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Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus (VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV's nonstructural protein 2 (nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the compounds. The results provide structural insights into a new class of potent nonpeptidic covalent inhibitors of nsP2 cysteine protease represented by compound 11 (VEEV TrD, EC 50 = 2.4 μM (HeLa), 1.6 μM (Vero E6)). These results may facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads., Competing Interests: The authors declare no competing financial interest.

Previous studies identified an adamantane dipeptide piperazine 3.47 that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann-Pick C1 (NPC1). The physicochemical properties of 3.47 limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified 3.47 derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC 50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.