 and VP4, which assemble to form viral capsid particles that protect its genetic material until it enters the host cell, whereupon“uncoating” takes place and the RNA is delivered to the translation and replication machinery.

Pleconaril is a fascinating preclinical compound, originally developed by pharmaceutical companies to treat Human rhinovirus (HRV). FDA-approval for pleconaril was ultimatelyrejected, but since then, a few research teams have been investigating the revival of thepleconaril series. This is not necessarily to find treatments for HRV, but to exploit its other antiviral activity, against NPEV of potentially broader concern.

It is easy to understate the drug discovery challenge for the EV-D68 capsid target. The pocket is adapted to fit a lipid from the environment, such as Sphingosine, and is lined with lipophilic residues. So, even if you find a compound that manages to fit cleanly into the pocket and produce an antiviral effect in-vitro, you also need to keep a close eye on its physicochemical properties (LogD, solubility) to make sure that you are developing something that has a chance of having a useful effect in-vivo.

Using publicly available capsid VP1 crystal and Cryo-EM structures, the authors of this publication carried out virtual screening and found a completely new compound that binds to the capsid, and displayed antiviral activity against EV-D68. Optimisation of this hit compound resulted in further analogues with potent antiviral activity, but importantly, good enough PK to carry out in-vivostudies. The antiviral activity spanned multiple clades of EV-D68, but dropped off for more dissimilar viruses, such as EV-A71 (although not as much aspleconaril). Synergistic effects were observed after treating cells simultaneously with a protease inhibitor, telaprevir.

Numerous in-vivoefficacy studies were conducted, measurements included viral titres from the spinal cord and muscle, reduction of paralysis caused by acute flaccid myelitis (AFM) according to the mouse model used, motor neuron counts and more. Highly promising in-vivo results and Cryo-EM data are presented here that will be significant for AViDD campaigns seeking desperately needed antivirals capable of treating EV-D68.

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