Since its discovery in 1976, Ebola virus has been responsible for numerous outbreaks, with case fatalities varying from 20% to 90% ^{1, 2} . From 2014 to 2016, West Africa was devastated with the largest Ebola outbreak in recorded history with six countries affected: Nigeria, Senegal, Guinea, Liberia, Mali and Sierra Leone. With no effective therapies or licensed vaccines available, little could be done to treat patients and prevent the spread of the virus ³ . Over a period of two years, Ebola virus was responsible for 28,646 reported infections, with 11,323 deaths ^{1, 3} .

Since the outbreak, a vaccine for Ebola virus (rVSV-EBOV) has been developed by the National Microbiological Laboratory in Winnipeg, Manitoba, Canada, and has been shown to be highly protective ⁴ . Undoubtedly, the rVSV-EBOV vaccine is to be a powerful defense against future Ebola outbreaks. However, it is unlikely to be sufficient to stop future outbreaks. As the natural host of Ebola virus remains unknown, eradication of Ebola reservoir populations is impossible ⁵ . Because of this, future spillover events are an inevitability ⁶ . Additionally, the unstable political environment of many affected African countries further complicates the effective administration of vaccines; as seen in the Democratic Republic of Congo during the current Ebola outbreak ^{2, 7} . These factors emphasize the need for acute infection therapies.

Previous publications have focused on potential drugs predicted to have efficacy against Ebola virus disease (EVD) and included drugs already approved and widely used that could perhaps be repurposed as treatments for EVD ^{8, 9} . Through a virtual screening process, Veljkovic et al. ⁸ identified 267 US Food and Drug Administration (FDA)-approved drugs with possible inhibitory effects against Ebola virus. Among these were the antimalarial quinoline drugs amodiaquine and chloroquine. Chloroquine has been shown to be protective against Ebola virus disease in an in vivo mouse model ⁹ . Additionally, amodiaquine has been identified as having inhibitory effects in a pseudo-type entry assay of Ebola virus ⁹ . Furthermore, retrospective analysis of relative risks for patients from the 2014 to 2016 West Africa outbreak identified amodiaquine in combination with artesunate as having therapeutic effects. Data have suggested that patients infected with Ebola virus that were prescribed artesunate-amodiaquine had a significantly reduced risk of death compared to those that were prescribed artemether-lumefantrine, the latter treatment not being statistically significantly different from no treatment ¹⁰ . This suggests that the quinoline derivative drug class may have therapeutic use in the treatment of EVD.

In this article, the efficacy of 36 novel quinolines ( Table 1) derivatives and previously approved quinoline compounds (amodiaquine and chloroquine) were examined for their ability to inhibit Ebola virus replication. Our results support previous reports that suggested that amodiaquine and chloroquine could be potential treatments for EVD. In addition, we identified additional, novel, quinolines that could be candidates for further study of their potential for inhibition of Ebola virus infection.

To assess the ability of quinolines to inhibit Ebola virus infection, we employed an established mini-genome model of Ebola replication, trVLP, allowing us to work under biosafety containment level 2 (CL 2) conditions ^{27– 31} .

In our first series of tests, the ability of amodiaquine and chloroquine to inhibit in vitro infection of Ebola virus replication was tested ( Figure 1). Following transfection with the required replication machinery and attachment receptor expression plasmids, HEK-293T cells were treated with the drugs at a concentration of 10 µM two hours pre-infection. Luciferase activity was measured 72 hours post-infection ²² . Both amodiaquine and chloroquine demonstrated significant reductions in luciferase activity, indicating inhibited viral transcription and replication ³² . Following these findings, dose-response experiments for amodiaquine and chloroquine were conducted. Inhibition activity was assessed at concentrations ranging from 10 µM to 0.31 µM ( Figure 2A and 2B). Amodiaquine exhibited a half maximal inhibitory concentration (IC ₅₀) of 1.45 µM, while chloroquine exhibited an IC ₅₀ of 3.95 µM. Cellular toxicity for both amodiaquine and chloroquine was measured using an MTT viability assay ( Figure 2A and 2B) to quantify drug toxicity along the concentration response ²² .

