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Step-by-step explanation:
he most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg−1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Main
The 2013–2016 outbreak of EVD in West Africa was the largest and most complex EBOV outbreak in the recorded history of the disease, with >28,000 EVD cases and >11,000 reported deaths1. Medical infrastructures in Guinea, Sierra Leone, and Liberia were seriously impacted by a loss of >500 healthcare workers1. Additionally, EVD-related sequelae (joint and muscle pain, as well as neurological, ophthalmic, and other symptoms) together with viral persistence and recrudescence in individuals who survived the acute disease have been documented2,3,4,5.
EBOV is a single-stranded negative-sense non-segmented RNA virus from the Filoviridae family. In addition to EBOV, other related viruses, namely Marburg, Sudan, and Bundibugyo viruses, have caused outbreaks with high fatality rates6. Although the efficacy of various experimental small molecules and biologics have been assessed in EVD animal models and in multiple clinical trials during the West African outbreak7,8,9,10,11,12,13,14,15,16,17,18, there are no therapeutics for which clinical efficacy and safety have been established for treatment of acute EVD or its sequelae. The availability of broadly effective antiviral(s) with a favourable benefit/risk profile would address a serious unmet medical need for the treatment of EBOV infection.
A 1′-cyano-substituted adenine C-nucleoside ribose analogue (Nuc) exhibits antiviral activity against a number of RNA viruses19. The mechanism of action of Nuc requires intracellular anabolism to the active triphosphate metabolite (NTP), which is expected to interfere with the activity of viral RNA-dependent RNA-polymerases (RdRp). Structurally, the 1′-cyano group provides potency and selectivity towards viral RNA polymerases, but because of slow first phosphorylation kinetics, modification of parent nucleosides with monophosphate promoieties has the potential to greatly enhance intracellular NTP concentrations20. GS-5734, the single Sp isomer of the 2-ethylbutyl L-alaninate phosphoramidate prodrug (Supplementary Information), effectively bypasses the rate-limiting first phosphorylation step of the Nuc (Fig. 1a). In human monocyte-derived macrophages, incubation with GS-5734 caused rapid loading of cells with high levels of NTP that persist with a half-life (t1/2) of 24 h following removal of GS-5734 (Extended Data Fig. 1a), resulting in up to 30-fold higher levels compared to incubation with Nuc (Fig. 1b). In cell-based assays, GS-5734 is active against a broad range of filoviruses including Marburg virus and several variants of EBOV (Fig. 1c). GS-5734 inhibits EBOV replication in multiple relevant human cell types including primary macrophages and human endothelial cells with half-maxi