To rapidly assess the inhibitory ability of the 36 novel quinoline derivatives ( Table 1), drugs were screened at a concentration of 10 µM as described above, with drug treatments occurring two hours pre-infection. From these tests, 2OHQ ¹² , 2NH2Q ¹³ , 2QQ ¹⁷ , 2Qi15 ¹⁸ , MD823, TOF411, DD2, DD1, and MBN91 were identified as having inhibitory activity in our in vitro assay ( Table 1). However, due to drug toxicity or availability of synthesized drug, only 2NH2Q was pursued for further study. A dose-response for 2NH2Q was conducted, with inhibition being assessed between 10 µM and 0.31 µM ( Figure 2C). Results indicated the IC ₅₀ of 2NH2Q to be 4.66 µM, slightly higher than either amodiaquine or chloroquine.

As outbreaks of EVD continue to occur, treatment and preventative strategies are urgently needed. In August 2018, the Ministry of Health of the Democratic Republic of the Congo declared a new outbreak of Ebola virus disease in North Kivu Province. As of January 19, 2019, there have been 636 confirmed cases of EVD and 370 confirmed deaths ^{2, 7} . Previously, it was suggested that anti-malarial drugs may be effective for the treatment of EVD ^{8– 10} . In the report, we have provided supporting evidence that antiparasitic drugs can inhibit Ebola virus replication in vitro, which could be a breakthrough in fighting EVD if proven by additional studies or clinical trials.

Quinoline is a heterocyclic aromatic organic compound and is the base structure for many small molecule drugs. Quinoline-based drugs are widely used for the treatment of malarial infections. As their production is cheap, they are easy to synthesize with high yields, and are chemically stable while being readily accessible, quinolines offer many advantages for the treatment of infectious disease in tropical climates, such as the climates found in Africa ^{8, 27} .

Our study aimed to test anti-malarial compounds against an Ebola replication model using the previously published trVLP in vitro assay, which has been shown to accurately reflect CL4 studies using intact infectious Ebola virus ²⁹ . We find amodiaquine to be effective at inhibiting Ebola viral replication. Amodiaquine is a useful anti-malarial agent in regions where Ebola virus outbreaks are possible, and this drug has been suggested to help with EVD outbreaks ¹⁰ . We also show using our assay that other antiparasitic malarial drugs can inhibit Ebola virus replication with low cellular toxicity. These include chloroquine, 3/35 novel synthetic quinolines, one termed 2NH2Q, with high efficacy in inhibiting Ebola virus replication and low toxicity, and 4/35 showing moderate efficacy in inhibiting Ebola virus replication while being relatively non-toxic ( Table 1).

Quinolines, such as amodiaquine or chloroquine, have been used in regions at risk for EVD to prevent malaria and some have stated that use of these compounds is correlated with reduced death from EVD, suggesting that quinolines could be useful as both an anti-malarial and EVD treatment/prevention drug.

In summary, 38 compounds selected for their antiparasitic activity were evaluated against Ebola virus and their cytotoxicity was also determined. Some of them had antileishmanial activity, such as 2PQsel ¹³ and others exhibited antiplasmodial activity, such as chloroquine, amodiaquine and BS460 ³³ . The most interesting compounds emerging from viral inhibition and cytotoxicity assays were chloroquine and amodiaquine, with the advantage of chloroquine having no toxicity at 10 µM. From the quinoline series, the most promising compounds were 2Qi15 and 2NH2Q, both of them being 2-substituted quinolines. When studying structure-activity relationships, the presence of an amino group at the extremity of the propylene chain (compound 2NH2Q) allowed a better cell viability (81%) than a hydroxyl group (compound 2OHQ, 31%), despite their similar capacity for viral inhibition (89%). Replacement by a carboxylic group (compound 2COOHQ) strongly reduced the antiviral inhibition but maintained good cell viability. The other substitutions performed on the quinoline scaffold were not really satisfactory in regard to their biological activity.

Our results support the hypothesis that antiparasitic quinolines active against malaria and leishmaniasis could be efficacious as a treatment for EVD. Further studies are necessary to confirm the utility of quinolines as anti- Ebola virus therapeutics, including consideration of these drugs when designing future Ebola drug trials in Africa.